KR102703683B1 - Novel genome editing TaRGET system and uses thereof - Google Patents

Abstract

The present invention relates to a novel ultracompact gene editing protein, a Cas12f1 mutant protein or a homolog protein thereof, and an ultracompact gene editing system (Hypercompact TaRGET system) comprising the same. The ultracompact gene editing system not only has increased gene editing efficiency, but also can be packaged into a single AAV vector, so that it can be efficiently delivered to a target site in a cell.
The ultra-small gene editing system for nucleic acid editing of the present invention expands the range of gene editing protein selection applicable to various target genes and nucleic acid forms, and can be usefully utilized for the treatment and research of genetic diseases through nucleic acid editing as a next-generation gene editing system that replaces existing large-sized gene editing systems that are difficult to deliver into cells.

Description

{Novel genome editing TaRGET system and uses thereof}

The present invention relates to a TaRGET (Tiny nuclease-augment RNA-based Genome Editing Technology) system, which is an ultra-small gene editing technology developed using a small-sized Cas12f1 variant or a homolog thereof and an engineered guide RNA. In particular, the present invention relates to a Cas12f1 variant or a homolog thereof which is smaller in size and has improved cleavage efficiency compared to existing Cas endonucleases, and a gene editing system using the variant and an engineered guide RNA.

Genome editing technology, which freely corrects the genetic information of living organisms as needed, enables changes to the genome to have the desired genetic information in bacteria, yeast, plants, and animal cells including humans. Currently, gene editing technology is evaluated as a core technology that will create new cutting-edge bio-field industries such as cell engineering, model animal production, transgenic plant production, and use in gene therapy for cancer, genetic diseases, and infectious diseases. Accordingly, gene editing technology has been rapidly developing recently, and various studies are being conducted.

Gene editing is performed through a gene editing system that can accurately find a target gene or target nucleic acid sequence and cut or modify that part, and the CRISPR/Cas system is a representative example. The gene editing system is a complex formed by Cas endonuclease and CRISPR RNA (crRNA) that recognizes the target gene sequence, and in some cases, a transactivating CRISPR RNA (tracrRNA) that binds to the Cas endonuclease may be added. A single guide RNA (sgRNA) in which the crRNA and tracrRNA are connected by a linker is mainly used, and this guide RNA accurately guides the Cas endonuclease of the gene editing CRISPR system to the double-stranded DNA base sequence of the target gene to be cut or modified. The Cas endonuclease located at the target gene site recognizes the protospacer adjacent motif (PAM) adjacent to the target gene sequence and then cleaves or modifies the internal or external base sequence (base pairs, bp) of the target nucleic acid sequence.

The target nucleic acid cut by the genome editing technology system is repaired through the DNA repair mechanism of homologous recombination (HDR) or non-homologous end joining (NHEJ). Through the DNA repair mechanism of non-homologous end joining (NHEJ), random base insertion (insertion and deletion, indel) occurs between the cut DNA regions, and as a result, frameshift mutation or premature mutation occurs in the coding part of the gene, and the target gene is removed (knocked out). Meanwhile, the DNA repair mechanism of homologous recombination (HDR) requires donor DNA to repair the cut DNA, and the sequence of the internal target gene is precisely replaced using the sequence of this donor DNA as a template, thereby completing gene editing (Jinek, M. et al., 2012).

There are various types of gene editing systems in nature, such as the CRISPR (CRISPR/Cas) system, and new CRISPR genes are continuously being discovered. Depending on the composition and number of Cas genes of the protein complex containing the Cas protein, which is a nuclease for gene editing of the CRISPR genome editing technology, it is largely divided into Class 1 and Class 2. Class 1 includes type I, type III, and type IV Cas nucleolytic proteins, and type II, type V, and type VI Cas nucleolytic proteins are classified into Class 2 (Koonin et al., 2017, Makarova et al., 2020). Among these, the Class 2 CRISPR/Cas system is characterized by its effector complex containing a large single protein with multiple domains. Cas9 (type II) derived from Streptococcus pyogenes , which has been most actively studied to date, and CRISPR/Cpf1 (type V), which is being actively studied for gene editing purposes, are representative Class 2 nucleolytic proteins (Chylinski et al., 2014, Shmakov et al., 2015).

However, the CRISPR/SpCas9 system, which has been the most actively studied and is known to be efficient to date, has been pointed out as a disadvantage that the size of the corresponding gene is very large. The SpCas9 gene alone is over 4.3 kb, and if you add the guide RNA and various gene expression components such as promoters and poly A sequences, it exceeds 5 kb. In this case, there is a problem that it cannot be delivered through adeno-associated virus (AAV), which has been proven to be safe and effective as a delivery vehicle for gene therapy. This is because the size of the gene that most AAVs can currently deliver is limited to 4.7 kb.

To overcome these problems, relatively small-sized CRISPR systems such as SaCas9 and CjCas9 have been discovered and developed and are being utilized as gene editing tools. However, the biggest problem pointed out is that these two gene editing tools show relatively inferior gene editing efficiency compared to SpCas9 or Cpf1. In addition, if these CRISPR systems are to be expanded to base editing, prime editing, and epigenetic regulation, they will face the same problem of AAV delivery limitations as SpCas9.

Therefore, it remains a very important task to apply various gene editing technologies and secure a gene editing system with higher efficiency. To solve this problem, a new ultra-small gene editing system is urgently needed that has a gene editing efficiency that is not inferior to that of Cas9 or Cpf1 proteins known to have excellent gene editing efficiency, but is smaller in molecular weight than SaCas9 and CjCas9, which are known as relatively small nucleolytic proteins.

KR 10-2015-0016588 A US 2020/0190494 A1

Jinek, M. et al., A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity, Science, Vol. 337, 816-821(2012) Koonin, E.V. et al., Mobile genetic elements and evolution of CRISPR-Cas system; All the way there and back, Genome Biol. Evol., Vol. 9, No. 10, 2812-2825(2017), Makarova, K.S. et al., Evolutionary classification of the CRISPR-Cas system: a burst of class 2 and derived variants, Nat. Rev. Microbiol., Vol. 18, 67-83(2020) Chylinski, K. et al., Classification and evolution of type II CRISPR-Cas system, Nucleic Acids Research, Vol. 42, no. 10, 6091-6105(2014) Shmakov, S. et al., Discovery and Functional Characterization of diverse class 2 CRISPR-Cas systems, Vol. 60, 385-397(2015) Karvelis, T. et al., PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage, Nucleic Acids Research, Vol. 48, no. 9, 5016-5023(2020) Harrington, L.B. et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science, Vol. 362, 839-842(2018) Takeda, S.N. et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021) Xiao, R. et al., Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease, bioRxiv, 1-20 (2020) Wang, D. et al., Adeno-associated virus vector as a platform for gene therapy delivery, Nat. Rev. Drug Discov., Vol. 18, no. 5, 358-378(2019)

The present invention aims to solve all of the problems described above.

The present invention aims to provide a variant protein of Cas12f1, a homolog protein thereof, or a small endonuclease comprising the same, which is distinct from Cas proteins that act as endonucleases in connection with conventional CRISPR systems.

Another object of the present invention is to provide a guide RNA capable of increasing the indel efficiency of the Cas12f1 mutant protein or a homolog protein thereof.

Another object of the present invention is to provide a gene editing system based on a Cas12f1 mutant protein.

Another object of the present invention is to provide a nucleic acid encoding a gene editing molecule based on a Cas12f1 mutant protein or a vector system for expressing the same.

Another object of the present invention is to provide a gene editing composition based on a Cas12f1 mutant protein.

Another object of the present invention is to provide a method for editing a gene using a Cas12f1 mutant protein.

The purpose of the present invention is not limited to the purpose mentioned above. The purpose of the present invention will become more apparent from the following description, and will be realized by the means and combinations thereof described in the claims.

A representative configuration of the present invention to achieve the above purpose is as follows.

According to one aspect of the present invention, a tiny endonuclease comprising a Cas12f1 variant protein or a homolog protein thereof and a polynucleotide encoding the same are provided.

According to another aspect of the present invention, a gene editing system is provided, comprising a small endonuclease comprising a Cas12f1 mutant protein or a homolog protein thereof; and a guide RNA.

According to another aspect of the present invention, a vector system is provided comprising a first nucleic acid construct operably linked to a nucleotide sequence encoding a small endonuclease comprising a Cas12f1 variant protein or a homolog protein thereof; and/or a second nucleic acid construct operably linked to a nucleotide sequence encoding a guide RNA.

According to another aspect of the present invention, a gene editing composition comprising the gene editing system, the vector system, or both systems is provided.

According to another aspect of the present invention, a gene editing method is provided, comprising a step of contacting the gene editing system, the vector system, or the gene editing composition with a target gene or a target nucleic acid.

The present invention provides an ultra-small genome editing system (termed "Hypercompact TaRGET system") comprising a novel Cas12f1 mutant-based CRISPR protein, which has not been previously known as an endonuclease, and an engineered guide RNA that exhibits excellent genome editing efficiency when used together with the mutant protein. The ultra-small genome editing system of the present invention has the advantage of being able to load all genome editing tools required for editing various genomes targeted at one adeno-associated virus (AAV) vector. Accordingly, the ultra-small genome editing system presents a novel genome editing system that solves the biggest limitation of using AAV vectors, which are clinically proven intracellular delivery vehicles, as packaging tools due to their size in genome editing systems including proteins such as Cas9 or Cpf1, which have been mainly used for chromosome editing in the past.

Above all, the ultra-small gene editing system according to the present invention exhibits excellent target gene editing efficiency by including a novel Cas12f1-based mutant CRISPR protein and a guide RNA suitably engineered therefor.

Figure 1 illustrates modification sites (MS) MS1 to MS5 for engineered guide RNA (hereinafter, “augment RNA”) according to one embodiment of the present invention.
FIGS. 2A and 2B illustrate exemplary structures representing various modification sites for producing engineered single guide RNAs (sgRNAs) according to embodiments of the present invention: FIG. 2A illustrates exemplary modification sites of a canonical sgRNA for a Cas12f1 variant. FIG. 2B illustrates exemplary modification sites of a mature form sgRNA for an engineered Cas12f1 variant according to an embodiment of the present invention.
FIGS. 3A to 3D are graphs showing the indel efficiency of Cas12f1, Cas12f1 variants, Cas12f1 variant v1, Cas12f1 variant v2, Cas12f1 variant v3, and Cas12f1 variants (SEQ ID NO: 1) having an amino acid added to the N-terminus or C-terminus by augment RNA according to an embodiment of the present invention (canonical sgRNA, wild-type guide RNA; Cas12f1_ge3.0, MS1/MS2/MS3 augment RNA; Cas12f1_ge4.0, MS2/MS3/MS4 augment RNA; Cas12f1_ge4.1, MS2/MS3/MS4/MS5 augment RNA): FIG. 3A shows the indel efficiency of Cas12f1 variants for the target sequence Target-1. The results of the measurement are shown. Fig. 3b shows the results of measuring the indel efficiency of Cas12f1 mutants for the target sequence Target-2. Fig. 3c shows the results of measuring the indel efficiency of Cas12f1 mutants for the target sequence Target-3. Fig. 3d shows the results of measuring the indel efficiency of Cas12f1 mutant proteins having an amino acid added to the N-terminus or C-terminus for the target sequences Target-1 and Target-2. Cas12f1_ge4.0 was used as the guide RNA.
Figure 4 shows the results of comparative measurements of the intracellular indel efficiency of existing gene editing proteins (SpCas9, AsCas12a) and Cas12f1 mutants.
Figures 5a and 5b illustrate the results of measuring the indel efficiency (%) of an ultra-small genome editing system comprising an augment RNA and a Cas12f1 variant having one or more modifications among MS1 to MS5 in each region of a wild-type guide RNA: Figure 5a shows the indel efficiency (%) for the target sequence Target-1. Figure 5b shows the indel efficiency (%) for the target sequence Target-2.
FIG. 6 illustrates the results of confirming the indel efficiency (%) for the target sequence Target-1 of the ultra-small gene editing system including augment RNA and Cas12f1 variant according to an embodiment of the present invention.
Figures 7a to 7d illustrate the results of measuring the indel efficiency (%) of the ultra-small genome editing system comprising an augment RNA and a Cas12f1 variant having one or more modifications among MS3 to MS5 in each region of the mature form sgRNA: Figures 7a and 7b are graphs showing the indel efficiency (%) for the target sequence Target-1, respectively. Figures 7c and 7d are graphs showing the indel efficiency (%) for the target sequence Target-2, respectively.
FIGS. 8A and 8B illustrate the results of measuring the indel efficiency (%) for the target sequence Target-1 of the ultra-small genome editing system comprising augment RNA and Cas12f1 variants according to embodiments of the present invention: FIG. 8A is a graph showing the indel efficiency (%) when augment RNA having a modification of MS3-3, MS3-3/MS4-3 or MS3-3/MS4-3/MS5-3 in mature form sgRNA was used. FIG. 8B is a graph showing the indel efficiency (%) when augment RNA having a modification of MS3-3, MS3-3/MS4-3 or MS3-3/MS4-3/MS5-3 and a modification of MS2 was used in mature form sgRNA.

The detailed description of the present invention which follows will be made with reference to specific embodiments in which the present invention may be practiced, with reference to the drawings, but the present invention is not limited thereto, but is limited only by the appended claims along with the full scope of equivalents to which such claims are entitled, if properly described. It should be understood that the various embodiments/embodiments of the present invention are different from each other, but are not necessarily mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be changed from one embodiment/embodiment to another, or embodiments/embodiments may be combined, without departing from the spirit and scope of the present invention. Technical and scientific terms used herein, unless otherwise defined, have the same meaning as commonly used in the art to which this invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and terms used in the singular will include the plural where appropriate, and vice versa.

I. Definition

The term "genome editing protein" or "nucleolytic protein" as used herein refers to an (endo)nuclease that can edit a target nucleic acid, DNA or RNA, or a protospacer adjacent motif (PAM) in a target gene so that double strand breaks (DSB) occur at an internal or external base sequence (base pair, bp) of the target nucleic acid sequence. In addition, the gene editing protein or nucleolytic protein is also referred to as an effector protein constituting a gene editing system or a nucleic acid construct for gene editing. Here, the effector protein may be a nucleolytic protein capable of binding to a guide RNA (gRNA) or an engineered RNA, or a peptide fragment capable of binding to a target nucleic acid or a target gene.

The term "gene editing CRISPR/Cas system" or "gene editing system" refers to a complex comprising a gene editing protein or a nucleolytic enzyme such as Cas endonuclease and a nucleic acid targeting molecule corresponding to the nucleolytic enzyme, which can bind to a target nucleic acid or a target gene to cleave or edit a target nucleic acid or a target region of the gene. Here, the nucleic acid targeting molecule may be represented by a guide RNA (gRNA), but is not limited thereto.

The term "Hypercompact TaRGET system" refers to a complex including a nucleolytic enzyme such as an ultracompact gene editing protein or a small endonuclease and a nucleic acid targeting molecule corresponding to the nucleolytic enzyme, and is used as a term to differentiate it from existing gene editing systems. Here, the nucleic acid targeting molecule may be represented by a guide RNA (gRNA), but is not limited thereto. The system refers to a complex capable of binding to a target nucleic acid or a target gene to cleave or edit a target region of the target nucleic acid or gene.

The term "nucleic acid construct" refers to a structure comprising a nucleotide sequence encoding a genome editing protein or a nucleic acid decomposition protein and/or a nucleotide sequence encoding a guide RNA as components, and may additionally comprise a nucleotide sequence encoding various types of (poly)peptides or linkers as needed. The nucleic acid construct can be used as a component of a vector for gene editing of the present invention or a hypercompact TaRGET system.

The term "target nucleic acid" or "target gene" refers to a gene or nucleic acid that is a target or target of gene editing by an ultra-compact genome editing system (e.g., Hypercompact TaRGET system). The target nucleic acid or target gene may be used interchangeably and may refer to the same target. Unless otherwise specified, the target gene may be a unique gene or nucleic acid of a target cell, a gene or nucleic acid derived from an external source, or a nucleic acid or gene synthesized artificially, and may refer to all of single-stranded DNA, double-stranded DNA, and/or RNA. The target gene or target nucleic acid is not particularly limited as long as it can be a target of gene editing by an ultra-compact genome editing system.

The term "target region" or "target sequence" refers to a sequence existing within a target nucleic acid or a target gene, and refers to a specific sequence that the ultra-small gene editing system of the present invention recognizes to cleave the target gene or target nucleic acid. The target region or target sequence may be appropriately selected depending on the purpose.

The term "guide RNA (gRNA)" refers to an RNA comprising a guide sequence capable of forming a complex with a gene editing protein or a nucleolytic protein, hybridizing with a target nucleic acid sequence, and having sufficient complementarity to the target nucleic acid sequence to cause sequence-specific binding of the complex to the target nucleic acid sequence. Guide molecule or guide RNA are used interchangeably herein.

The term "scaffold region" refers collectively to the portion of a guide RNA (gRNA) that can interact with a genome editing protein or a nucleolytic protein, and can refer to the remaining portion of a guide RNA found in nature, excluding the spacer.

The term "spacer sequence" refers to a polynucleotide that hybridizes with a portion of a target sequence in an ultra-small genome editing system. For example, the spacer sequence refers to 10 to 50 contiguous nucleotides near the 3'-end of a crRNA of a guide RNA in an ultra-small genome editing system.

The terms "tracrRNA" and "crRNA" include all meanings that would be recognized by a person skilled in the art of genome editing technology. They can be used as terms referring to each molecule of a dual guide RNA found in nature, and can also be used to refer to each corresponding portion of a single guide RNA connecting the tracrRNA and crRNA with a linker. Unless otherwise stated, when they are written as tracrRNA and crRNA alone, they mean the tracrRNA and crRNA constituting the genome editing system.

The term "vector", unless otherwise specified, refers to all materials capable of transporting genetic material into a cell. For example, the vector may be a DNA molecule containing a nucleic acid encoding an effector protein of a genome editing system, which is the genetic material to be delivered, and/or a nucleic acid encoding a guide RNA (gRNA), but is not limited thereto. In addition, the "vector" in the present invention may be an "expression vector" containing essential regulatory elements operably linked so that the inserted gene is normally expressed. The term "operably linked" means, in gene expression technology, that a specific structure is linked to another structure so that the specific structure can function in the intended manner.

The term "engineered" is a term used to distinguish it from substances, molecules, etc. that already have a composition that exists in nature, and means that an artificial modification has been applied to said substances, molecules, etc. For example, in the case of "engineered guide RNA", a guide RNA (gRNA) that has an artificial modification applied to the composition of a guide RNA (gRNA) that exists in nature may be referred to as augment RNA in this specification.

The terms "polynucleotide" and "nucleic acid" may be used interchangeably and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, the terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, unnatural or derivatized nucleotide bases. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides, as applicable to the embodiments described herein.

The terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to a polymeric form of amino acids of any length which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The terms include fusion proteins, including but not limited to, fusion proteins with heterologous amino acid sequences, with or without an N-terminal methionine residue, fusions with heterologous and homologous leader sequences; immunologically tagged proteins, and the like.

The terms "A, T, C, G and U" may be appropriately interpreted as a base, a nucleoside or a nucleotide in DNA or RNA, depending on the context and technology. For example, when referring to a base, it may be interpreted as one selected from adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), respectively. When referring to a nucleoside, it may be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G) or uridine (U), respectively, and when referring to a nucleotide in a sequence, it should be interpreted as meaning a nucleotide containing each of the above nucleosides.

The term "about" means an amount, level, value, number, frequency, percent, dimension, size, quantity, weight or length that varies by 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 percent of the reference amount, level, value, number, frequency, percent, dimension, size, quantity, weight or length.

All technical terms used in the present invention, unless otherwise defined, include all meanings that can be recognized by a person skilled in the art, are used with the same meaning as commonly understood, and can be appropriately interpreted according to the context. In addition, although preferred methods or samples are described in this specification, similar or equivalent ones are also included in the scope of the present invention.

II. High-efficiency, ultra-small gene editing system/composition

The present inventors have confirmed that the TnpB protein, known as a factor constituting the transposase derived from (bacterial species), has an amino acid sequence similar to the Cas12f1 protein, has a molecular weight about 1/3 smaller than that of existing nucleic acid-degrading proteins including the Cas9 protein, which has been studied the most to date, and has a much higher nucleic acid cleavage efficiency for target nucleic acids or target genes, thereby defining the protein as a Cas12f1 mutant protein and elucidating for the first time that the protein exhibits highly efficient ultra-small gene editing protein activity.

In addition, the present inventors have created a novel ultra-small gene editing system comprising a small endonuclease including a Cas12f1 mutant protein or a homolog protein thereof, which is an ultra-small gene editing protein, and an augment RNA showing high indel efficiency for the Cas12f1 mutant protein, as a system that can be easily loaded onto an adeno-associated virus (AAV) vector and can be effectively delivered into cells (in vivo) to edit target nucleic acids or target genes within a cell.

The present inventors have confirmed for the first time that more efficient and applicable gene editing is possible by using a novel ultra-small gene editing protein, Cas12f1 mutant protein, rather than the previously known Cas endonuclease, such as Cas9 or Cpf1, and have completed the present invention based on the fact that the novel ultra-small gene editing system can be utilized for various genome editing.

Therefore, the present invention relates to an ultra-compact gene editing system (Hypercompact TaRGET system) comprising a small endonuclease comprising a Cas12f1 mutant protein or a homolog protein thereof; and a guide RNA, for use in specifically and highly efficiently editing a target nucleic acid or a target gene.

Furthermore, the present invention relates to a construct or vector for ultra-small nucleic acid editing comprising a small endonuclease comprising a Cas12f1 mutant protein or a homolog protein thereof, a method for editing a target nucleic acid or a target site in a target gene using the same, and a composition therefor.

The above-described ultra-small gene editing system according to the present invention is a meaningful result that overcomes the limitation of loading most of the Cas endonucleases and gene scissors systems containing them, which have been studied previously, onto adeno-associated virus (AAV) vectors approved by the FDA as intracellular delivery vehicles due to their size.

Furthermore, since the above-described ultra-compact gene editing system (Hypercompact TaRGET system) has high gene editing specificity and editing efficiency for cutting a specific target region of a target nucleic acid or a target gene, the ultra-compact gene editing system (Hypercompact TaRGET system) according to the present invention has a wide range of applications in editing technology research for various editing of target nucleic acids and as a new treatment for gene-related diseases.

Hereinafter, each component of the ultra-small gene editing system/composition provided in the present invention and its manufacturing method are described in detail.

1. Cas12f1 mutant protein and its homolog protein

According to one aspect of the present invention, a Cas12f1 mutant protein or a homolog protein thereof or a (small) endonuclease comprising them is provided, which exhibits excellent activity in cleaving a target site of a target nucleic acid and is characterized in that the size of the nucleic acid-degrading protein is significantly smaller by about 1/3 compared to the existing CRISPR/Cas9 system.

The Cas12f1 variant protein includes both a Cas12f1 variant found in nature and an engineered Cas12f1 variant. Specifically, the Cas12f1 variant protein includes an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 1 to 28 amino acids deleted or substituted from the N-terminus based on the sequence (except for a Cas12f1 protein consisting of an amino acid sequence of SEQ ID NO: 5).

In some embodiments, the Cas12f1 variant protein may be or include a sequence derived from a transposase accessory protein TnpB protein of the IS200/IS605 family, which is similar in size to the Cas12f1 protein belonging to the V-F subtype of Class 2, type V CRISPR/nucleolytic proteins. The TnpB protein is a protein conventionally known as a transposase. Until now, the TnpB protein has only been known as a transposon-encoded nuclease encoding a transposon, and it is not known whether the TnpB protein has Cas endonuclease activity. In addition, the guide RNA for the TnpB protein is also not known. The present invention was completed by first confirming that a Cas12f1 variant or an engineered Cas12f1 variant based in part on the TnpB protein sequence has excellent endonuclease activity for targeting and editing a target nucleic acid or a target gene while being similar in size to the Cas12f1 protein, which belongs to the group with the smallest molecular weight among nucleolytic proteins, and by producing a guide RNA that exhibits excellent editing efficiency when used together with the Cas12f1 variant protein. The Cas12f1 variant protein belongs to the group with the smallest molecular weight among the currently existing nucleolytic proteins, and forms a complex with the engineered short guide RNA (gRNA) of the present invention to have an excellent effect for targeting and editing a target nucleic acid or a target gene, and therefore has a great advantage in producing an ultra-small gene editing system for intracellular application. In addition, unlike Cas9 which has 5'-NGG-3' as a PAM, the Cas12f1 mutant protein or its homolog protein, which is an ultra-small gene editing protein, has a T-rich PAM such as 5'-TTTA-3' or 5'-TTTG-3' as a PAM, thereby enabling the selection of a sequence rich in thymine (T) as a target nucleic acid or target gene, thereby expanding the range of choices of nucleic acid-decomposing proteins for genome editing.

In some embodiments, the Cas12f1 variant protein can be a variant protein comprising or consisting of the amino acid sequence of SEQ ID NO: 1. Furthermore, the Cas12f1 variant protein can be a Cas12f1 variant protein comprising or consisting of an amino acid sequence in which 1 to 28 amino acids are deleted or substituted at the N-terminus of the amino acid sequence of SEQ ID NO: 1. In this case, a Cas12f1 protein comprising the amino acid sequence of SEQ ID NO: 5 is not included. Specifically, the Cas12f1 variant protein can be a Cas12f1 variant protein comprising or consisting of any one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 4.

In another embodiment, the Cas12f1 variant protein can be a Cas12f1 protein comprising an amino acid sequence of SEQ ID NO: 5, which further comprises one or more amino acids. Specifically, the Cas12f1 variant protein can comprise or consist of a Cas12f1 variant v1 protein (SEQ ID NO: 2) comprising an N-terminal 26aa of CasX at the N-terminus of the Cas12f1 protein, a Cas12f1 variant v2 protein (SEQ ID NO: 3) comprising a 28aa random sequence, or a Cas12f1 variant v3 protein (SEQ ID NO: 4) comprising a 26aa random sequence.

In some embodiments, a homolog protein of a Cas12f1 variant protein can be a TnpB protein from a variety of organisms or a variant derived therefrom. Specifically, the homolog protein can comprise any one amino acid sequence selected from the group consisting of SEQ ID NO: 141 to SEQ ID NO: 232. A homolog protein means a protein that shares the same in vivo activity (i.e., endonuclease activity) as a Cas12f1 variant protein, and regardless of their sequence similarity (or identity), means a protein that has preserved characteristics derived from a common ancestor without being lost.

In some implementations, the Cas12f1 variant protein may be comprised of an amino acid sequence having 1 to 600 amino acids added to the N-terminus or C-terminus based on the amino acid sequence of SEQ ID NO: 1. Specifically, the amino acid sequence added to the N-terminus or C-terminus may be the amino acid sequence of SEQ ID NO: 233 or SEQ ID NO: 234. An NLS sequence may further be included between the additional sequence and the Cas12f1 variant protein.

In addition, the Cas12f1 variant protein may have the same function as the wild-type Cas12f1 protein, or may have a changed function compared to the wild-type Cas12f1 protein. More specifically, the change includes a modification of all or part of the function, a loss of all or part of the function, and/or the addition of an additional function. The Cas12f1 variant protein may include any change without particular limitation, as long as it is a change that a person skilled in the art can apply to the nucleolytic protein of the ultra-small genome editing system. For example, the Cas12f1 variant protein may have an activity to cleave a DNA double-strand, as well as an activity to cleave a single-stranded DNA or RNA, or a hybrid double-strand of DNA and RNA, and to perform base editing or prime editing.

In some embodiments, since the miniature genome editing system of the present invention cleaves a nucleic acid at a target nucleic acid or a target site of a target gene, the target site may be characterized in that it is located in the nucleus of a cell. Accordingly, the Cas12f1 variant protein or a homolog thereof used in the miniature genome editing system of the present invention may include one or more nuclear localization signal (NLS) sequences that localize it into the nucleus. For example, the one or more nuclear localization signal sequences may have a sufficient amount or activity strength to induce the Cas12f1 variant protein or a homolog thereof to be targeted into the nucleus of a eukaryotic cell (including a mammalian cell) in a detectable amount in the nucleus. For example, the difference in the activity strength may result from the number of NLSs included in the Cas12f1 variant protein or a homolog thereof, the type of specific NLS(s) used, or a combination of these factors.

Additionally, in other embodiments, the variant protein or an analog thereof can comprise variously selected from about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more NLSs at or near the amino-terminus (N-term), about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more NLSs at or near the carboxy-terminus (C-term), or combinations thereof. For example, the protein can comprise zero or at least one NLS sequence at the amino-terminus (N-term) and/or zero or one NLS sequence at the carboxy-terminus (C-term). When more than one NLS sequence is present, each NLS sequence can be selected independently of the others, such that a single NLS can be present in more than one copy, and can be present in combination with more than one other NLS that is present in more than one copy.

In some embodiments, the NLS sequence is heterologous to the protein, including but not limited to the following NLS sequences. For example, the NLS can be the NLS of the SV40 virus large T-antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 54), the nucleoplasmin bipartite NLS having the NLS sequence KRPAATKKAGQAKKKK (SEQ ID NO: 55) from nucleoplasmin, the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 56) or RQRRNELKRSP (SEQ ID NO: 57). Additionally, it may be an NLS sequence derived from the hRNPA1 M9 NLS sequence, the NLS sequence of the IBB domain from importin-alpha, the NLS sequence of myoma T protein, the NLS sequence of human p53, the NLS sequence of mouse c-abl IV, the NLS sequence of influenza virus NS1, the NLS sequence of hepatitis virus delta antigen, the NLS sequence of mouse Mx1 protein, the NLS sequence of human poly(ADP-ribose) polymerase or the NLS sequence of the steroid hormone receptor (human) glucocorticoid.

In addition, the Cas12f1 variant protein or its homolog protein may be a fusion of various enzymes that can be involved in the gene expression process within a cell. At this time, the Cas12f1 analog protein to which the enzyme is fused may cause various quantitative and/or qualitative changes in gene expression within a cell. For example, the various enzymes to be additionally coupled may be DNMT, TET, KRAB, DHAC, LSD, p300, Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase or a variant thereof. At this time, the Cas12f1 variant protein or its homolog protein to which the reverse transcriptase is fused may also function as a prime editor.

2. PAM sequence of Cas12f1 mutant protein

In some implementations, the following two conditions are required for the miniature gene editing system to be positioned at the target nucleic acid or target site of the target gene and to accurately cleave the target site nucleic acid.

First, there must be a base sequence of a certain length that can be recognized by the Cas12f1 variant protein or its homolog protein in the target nucleic acid or target gene. In addition, there must be a sequence that can complementarily bind to a spacer sequence included in a guide RNA (gRNA) for the Cas12f1 variant protein or its homolog protein around the base sequence of the certain length. In other words, when the Cas12f1 variant protein or its homolog protein recognizes the base sequence of the certain length, and the spacer sequence portion included in the guide RNA (gRNA) complementarily binds to a sequence portion around the base sequence of the certain length, the target nucleic acid or the target site nucleic acid of the target gene can be accurately cut or edited. At this time, the base sequence of a certain length recognized by the Cas12f1 variant protein or its homolog protein is called a protospacer adjacent motif (PAM) sequence. The PAM sequence is a unique sequence determined by the Cas12f1 variant protein or its homolog protein, which is an ultra-small gene editing protein. This means that when determining the target sequence of the Cas12f1 variant protein or its homolog protein complex in the ultra-small gene editing system, the target sequence must be determined within the sequence adjacent to the PAM sequence.

The PAM sequence of the Cas12f1 variant protein or a homolog protein thereof may be a T-rich sequence. More specifically, the PAM sequence of the Cas12f1 variant protein or a homolog protein thereof may be 5'-TTTN-3', wherein N is one of deoxythymidine (T), deoxyadenosine (A), deoxycytidine (C), or deoxyguanosine (G).

In one embodiment, the PAM sequence of the Cas12f1 variant protein or a homolog protein thereof can be 5'-TTTA-3', 5'-TTTT-3', 5'-TTTC-3' or 5'-TTTG-3'. Preferably, the PAM sequence of the Cas12f1 variant protein or a homolog protein thereof can be 5'-TTTA-3' or 5'-TTTG-3'.

In another embodiment, the PAM sequence of the Cas12f1 variant protein or a homologous protein thereof may be different from the PAM sequence of the wild-type Cas12f1 variant protein (or a protein comprising a sequence derived from TnpB).

3. Engineered guide RNA (augment RNA) for Cas12f1 mutant protein

(1) Overview

The embodiments of the present invention have been derived to overcome the limitations of intracellular delivery due to the protein molecular weight of the Cas9 of the prior art. Therefore, in addition to selecting a Cas12f1 variant protein or a homolog protein having a small molecular weight as a gene editing protein included in the ultra-small gene editing system of the present invention, a guide RNA (gRNA) for the Cas12f1 variant or a homolog thereof was artificially engineered to be much shorter than that existing in nature to achieve size minimization and at the same time produce an engineered guide RNA (augment RNA) with increased indel efficiency for the target.

First, since a guide RNA (gRNA) existing in nature has not been found for the Cas12f1 mutant, which is an ultra-small gene editing protein of the present invention, an optimal guide RNA (gRNA) exhibiting high-efficiency targeting and editing activity for the Cas12f1 mutant protein was created. From this perspective, the guide RNA (gRNA) existing in nature for the Cas12f1 mutant protein may be a guide RNA (gRNA) found in nature for a Cas12f1 protein having a size similar to that of the Cas12f mutant protein. Specifically, the guide RNA (gRNA) may have a base sequence of SEQ ID NO: 6.

In one embodiment, the guide RNA (gRNA) for the Cas12f1 mutant protein is characterized by being an engineered guide RNA (engineered gRNA or augmented RNA) that adds a new structure to a guide RNA (gRNA) found in nature or modifies part of its structure, and includes a new structure, a U-rich tail, at the 3'-terminus of the guide RNA (gRNA). As an example, the guide RNA is an engineered guide RNA in which one or more nucleotide sequences are deleted, substituted, or added to a wild-type guide RNA consisting of a nucleotide sequence of SEQ ID NO: 6, and the spacer portion of the engineered guide RNA complementary to the target sequence may be composed of a nucleotide sequence of 15 to 50 nucleotides.

In some embodiments, the engineered guide RNA can comprise an engineered tracrRNA sequence comprising the first to fourth regions of the scaffold and/or an engineered crRNA comprising the fifth to sixth regions of the scaffold. In addition, the engineered guide RNA can comprise a seventh region, a U-rich tail sequence, at the 3' end of the crRNA. In some embodiments, the engineered guide RNA can be engineered at one or more modification sites selected from modification sites MS1 to MS5. Figure 1 illustrates modification sites MS1 to MS5 comprised by an engineered guide RNA (engineered gRNA) according to an embodiment of the present invention. In addition, the engineered guide RNA can comprise an engineered tracrRNA sequence comprising a scaffold in which one or more regions of the first to fourth regions of the scaffold are modified, an engineered crRNA sequence comprising a scaffold in which one or more regions of the fifth to sixth regions of the scaffold are modified, and/or a U-rich tail sequence which is a modified seventh region. The fourth region of tracrRNA and the fifth region of crRNA are complementary binding sites and include modification site 1 (MS1) and modification site 4 (MS4) of the guide RNA (gRNA). In addition, the seventh region, the U-rich tail sequence, corresponds to modification site 2 (MS2). The first region is modification site 3 (MS3), and the second region includes modification site 5 (MS5). The engineered guide RNA includes a modification in any one of MS1 to MS5, and may include any combination of one or more modifications selected from them.

In some embodiments, the ultra-small genome editing system may be engineered to optimize the length of tracrRNA and crRNA constituting the guide RNA, and to remove unnecessary scaffold sequences to produce highly efficient guide RNAs. The manipulation in the scaffold sequence enables the production of short guide RNAs, resulting in reduced guide RNA synthesis costs and securing additional loading space when inserted into a viral vector. Above all, the ultra-small genome editing system comprising an engineered guide RNA optimized for the Cas12f1 variant protein of the present invention significantly improves the efficiency of cleavage or editing of a target nucleic acid or a target gene, and further makes it more advantageous to use it as a therapeutic agent by loading it into an adeno-associated virus (AAV) vector.

Wild-type tracrRNA (SEQ ID NO: 58; 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAA-3'), which can be a naturally occurring guide RNA for a Cas12f1 mutant protein or a homolog protein thereof, contains five consecutive uridine (U) sequences therein. This has a problem in that, when the wild-type tracrRNA is attempted to be expressed in a cell using a vector, etc., the sequence acts as a transcription termination signal under certain conditions. Therefore, when the five consecutive uridine (U) sequences function as a termination signal, the normal expression of the tracrRNA is suppressed, and the formation of normal guide RNA is also inhibited, resulting in a decrease in the cleavage or editing efficiency of the target nucleic acid or target gene of the ultra-small gene editing system of the present invention. In one embodiment, the inventors of the present invention developed a tracrRNA in which at least one uridine (U) base among the five consecutive uridine (U) sequences of the wild-type tracrRNA (SEQ ID NO: 58) was artificially modified to another base, A, C, T, or G.

In addition, in one embodiment, the engineered guide RNA is characterized by adding a new structure to a guide RNA found in nature and removing or modifying part of its structure, and including a new structure, a U-rich tail, at the 3'-terminus. The engineered guide RNA including the U-rich tail serves to increase the nucleic acid cleavage or editing rate for a target nucleic acid or a target gene of an ultra-small gene editing system. The present inventors produced a highly efficient guide RNA having increased cleavage or editing efficiency of a target nucleic acid or a target gene by forming a complex with a Cas12f1 mutant protein or a homolog protein thereof, among the engineered guide RNAs (engineered gRNA), and completed an ultra-small gene editing system including the same.

In another embodiment, the engineered guide RNA is characterized in that at least a portion of the scaffold region that interacts with the Cas12f1 variant protein in its composition is modified. The scaffold region includes portions of the tracrRNA and the crRNA, and does not necessarily refer to one molecule of RNA. For example, the sequence of the engineered guide RNA can include an engineered tracrRNA sequence comprising a modification in one or more of the first to fourth regions of the scaffold and/or an engineered crRNA sequence comprising a modification in one or more of the fifth to sixth regions of the scaffold, and further include a modified seventh region, a U-rich tail sequence.

Additionally, the engineered guide RNA may further include a linker or tag as needed.

In one embodiment, the engineered scaffold region may be a combination of modifications in any one or more of the first to seventh regions described above in a scaffold region found in nature. In this case, the engineered tracrRNA may be a tracrRNA that is modified not to include five or more consecutive uridine sequences (MS1 modification). In addition, the engineered tracrRNA may be a tracrRNA that is modified not to include five or more consecutive uridine sequences and is modified to be shorter than a wild-type tracrRNA. In addition, the engineered tracrRNA may include the first region, the second region, the third region, and the fourth region (including the MS1 modification) in the order from the 5'-end to the 3'-end. In addition, the engineered crRNA may include the fifth region, the sixth region, and the spacer sequence, which is a guide sequence, in the order from the 5'-end to the 3'-end. The fourth region can comprise any polynucleotide having sufficient complementarity to bind to the direct repeat sequence of the crRNA.

The first region (MS3 portion, regions 1-21) can be a 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 7) sequence or a partial sequence of the SEQ ID NO: 7 sequence. The partial sequence of the SEQ ID NO: 7 sequence can be a sequential partial sequence from the 3'-terminal end of the SEQ ID NO: 7 sequence, wherein the 5'-terminal sequence is sequentially removed. More specifically, the first region can be 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 8), 5'-UGGAGAA-3' or 5'-A-3'. Alternatively, the engineered tracrRNA can be one in which the sequence corresponding to the first region (region 1-21) is completely removed.

The second region (MS5 portion, region 22-71) may be a 5'-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 9) sequence or a partial sequence of the SEQ ID NO: 9 sequence. The partial sequence of the SEQ ID NO: 9 sequence may be a sequence in which at least one pair of nucleotides forming a complementary bond and/or at least one nucleotide not forming a complementary bond in the SEQ ID NO: 9 sequence is deleted. In one embodiment, the second region may be a 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGUG-3' (SEQ ID NO: 10) sequence, a 5'-CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 11) sequence, or a 5'-CCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 12) sequence. In this case, the loop portion included in any one of the sequences of SEQ ID NO: 9 to SEQ ID NO: 12 is a 5'-UUAG-3' sequence, which may be replaced with a 5'-GAAA-3' sequence as needed.

The third region (front part of MS4, region 72-129) may be the sequence 5'-GGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAA-3' (SEQ ID NO: 13) or a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 13.

The fourth region (MS4 portion including MS1, portions 130-161) can be a 5'-CAAAUUCANNNNNCCUCUCCAAUUCUGCACAA-3' (SEQ ID NO: 14) sequence or a portion of the SEQ ID NO: 14 sequence. In the SEQ ID NO: 14 sequence, the internal 5'-NNNNN-3' portion is the MS1 portion, wherein each N can be A, C, G or U. In one embodiment, the portion of the SEQ ID NO: 14 sequence can be a sequence including the 5'-CAAAUUCANNNNN-3' (SEQ ID NO: 15) sequence of the SEQ ID NO: 14 sequence but not including a portion of the 3'-terminal sequence. Specifically, it can be a 5'-CAAAUUCANNNNNCCUCUCCAAUUC-3' (SEQ ID NO: 16) sequence, a 5'-CAAAUUCANNNNNCCUCUC-3' (SEQ ID NO: 17) sequence or a 5'-CAAAUUCANNNNN-3' (SEQ ID NO: 15) sequence. In addition, the fourth region can include a 5'-NNNNN-3' site is replaced with 5'-NNNVN-3' or 5'-NVNNN-3', wherein each N is independently A, C, G or U, and V can be A, C or G. Preferably, the fourth region can be a sequence including a 5'-CAAAUUCANNNCN-3' (SEQ ID NO: 18) sequence but not including a portion of the 3'-terminal sequence. Wherein each N is independently A, C, G or U. In one embodiment, the crRNA can be a wild-type crRNA or an engineered crRNA. The above wild-type crRNA may include a spacer sequence, which is a wild-type repeat sequence and a guide sequence, in order from the 5'-terminal to the 3'-terminal. The wild-type repeat sequence may be a sequence of 5'-GUUGCAGAACCCGAAUAGACGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 19).

Meanwhile, the engineered guide RNA includes an engineered tracrRNA (transactivating CRISPR RNA) or an engineered crRNA (CRISPR RNA), wherein the engineered tracrRNA is a tracrRNA modified not to include five or more consecutive uridine sequences and having a shorter nucleotide sequence length than a wild-type tracrRNA, and the engineered crRNA may include the nucleotide sequence of SEQ ID NO: 19 or a portion thereof.

Specifically, in the engineered crRNA, the fifth region can be a 5'- GUUGCAGAACCCGAAUAGNNNNNUGAAGGA-3' (SEQ ID NO: 20) sequence or a portion of the SEQ ID NO: 20 sequence. Each N can independently be A, C, G or U. The portion of the SEQ ID NO: 20 sequence can be a sequence that includes the 5'-NNNNNUGAAGGA-3' (SEQ ID NO: 21) sequence of SEQ ID NO: 20 sequence but does not include a portion of the 5'-terminal sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 nucleotide sequence). Preferably, the fifth region may comprise the sequence 5'-NBNNNUGAAGGA-3' (SEQ ID NO: 22), wherein each N may independently be A, C, G or U, and wherein B may be U, C or G.

Additionally, the sixth region may be a 5'-AUGCAAC-3' (SEQ ID NO: 23) sequence or a sequence having at least 70% sequence identity or sequence similarity to the 5'-AUGCAAC-3' sequence.

Additionally, the engineered crRNA can further include a U-rich tail sequence as a seventh region at the 3'-end of the crRNA. The U-rich tail sequence can be a 5'-(UaN)dUe-3' sequence, a 5'-UaVUaVUe-3' sequence or a 5'-UaVUaVUaVUe-3' sequence. Here, N can be A, C, G or U, respectively, and V can be independently A, C or G. Additionally, a can be an integer from 0 to 4, and d can be an integer from 0 to 3. e can be an integer from 0 to 10. In one embodiment, the seventh region can be U ₄ AU ₄ .

Additionally, in one embodiment, the engineered crRNA can include a fifth region (including the MS1 modification), a sixth region, and a guide sequence (spacer sequence) in that order from the 5'-end to the 3'-end. Specifically, the fifth region can be a 5'-GUUGCAGAACCCGAAUAGNBNNNUGAAGGA-3' (SEQ ID NO: 59) sequence or a portion of the SEQ ID NO: 59 sequence. Here, N can independently be A, C, G, or U, and B can be U, C, or G. The portion of the SEQ ID NO: 59 sequence can be a sequence that includes the 5'-NBNNNUGAAGGA-3' (SEQ ID NO: 60) sequence among the SEQ ID NO: 59 sequences but does not include a portion of the 3'-end sequence. Here, N can independently be A, C, G, or U. B can be U, C, or G.

The engineered guide RNA (engineered gRNA) can be a dual guide RNA or a single guide RNA. When the engineered guide RNA is a single guide RNA, the engineered guide RNA can further include a linker sequence. At this time, the linker sequence can be located between the engineered tracrRNA and the crRNA, and the linker sequence can be located between the engineered tracrRNA and the crRNA, and can be 5'-GAAA-3' or 5'-UUAG-3'. More specifically, the engineered tracrRNA has the sequence 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUCCAAUUCUGCACAA-3' (SEQ ID NO: 24), 5'-ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUCCAAUUCUGCACAA-3' (SEQ ID NO: 25), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAUUAGUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUCCAAUUCUGCACAA-3' (SEQ ID NO: 26) sequence or 5'-ACCGCUUCACCAAUUAGUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUCCAAUUCUGCACAA-3' (SEQ ID NO: 27).

Additionally, the engineered crRNA can comprise the sequence 5'-GUUGCAGAACCCGAAUAGNGNNNUGAAGGAAUGCAAC-3' (SEQ ID NO: 28), wherein each N can independently be A, C, G, or U.

Preferably, the engineered tracrRNA may comprise or consist of any one of the base sequences selected from SEQ ID NO: 29 to SEQ ID NO: 32, wherein the internal 5'-NNNCN-3' sequence is replaced with the 5'-GUGCU-3' sequence in any one of the base sequences of SEQ ID NO: 24 (MS1), SEQ ID NO: 25 (MS1/MS3), SEQ ID NO: 26 (MS1/MS5-3), or SEQ ID NO: 27 (MS1/MS3/MS5-3).

Additionally, the engineered crRNA may include or consist of a base sequence of 5'-GUUGCAGAACCCGAAUAG NGNNN UGAAGGAAUGCAAC-3' (SEQ ID NO: 33), in which the 5'-NGNNN-3' sequence within the base sequence of 5'-GUUGCAGAACCCGAAUAG NGNNN UGAAGGAAUGCAAC-3' (SEQ ID NO: 28) is replaced with the 5'-AGCAA-3' sequence.

As another example, the engineered tracrRNA has the sequence 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUC-3' (SEQ ID NO: 34), 5'-ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUC-3' (SEQ ID NO: 35), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAUUAGUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUG UCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUC-3' (SEQ ID NO: 36) sequence or 5'-ACCGCUUCACCAAUUAGUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUC-3' (SEQ ID NO: 37) sequence.

At this time, the engineered crRNA can include the sequence 5'-GAAUAGNGNNNUGAAGGAAUGCAAC-3' (SEQ ID NO: 38) and a guide sequence, wherein each N can independently be A, C, G, or U.

Preferably, the engineered tracrRNA may comprise or consist of any one of the base sequences selected from SEQ ID NO: 39 to SEQ ID NO: 42, wherein the internal 5'-NNNCN-3' sequence is replaced with the 5'-GUGCU-3' sequence in any one of the base sequences of SEQ ID NO: 34 (MS1/MS4-2), SEQ ID NO: 35 (MS1/MS3/MS4-2), SEQ ID NO: 36 (MS1/MS5-3/MS4-2), or SEQ ID NO: 37 (MS1/MS3/MS5-3/MS4-2).

Additionally, the engineered crRNA may comprise or consist of a sequence of 5'-GAAUAG AGCAA UGAAGGAAUGCAAC-3' (SEQ ID NO: 43), wherein the 5'-NGNNN-3' sequence within the base sequence of 5'-GAAUAG NGNNN UGAAGGAAUGCAAC-3' (SEQ ID NO: 38) is replaced with the sequence 5'-AGCAA-3'.

The engineered tracrRNA may be a tracrRNA in which the first stem-loop structure and/or the second stem-loop structure (the first region comprising the MS3 modification and the second region comprising the MS5 modification herein) from the 5'-end of the wild-type tracrRNA is removed. Here, the removal of the second stem-loop structure may still have at least one double-stranded duplex structure composed of at least two or more nucleotides, and the loop structure may not be removed. In addition, the engineered tracrRNA may be a tracrRNA modified such that it does not contain five or more consecutive uridine sequences at any polynucleotide portion (the fourth region comprising the MS1 and/or MS4 modification herein) that has sufficient complementarity to bind to the crRNA sequence. Additionally, the engineered tracrRNA can comprise any nucleotide sequence of 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 1 to 6, 2 to 6, or 3 to 6 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) that has sufficient complementarity to bind to the crRNA sequence.

In one embodiment, the engineered guide RNA can comprise a sequence selected from the group consisting of SEQ ID NO: 44 to SEQ ID NO: 48 and SEQ ID NO: 105 to SEQ ID NO: 137; or a sequence selected from the group consisting of SEQ ID NO: 125 to 137, wherein a U-rich tail sequence is added to the 3'-end. For example, the engineered guide RNA (augment RNA) for the ultra-small gene editing protein Cas12f1 variant protein of the present invention or a homolog protein thereof may be an augment RNA having a modification in MS1 (SEQ ID NO: 44), an augment RNA having a modification in MS1/MS2 (SEQ ID NO: 45), an augment RNA Cas12f1_ge3.0 having a modification in MS1/MS2/MS3 (SEQ ID NO: 46), an augment RNA Cas12f1_ge4.0 having a modification in MS2/MS3/MS4 (SEQ ID NO: 47), and/or an augment RNA Cas12f1_ge4.1 having a modification in MS2/MS3/MS4/MS5 (SEQ ID NO: 48).

Meanwhile, the contents related to the engineered guide RNA (augment RNA) of the present invention, the engineered guide RNA, the engineered tracrRNA (transactivating CRISPR RNA) and the engineered crRNA (CRISPR RNA) disclosed in the applications PCT/KR2020/014961, PCT/KR2021/013923, PCT/KR2021/013933 and PCT/KR2021/013898 are all cited. Hereinafter, when describing the contents of the engineered guide RNA (augment RNA) in the present specification, it should be understood that all contents disclosed in the above applications are included.

(2) Scaffold area

(2-1) Structure and function

When the sequence of the engineered guide RNA according to the embodiment of the present invention is functionally divided, it can be divided into a sequence portion that interacts with the Cas12f1 variant protein to form a guide RNA and Cas12f1 variant protein complex, a sequence portion that allows the guide RNA and Cas12f1 variant protein complex to find a target nucleic acid, and a U-rich tail sequence portion. At this time, the sequence portion that interacts with the Cas12f1 variant protein to form a guide RNA and Cas12f1 variant protein complex may be referred to as a scaffold sequence. Specifically, the scaffold sequence may include sequences of two or more RNA molecules, namely, tracrRNA and crRNA.

In one embodiment, when the engineered guide RNA is a dual guide RNA, the scaffold sequence may include a tracrRNA sequence among the augment RNA sequences and a CRISPR RNA repeat sequence included in the crRNA. As an example, the tracrRNA sequence may be a modified version of all or a portion of a tracrRNA sequence found in nature. Additionally, the CRISPR RNA repeat sequence may be a modified version of all or a portion of a CRISPR RNA repeat sequence found in nature.

Additionally, when the engineered guide RNA is a single guide RNA, the scaffold sequence may include the engineered tracrRNA sequence, the linker sequence, and the CRISPR RNA repeat sequence included in the engineered crRNA sequence. In one embodiment, the tracrRNA sequence may be a modified version of all or part of a tracrRNA sequence found in nature.

In addition, in one embodiment, the scaffold region includes a portion of tracrRNA and crRNA, and does not necessarily refer to one molecule of RNA. The scaffold region can be further subdivided into a first region, a second region, a third region, a fourth region, a fifth region, and a sixth region. When the subdivided regions are described according to the portions of tracrRNA and crRNA, the first to fourth regions are included in tracrRNA, and the fifth to sixth regions are included in crRNA, i.e., the crRNA repeat sequence portion.

In one embodiment, the engineered scaffold region is different from the scaffold region of a guide RNA found in nature, characterized in that a portion of the scaffold portion is modified. For example, the engineered scaffold region may be a scaffold region of a guide RNA found in nature with a portion of the scaffold region removed. In another example, the engineered scaffold region may be a scaffold region of a guide RNA found in nature with one or more nucleotides removed. The scaffold region is a region comprising a portion of tracrRNA and crRNA, which interacts with the Cas12f1 variant protein or a homolog protein thereof.

(2-2) Scaffold area 1

The scaffold first region is a region including the 5'-end of tracrRNA, wherein the first region includes nucleotides forming a Stem structure within the guide RNA and Cas12f1 variant protein complex, and may include nucleotides adjacent thereto. The first region may include a region that does not interact with the Cas12f1 variant protein in the guide RNA and Cas12f1 variant protein complex.

In some implementations, the first region can mean the 1st nucleotide to the 21st nucleotide from the 5'-terminus of a wild-type tracrRNA comprising a base sequence of SEQ ID NO: 58. For example, the sequence of the first region can be 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 7). Additionally, the sequence of the first region can be a partial sequence of the sequence of SEQ ID NO: 7. The partial sequence of the sequence of SEQ ID NO: 7 can be a sequential partial sequence from the 3'-terminus of the sequence of SEQ ID NO: 7 after the sequence of the 5'-terminus of the sequence of SEQ ID NO: 7 is sequentially removed. More specifically, the first region can be 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 8), 5'-UGGAGAA-3' or 5'-A-3'. Alternatively, the engineered tracrRNA can be one in which the sequence corresponding to the first region (region 1-21) is completely removed.

(2-3) Scaffold area 2

The scaffold second region refers to a region located at the 3'-end of the first region in the tracrRNA. The second region comprises nucleotides that form a Stem structure within the guide RNA and the Cas12f1 variant protein complex, and may comprise nucleotides adjacent thereto. In this case, the Stem structure is different from the Stem included in the first region. The second region comprises a Stem 2 portion (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)). The second region may comprise one or more nucleotides adjacent to the Stem 2 portion. The second region may comprise a region that does not interact with the Cas12f1 variant protein in the guide RNA and the Cas12f1 variant protein complex.

In one embodiment, the second region may refer to a region from the 22nd nucleotide to the 71st nucleotide from the 5'-end of a wild-type tracrRNA comprising a base sequence of SEQ ID NO: 58. Specifically, the sequence of the second region may be 5'-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 9). In addition, the second region may be a partial sequence of the SEQ ID NO: 9 sequence. The partial sequence of the SEQ ID NO: 9 sequence may be a sequence in which at least one pair of nucleotides forming a complementary bond and/or at least one nucleotide not forming a complementary bond in the SEQ ID NO: 9 sequence is deleted. For example, the sequence may be 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGUG-3' (SEQ ID NO: 10), 5'-CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 11) or 5'-CCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 12). In this case, the loop portion included in any one of the sequences of SEQ ID NOs: 9 to 12 is a 5'-UUAG-3' sequence, which may be substituted with a 5'-GAAA-3' sequence as needed.

(2-4) Scaffold 3rd area

The scaffold third region refers to a region located at the 3'-end of the second region in tracrRNA. The third region includes nucleotides that form a Stem structure within the guide RNA and Cas12f1 protein complex and nucleotides that form a complementary bond with some of the nucleotides included in crRNA, and may include nucleotides adjacent thereto.

In one embodiment, the third region may refer to a region from the 72nd nucleotide to the 129th nucleotide from the 5'-terminus of the wild-type tracrRNA comprising the base sequence of SEQ ID NO: 58. In one embodiment, the sequence of the third region may be a sequence of 5'-GGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAA-3' (SEQ ID NO: 13) or a sequence having at least 70% sequence homology to the sequence of SEQ ID NO: 13.

(2-5) Scaffold Area 4

The fourth region of the scaffold refers to a region located at the 3'-end of the third region of tracrRNA. The fourth region comprises nucleotides capable of forming complementary bonds with some nucleotides included in the crRNA within the guide RNA and Cas12f1 variant protein complex, and may comprise nucleotides adjacent thereto. The fourth region may comprise one or more nucleotides that complementarily bind with one or more nucleotides included in the fifth region of the crRNA. The fourth region may comprise a region that does not interact with the Cas12f1 variant protein in the guide RNA and Cas12f1 variant protein complex.

In one embodiment, the fourth region may mean a region from the 130th nucleotide to the 161st nucleotide from the 5'-terminus of a wild-type tracrRNA including a base sequence of SEQ ID NO: 58. The fourth region may be an MS4 portion including MS1, a 5'-CAAAUUCANNNNNCCUCUCCAAUUCUGCACAA-3' (SEQ ID NO: 14) sequence or a partial sequence of the SEQ ID NO: 14 sequence. In the SEQ ID NO: 14 sequence, the internal 5'-NNNNN-3' portion is the MS1 portion, and each of the Ns may be A, C, G, or U. As an example, the partial sequence of the SEQ ID NO: 14 sequence may be a sequence including the 5'-CAAAUUCANNNNN-3' (SEQ ID NO: 15) sequence among the SEQ ID NO: 14 sequence, but not including a partial sequence of the 3'-terminal portion. Specifically, it may be a 5'-CAAAUUCANNNNNCCUCUCCAAUUC-3' (SEQ ID NO: 16) sequence, a 5'-CAAAUUCANNNNNCCUCUC-3' (SEQ ID NO: 17) sequence or a 5'-CAAAUUCANNNNN-3' (SEQ ID NO: 15) sequence. In addition, the fourth region may include a 5'-NNNNN-3' portion substituted with 5'-NNNVN-3' or 5'-NVNNN-3'. Here, N may each independently be A, C, G or U, and V may be A, C or G. Preferably, the fourth region may be a sequence including a 5'-CAAAUUCANNNCN-3' (SEQ ID NO: 18) sequence but not including a part of the 3'-terminal sequence. Here, N may each independently be A, C, G or U.

(2-6) Scaffold Area 5

The fifth region of the scaffold refers to a region comprising the 5'-end of the crRNA. The fifth region comprises a nucleotide that forms a complementary bond with one or more nucleotides of the fourth region within the guide RNA and Cas12f1 variant protein complex, and may comprise nucleotides adjacent thereto. The fifth region may comprise one or more nucleotides that complementarily bind with one or more nucleotides included in the fourth region. The fifth region may comprise a region that does not interact with the Cas12f1 variant protein in the guide RNA and Cas12f1 variant protein complex.

The fifth region may refer to a region from the 1st nucleotide to the 30th nucleotide from the 5'-terminus of the wild-type crRNA repeat sequence including the base sequence of SEQ ID NO: 19. For example, in the engineered crRNA, the fifth region may be a 5'- GUUGCAGAACCCGAAUAGNNNNNUGAAGGA-3' (SEQ ID NO: 20) sequence or a partial sequence of the SEQ ID NO: 20 sequence. Here, N may independently be A, C, G, or U. The partial sequence of SEQ ID NO: 20 may be a sequence including the 5'-NNNNNUGAAGGA-3' (SEQ ID NO: 21) sequence among the base sequences of SEQ ID NO: 20 but not including a partial sequence of the 5'-terminal part. Preferably, the fifth region may include a 5'-NBNNNUGAAGGA-3' (SEQ ID NO: 22) sequence. Here, N can be independently A, C, G or U, and B can be U, C or G.

Additionally, the sequence of the fifth region can be 5'-GUUGCAGAACCCGAAUAGNBNNNUGAAGGA-3' (SEQ ID NO: 59). Here, N can be A, C, G or U, and B can be U, C, or G. Preferably, it can be 5'-GUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 65). In one embodiment, the fifth region can mean the 21st nucleotide to the 30th nucleotide from the 5'-end of the wild-type crRNA repeat sequence including the base sequence of SEQ ID NO: 19. In one embodiment, the sequence of the fifth region can be 5'-GAAUGAAGGA-3' (SEQ ID NO: 66).

(2-7) Scaffold Area 6

The scaffold region 6 refers to a region located in the 3'-terminal direction of the region 5 in crRNA. It may mean from the 31st nucleotide to the 37th nucleotide from the 5'-terminus of the wild-type crRNA repeat sequence including the base sequence of SEQ ID NO: 19.

In one embodiment, the sixth region can be a 5'-AUGCAAC-3' sequence (SEQ ID NO: 23) or a sequence having at least 70% sequence identity to the 5'-AUGCAAC-3' sequence. The sixth region comprises a nucleotide that forms a complementary bond with one or more nucleotides of the third region within the guide RNA and Cas12f1 variant protein complex, and can comprise nucleotides adjacent thereto.

(2-8) Scaffold Area 7

In addition, the engineered crRNA according to the embodiment of the present invention can further include a U-rich tail sequence as a seventh region at the 3'-end of the crRNA. This is in addition to the engineered scaffold region that can be introduced to improve the gene editing efficiency of the ultra-small gene editing system including the guide RNA and Cas12f1 variant protein complex of the present invention. The engineered scaffold region synergizes with the above-mentioned U-rich tail to improve the gene editing efficiency of the ultra-small gene editing system using the engineered guide RNA. The U-rich tail sequence can be 5'-(UaN)dUe-3', 5'-UaVUaVUe-3' or 5'-UaVUaVUaVUe-3'. Here, N can be A, C, G or U, and V can be A, C or G independently. Additionally, a can be an integer from 0 to 4, d can be an integer from 0 to 3, and e can be an integer from 0 to 10. As an example, the seventh region can be U ₄ AU ₄ .

In another embodiment, the engineered guide RNA may include a U-rich tail enriched with uridine (U) at the 3'-terminal portion. The U-rich tail sequence is basically enriched with uridine and includes a sequence of one or more consecutive uridines. The U-rich tail sequence may further include additional bases other than uridine depending on the actual usage environment and expression environment of the engineered miniature genome editing system, for example, the environment inside a eukaryotic cell or a prokaryotic cell.

In an embodiment of the present invention, the U-rich tail sequence provided may more preferably include a modified uridine repeat sequence in which one ribonucleoside other than uridine (A, C, G) is included for every 1 to 5 repetitions of uridine (U). The modified uridine contiguous sequence is particularly useful when designing a vector expressing an engineered crRNA. In one embodiment, the U-rich tail sequence may include a sequence in which UV, UUV, UUUV, UUUUV and/or UUUUUV are repeated one or more times. In this case, V is one of adenosine (A), cytidine (C), and guanosine (G).

In one implementation, the sequence of the U-rich tail can be represented as (UaN)bUc, where N is one of A, U, C, or G, a, b, and c are integers, a can be 1 or more and 5 or less, b can be 0 or more and 2 or less, and c can be 1 or more and 10 or less. In one embodiment, the sequence of the U-rich tail is 5'-U-3', 5'-UU-3', 5'-UUU-3', 5'-UUUU-3', 5'-UUUUU-3', 5'-UUUUU-3', 5'-UUUUUU-3', 5'-UUURUUU-3' (SEQ ID NO: 67), 5'-UUURUUUUU-3' (SEQ ID NO: 68), 5'-UUUURU-3' (SEQ ID NO: 69), 5'-UUUURUU-3' (SEQ ID NO: 70), 5'-UUUURUUU-3' (SEQ ID NO: 71), 5'-UUUURUUUU-3' (SEQ ID NO: 72), 5'-UUUURUUUUU-3' (SEQ ID NO: 73) or It can be 5'-UUUURUUUUU-3' (SEQ ID NO: 74), where R can be A or G.

Preferably, the sequence of the U-rich tail may comprise or consist of any one base sequence selected from the group consisting of SEQ ID NOs: 75 to 82, wherein R is A in any one of the base sequences of SEQ ID NOs: 67 to 74. In addition, the sequence of the U-rich tail may comprise or consist of any one base sequence selected from the group consisting of SEQ ID NOs: 83 to 90, wherein R is G in any one of the base sequences of SEQ ID NOs: 67 to 74. Most preferably, the sequence of the U-rich tail may be 5'-UUUUAUUUU-3' (SEQ ID NO: 80), 5'-UUUUAUUUUUU-3' (SEQ ID NO: 82), 5'-UUUUGUUUUUU-3' (SEQ ID NO: 90) or 5'-UUUUUUU-3' (SEQ ID NO: 91).

(2-9) Connection relationship of scaffold area

In one embodiment, the sequence of the tracrRNA within the scaffold region can comprise or consist of 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3' (SEQ ID NO: 61) or 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNNNCCUCUCCAAUUCUGCACAA-3' (SEQ ID NO: 62). Wherein, N can be A, C, G or U.

In one embodiment, the tracrRNA comprises a first region, a second region, a third region, and a fourth region. In one embodiment, the tracrRNA comprises a first region, a second region, a third region, and a fourth region sequentially linked from the 5'-end to the 3'-end.

Also, in one embodiment, the sequence of the crRNA in the scaffold region includes a crRNA repeat sequence and a spacer sequence. At this time, the crRNA repeat sequence can be 5'-GAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 63) or 5'-GGAAUGCAAC-3' (SEQ ID NO: 64). The crRNA repeat sequence can include a fifth region and a sixth region. The spacer sequence can vary depending on the target sequence and generally includes 10 to 50 nucleotides. In one embodiment, the crRNA is a fifth region, a sixth region, and a spacer sequentially linked from the 5'-end to the 3'-end.

In one embodiment, the crRNA can be a wild-type crRNA or an engineered crRNA. The crRNA can include a spacer sequence, which is a wild-type repeat sequence and a guide sequence, in order from the 5'-end to the 3'-end. The wild-type repeat sequence can be a sequence of 5'-GUUGCAGAACCCGAAUAGACGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 19).

(3) Spacer sequence

The spacer sequence is a sequence complementary to a target site sequence in a target nucleic acid or a target gene, and is linked to the 3'-end of the crRNA repeat sequence. The spacer sequence is a sequence homologous to a protospacer sequence adjacent to a PAM (Protospacer Adjacent Motif) sequence recognized by the Cas12f1 variant protein, and has a sequence in which thymidine (T) of the protospacer sequence is substituted with uridine (U). At this time, the target sequence and the protospacer sequence are determined within the sequence adjacent to the PAM sequence included in the target nucleic acid, and the spacer sequence is determined accordingly.

In one embodiment, the spacer sequence portion of the crRNA can complementarily bind to the target nucleic acid. In one embodiment, the spacer sequence portion of the crRNA can complementarily bind to the target sequence portion of the target nucleic acid. For example, when the target nucleic acid is a double-stranded DNA, the spacer sequence may be a sequence complementary to a target sequence included in a target strand of the double-stranded DNA. Here, when the target nucleic acid is a double-stranded DNA, the spacer sequence may include a sequence homologous to a protospacer sequence included in a non-target strand of the double-stranded DNA. Specifically, the spacer sequence may have the same base sequence as the protospacer sequence, but may have a sequence in which each of the thymidines (T) included in the base sequence is substituted with uridine (U). For example, the spacer sequence may include an RNA sequence corresponding to the DNA sequence of the protospacer.

In one embodiment, the spacer sequence may be from 10 nucleotides to 50 nucleotides in length. Preferably, the spacer sequence may be from 17 nucleotides to 30 nucleotides in length. More preferably, the spacer sequence may be from 17 nucleotides to 25 nucleotides in length.

(4) Single guide RNA or dual guide RNA

The engineered guide RNA according to an embodiment of the present invention may be a single guide RNA or a dual guide RNA. Dual guide RNA means that the guide RNA is composed of two RNA molecules, tracrRNA and crRNA. Single guide RNA (sgRNA) means that the 3'-end of the engineered tracrRNA and the 5'-end of the engineered crRNA are connected via a linker.

In one embodiment, the engineered single guide RNA (sgRNA) additionally comprises a linker sequence, and the tracrRNA sequence and the crRNA sequence can be connected via the linker sequence. Preferably, the 3'-terminus of the fourth region and the 5'-terminus of the fifth region included in the engineered scaffold can be connected via a linker. More preferably, the 3'-terminus of the fourth region and the 5'-terminus of the fifth region can be connected via a linker 5'-GAAA-3'.

In one embodiment, the sequence of the single guide RNA is sequentially connected from the 5'-end to the 3'-end, including a tracrRNA sequence, a linker sequence, a crRNA sequence, and a U-rich tail sequence. A portion of the tracrRNA sequence and all or a portion of the CRISPR RNA repeat sequence included in the crRNA sequence have sequences complementary to each other. More specifically, the single guide RNA may have a sequence selected from the group consisting of SEQ ID NOs: 44 to 48.

In addition, the engineered guide RNA according to the embodiment of the present invention may be a dual guide RNA in which tracrRNA and crRNA form separate RNA molecules. At this time, a part of the tracrRNA and a part of the crRNA may have complementary sequences to each other to form a double-stranded RNA. More specifically, in the dual guide RNA, a part including the 3'-end of the tracrRNA and a part including the CRISPR RNA repeat sequence of the crRNA may form a double strand. The engineered guide RNA may bind to a Cas12f1 variant protein to form a guide RNA and Cas12f1 variant protein complex, and recognize a target sequence complementary to a spacer sequence included in the crRNA sequence to edit a target nucleic acid including the target sequence.

In one embodiment, the sequence of the tracrRNA can comprise a complementary sequence having 0 to 20 mismatches with the CRISPR RNA repeat sequence. Preferably, the tracrRNA sequence can comprise a complementary sequence having 0 to 8 or 8 to 12 mismatches with the CRISPR RNA repeat sequence.

(5) Modification to create single guide RNA (sgRNA)

(5-1) Overview

The engineered guide RNA provided in the present invention may be a single guide RNA (sgRNA) molecule. Accordingly, the engineered scaffold region may have at least one of each region modified, and additionally, the 3'-end of the tracrRNA fourth region and the 5'-end of the crRNA fifth region may be connected via a linker.

In one embodiment, the engineered scaffold region may be a scaffold region found in nature that is modified at one or more sites, and the 3'-end of the fourth region and the 5'-end of the fifth region may be connected via a linker, wherein the linker may be 5'-GAAA-3'.

Also, in one embodiment, the engineered scaffold region includes regions corresponding to each portion of a scaffold region found in nature. Specifically, the engineered scaffold region includes a first region, a second region, a third region, a fourth region, a fifth region, and a sixth region, which correspond to regions first to sixth included in a scaffold region found in nature, respectively.

In another embodiment, the engineered scaffold region may not include a region corresponding to the first region and/or the second region among the scaffold regions found in nature. Specifically, in the micro genome editing system, the micro genome editing protein may be a protein comprising or consisting of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, and the guide RNA may comprise or consist of any one base sequence selected from the group consisting of SEQ ID NO: 44 to SEQ ID NO: 48.

Preferably, the ultra-small gene editing protein is a protein comprising or consisting of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, and the guide RNA may be any one selected from the group consisting of augment RNA having a modification in MS1 (SEQ ID NO: 44), augment RNA having a modification in MS1/MS2 (SEQ ID NO: 45), augment RNA Cas12f1_ge3.0 having a modification in MS1/MS2/MS3 (SEQ ID NO: 46), augment RNA Cas12f1_ge4.0 having a modification in MS2/MS3/MS4 (SEQ ID NO: 47), and augment RNA Cas12f1_ge4.1 having a modification in MS2/MS3/MS4/MS5 (SEQ ID NO: 48).

For example, the guide RNA is MS1/MS3-1 augment RNA (SEQ ID NO: 105), MS1/MS3-2 augment RNA (SEQ ID NO: 106), MS1/MS3-3 augment RNA (SEQ ID NO: 107), MS1/MS4 ^* -1 augment RNA (SEQ ID NO: 108), MS1/MS4 ^* -2 augment RNA (SEQ ID NO: 109), MS1/MS4 ^* -3 augment RNA (SEQ ID NO: 110), MS1/MS5-1 augment RNA (SEQ ID NO: 111), MS1/MS5-2 augment RNA (SEQ ID NO: 112), MS1/MS5-3 augment RNA (SEQ ID NO: 113), MS1/MS2/MS4 ^* -2 augment RNA (SEQ ID NO: 114), MS1/MS3-3/MS4 ^* -2 augment RNA (SEQ ID NO: 115), MS1/MS2/MS5-3 augment RNA (SEQ ID NO: 116), It can be MS1/MS3-3/MS5-3 sgRNA (SEQ ID NO: 117), MS1/MS4 ^* -2/MS5-3 augment RNA (SEQ ID NO: 118), MS1/MS2/MS3-3/MS4 ^* -2 augment RNA (SEQ ID NO: 119), MS1/MS2/MS3-3/MS5-3 augment RNA (SEQ ID NO: 120), MS1/MS2/MS4 ^* -2/MS5-3 augment RNA (SEQ ID NO: 121), MS1/MS3-3/MS4 ^* -2/MS5-3 augment RNA (SEQ ID NO: 122), or MS1/MS2/MS3-3/MS4 ^* -2/MS5-3 sgRNA (SEQ ID NO: 123).

Additionally, the guide RNA may be 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA UUU gaaa GAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN-3' (SEQ ID NO: 124), which is a mature form sgRNA.

For example, the guide RNA may be an augment RNA having some modifications in the nucleic acid sequence of the Mature form sgRNA. Specifically, it may be MS3-1 augment RNA (SEQ ID NO: 125), MS3-2 augment RNA (SEQ ID NO: 126), MS3-3 augment RNA (SEQ ID NO: 127), MS4-1 augment RNA (SEQ ID NO: 128), MS4-2 augment RNA (SEQ ID NO: 129), MS4-3 augment RNA (SEQ ID NO: 130), MS5-1 augment RNA (SEQ ID NO: 131), MS5-2 augment RNA (SEQ ID NO: 132), MS5-3 augment RNA (SEQ ID NO: 133), MS3-3/MS4-3 augment RNA (SEQ ID NO: 134), MS3-3/MS5-3 augment RNA (SEQ ID NO: 135), MS4-3/MS5-3 augment RNA (SEQ ID NO: 136), or MS3-3/MS4-3/MS5-3 augment RNA (SEQ ID NO: 137).

As another example, the augment RNA having a partial modification of the nucleic acid sequence in the Mature form sgRNA may be an augment RNA in which the MS2 modification of the present invention is added to each of the engineered augment RNAs consisting of the base sequences of SEQ ID NOS: 125 to 137. Here, the MS2 modification is a U-rich tail sequence, and the sequence may be a 5'-(UaN)dUe-3' sequence, a 5'-UaVUaVUe-3' sequence, or a 5'-UaVUaVUaVUe-3' sequence. Here, N may be A, C, G, or U. Each V may independently be A, C, or G. The a may be an integer from 0 to 4. d may be an integer from 0 to 3, and e may be an integer from 0 to 10. Preferably, it may be U ₄ AU ₄ .

Below we detail the deformation in the engineered scaffold area.

(5-2) Deformation in the first area of the scaffold

In one embodiment, the engineered scaffold region comprised in the engineered guide RNA for the Cas12f1 variant protein or a homolog protein thereof can comprise one or more nucleotides from the first region of the scaffold region removed. More specifically, the removed nucleotide can be a nucleotide comprised in a portion of the first region that forms the Stem structure in the guide RNA and Cas12f1 variant protein complex.

In one embodiment, the removed nucleotide may be a nucleotide belonging to Stem 1 (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) in the first region. In one example, the removed nucleotide may be a nucleotide that does not interact with the Cas12f1 variant protein in the guide RNA and Cas12f1 variant protein complex in the first region.

In one embodiment, the modified first region (MS3 portion, regions 1-21) can be 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 7) or a portion of the sequence of SEQ ID NO: 7. The portion of the sequence of SEQ ID NO: 7 can be a sequence in which at least 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides are sequentially removed from the 5'-end of SEQ ID NO: 7. More specifically, from the 5'-terminal to the 3'-terminal, a portion of the sequence of SEQ ID NO: 7 may be a sequential portion of the sequence at the 3'-terminal end where the 5'-terminal sequence of SEQ ID NO: 7 is sequentially removed, such as 5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5'-UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3'. As an example, the first region may be 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 8), 5'-UGGAGAA-3', or 5'-A-3'. Alternatively, the entire first region may be removed.

(5-3) Deformation in the second area of the scaffold

In another embodiment, the engineered scaffold region can comprise a modified second region, wherein the modified second region has one or more nucleotides removed from the second region of the scaffold region, wherein the removed nucleotides are nucleotides selected from the region that forms the Stem structure in the guide RNA and Cas12f1 variant protein complex.

In one embodiment, the removal of the nucleotide occurs in a portion forming the Stem structure in the second region, and the nucleotide may be removed in base pair units. In one embodiment, the removed nucleotide may be a nucleotide included in a portion forming the Stem structure in the guide RNA and Cas12f1 variant protein complex in the second region.

In one embodiment, the modified second region (MS5 portion, region 22-71) of the engineered scaffold region can be the sequence 5'-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 9) or a portion of the sequence SEQ ID NO: 9. It can be a second region of the scaffold region found in nature having 1 to 50 nucleotides removed.

A part of the sequence of SEQ ID NO: 9 may be a sequence in which at least one pair of nucleotides forming a complementary bond and/or at least one nucleotide not forming a complementary bond in SEQ ID NO: 9 is deleted. In this case, the 5'-UUAG-3' sequence of the loop portion included in the part of the sequence of SEQ ID NO: 9 may be optionally replaced with the 5'-GAAA-3' sequence. In addition, the second region may be one in which the loop is removed.

For example, the modified second region may be a second region of the scaffold region found in nature, in which at least one of the 1st to 22nd nucleotides and/or the 27th to 50th nucleotides from the 5'-end based on the base sequence of SEQ ID NO: 9 is removed. In addition, the modified second region may be a second region of the scaffold region found in nature, in which at least one of the 1st to 22nd nucleotides and/or the 27th to 50th nucleotides from the 5'-end based on the sequence of SEQ ID NO: 11 is removed, and the 23rd to 26th nucleotides are replaced with another one. Specifically, it may be a 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGUG-3' (SEQ ID NO: 10) sequence, a 5'-CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 11) sequence, or a 5'-CCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 12) sequence.

In addition, the engineered scaffold region provided by the present invention may be a scaffold region found in nature from which a second region is removed. In one embodiment, the engineered scaffold region may not have a region corresponding to the second region of a scaffold region found in nature. In one example, the sequence of the engineered scaffold region from which the second region is removed may be 5'-CUUCACUGAUAAAGUGGAGAAGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 92).

(5-4) Deformation in the third area of the scaffold

In one embodiment of the present invention, the engineered scaffold region can comprise a modified third region, wherein the modified third region is one or more nucleotides removed from the third region of the scaffold region found in nature. Wherein the removed nucleotides are nucleotides selected from the region forming the Stem structure in the guide RNA and Cas12f1 variant protein complex.

In one embodiment, the modified third region (MS1 front portion, regions 72-129) of the engineered scaffold region can be 5'-GGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAA-3' (SEQ ID NO: 13) or a sequence having at least 70% (e.g., 70%, 80% or 90%) sequence identity to SEQ ID NO: 13. It can be a third region of the scaffold region found in nature having 1 to 20 nucleotides removed. In one embodiment, the modified third region may be a third region of the scaffold region found in nature, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides are removed from the 5'-terminus among the 28th to 37th nucleotides and/or the 42nd to 51st nucleotides based on the nucleotide sequence of SEQ ID NO: 13. In one embodiment, the modified third region may be a third region of the scaffold region found in nature, wherein one or more pairs of nucleotides that form base pairs in the guide RNA and Cas12f1 variant protein complex are removed from the 27th to 36th nucleotides and the 42nd to 51st nucleotides based on the nucleotide sequence of SEQ ID NO: 13. In one embodiment, the modified third region may be a third region of a scaffold region found in nature, wherein one or more pairs of nucleotides that form base pairs and/or one or more nucleotides that do not form base pairs in the guide RNA and Cas12f1 variant protein complex are removed from the 27th to 36th nucleotides and the 42nd to 51st nucleotides from the 5'-end based on the nucleotide sequence of SEQ ID NO: 13. In one example, the modified third region is characterized by comprising the sequence 5'-GCUGCUUGCAUCAGCCUAAUGUCGAG-3' (SEQ ID NO: 93), 5'-UUCG-3', and 5'-CUCGA-3'.

(5-5) Deformation in the 4th and/or 5th area of the scaffold

The engineered scaffold region provided herein may be a scaffold region found in nature in which the fourth region and the fifth region are modified. Since the fourth region and the fifth region include a portion that hybridizes with each other within the guide RNA and the Cas12f1 variant protein complex to form the Stem, these portions may be modified together to form the engineered scaffold region. The modified fourth region is characterized in that one or more nucleotides are removed from the fourth region of the scaffold region found in nature. The modified fifth region is characterized in that one or more nucleotides are removed from the fifth region of the scaffold region found in nature.

In one embodiment, the modified fourth region is characterized by having a 5'-CAAA-3' or 5'-AACAAA-3' sequence in the 5'-terminal direction. In one embodiment, the modified fifth region is characterized by having a 5'-GGA-3' sequence in the 3'-terminal direction. In one embodiment, the modified fourth region of the engineered scaffold region can be a fourth region of a scaffold region found in nature that has 1 to 7 nucleotides removed. In one embodiment, the modified fourth region of the engineered scaffold region can be a fourth region of a scaffold region found in nature that has 1 to 28 nucleotides removed.

Additionally, the modification in the fourth region may be such that one or more of the 9th to 15th nucleotides from the 5'-terminus, based on the base sequence of SEQ ID NO: 27, in the fourth region of the scaffold region found in nature are removed. In one embodiment, the modified fourth region may be such that one or more of the 9th to 36th nucleotides from the 5'-terminus, based on the base sequence of SEQ ID NO: 27, in the fourth region of the scaffold region found in nature are removed. Specifically, the modification in the fourth region can be a sequence that includes 5'-CAAAUUCANNNNN-3' (SEQ ID NO: 15) of the above SEQ ID NO: 14, but does not include a portion of the sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide sequence) from the 3'-end. For example, it can be 5'-CAAAUUCANNNNNCCUCUCCAAUUC-3' (SEQ ID NO: 16), 5'-CAAAUUCANNNNNCCUCUC-3' (SEQ ID NO: 17) or 5'-CAAAUUCANNNNN-3' (SEQ ID NO: 15). Additionally, the fourth region may include a sequence in which the 5'-NNNNN-3' portion is replaced with 5'-NNNVN-3' or 5'-NVNNN-3', wherein each of said Ns is independently A, C, G or U, and wherein said V can be A, C or G. Preferably, the fourth region may be a sequence comprising 5'-CAAAUUCANNNCN-3' (SEQ ID NO: 18) and not including a portion of the sequence (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide sequence) from the 3'-end. Wherein each of said Ns is independently A, C, G or U.

In one embodiment, the modified fifth region may be a fifth region of a scaffold region found in nature, wherein one or more of the 1st to 7th nucleotides from the 5'-terminus, based on the nucleotide sequence of SEQ ID NO: 19, are removed. In one embodiment, the modified fifth region may be a fifth region of a scaffold region found in nature, wherein one or more of the 1st to 27th nucleotides from the 5'-terminus, based on the nucleotide sequence of SEQ ID NO: 19, are removed.

In one embodiment, the modified fourth and fifth regions may be those in which one or more pairs of nucleotides that form base pairs and/or one or more non-base paired nucleotides in the guide RNA and Cas12f1 variant protein complex are removed from the fourth and fifth regions of the scaffold region found in nature, the 9th to 15th nucleotides from the 5'-terminus based on the nucleotide sequence of SEQ ID NO: 14 and the 1st to 7th nucleotides from the 5'-terminus based on the nucleotide sequence of SEQ ID NO: 19.

In addition, the modified fourth and fifth regions may be those in which one or more pairs of nucleotides forming base pairs and/or one or more pairs of nucleotides that are mismatched in the guide RNA and Cas12f1 variant protein complex are removed from the 9th to 15th nucleotides from the 5'-terminus based on the nucleotide sequence of SEQ ID NO: 14 and from the 1st to 7th nucleotides from the 5'-terminus based on the nucleotide sequence of SEQ ID NO: 19 in the fourth and fifth regions of the scaffold region found in nature.

The above modified fourth and fifth regions may be those in which one or more pairs of nucleotides that form base pairs and/or one or more nucleotides that do not form base pairs in the guide RNA and Cas12f1 variant protein complex are removed from the 9th to 36th nucleotides from the 5'-end based on the nucleotide sequence of SEQ ID NO: 14 and from the 1st to 27th nucleotides based on the nucleotide sequence of SEQ ID NO: 19 in the fourth and fifth regions of the scaffold region found in nature.

In one embodiment, the sequence of the modified fourth region can be or comprises 5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', 5'-AACAAAUUCA-3' (SEQ ID NO: 94), 5'-AACAAAUUCAU-3' (SEQ ID NO: 95), 5'-AACAAAUUCAUU-3' (SEQ ID NO: 96), 5'-CAAA-3', 5'-CAAAU-3', 5'-CAAAUU-3', 5'-CAAAUUC-3', 5'-CAAAUUCA-3', 5'-CAAAUUCAU-3' or 5'-CAAAUUCAUU-3'.

Additionally, the sequence of the modified fifth region may be or include 5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3', 5'-AUGAAGGA-3' or 5'-AAUGAAGGA-3'.

Preferably, the engineered scaffold region wherein the fourth region and the fifth region are modified comprises, from the 5'-end to the 3'-end, one or more sequences selected from the group consisting of 5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', 5'-AACAAAUUCA-3' (SEQ ID NO: 94), 5'-AACAAAUUCAU-3' (SEQ ID NO: 95) and 5'-AACAAAUUCAUU-3' (SEQ ID NO: 96); And it may be a nucleic acid comprising a sequence in which, from the 5'-terminal to the 3'-terminal direction, at least one sequence selected from the group consisting of 5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3', 5'-AUGAAGGA-3' and 5'-AAUGAAGGA-3' is linked.

(5-6) Deformation in the 6th area of the scaffold

In an engineered scaffold according to an embodiment of the present invention, the sixth region is a region comprising nucleotides belonging to crRNA in the PK(R:AR-1) portion. The sixth region of the engineered scaffold may be identical to the sixth region of a scaffold found in nature, or may be modified as long as the function of the sixth region is not impaired. As an example, the sixth region may be 5'-AUGCAAC-3' (SEQ ID NO: 23) or a sequence having at least 70% sequence homology thereto.

(5-7) Modification in the 7th region of the guide RNA

In an embodiment of the present invention, the modification in the 7th region of the guide RNA includes providing a U-rich tail sequence at the 3'-end of the crRNA to improve the gene editing efficiency of the gene editing system of the present invention. The U-rich tail sequence is characterized by being basically rich in uridine and includes a sequence of one or more consecutive uridines.

In some implementations, the engineered crRNA can further comprise a U-rich tail sequence as a seventh region at the 3'-end of the crRNA. The U-rich tail sequence can be 5'-(UaN)dUe-3', 5'-UaVUaVUe-3' or 5'-UaVUaVUaVUe-3'. Each of N can be A, C, G or U. Each of V can independently be A, C or G. a can be an integer from 0 to 4. d can be an integer from 0 to 3. e can be an integer from 0 to 10.

In one embodiment, the U-rich tail sequence may include 1 to 10 uridine repeat sequences. The U-rich tail sequence may further include additional bases other than uridine depending on the actual usage environment and expression environment of the gene editing system including the engineered guide RNA, for example, the internal environment of a eukaryotic cell or a prokaryotic cell. As an example, the U-rich tail sequence may include a sequence in which UV, UUV, UUUV and/or UUUUV are repeated one or more times. In this case, V is one of adenosine (A), cytidine (C), and guanosine (G). The U-rich tail sequence is characterized in that it is linked to the 3'-end of the crRNA sequence included in the ultra-small gene editing system of the present invention.

The above U-rich tail sequence serves to increase the cleavage efficiency of the augment RNA and Cas12f1 mutant protein complex provided in the present invention for the target nucleic acid. At this time, the target nucleic acid may be single-stranded DNA, double-stranded DNA, and/or RNA. The term "tail sequence" used herein may mean not only the RNA sequence itself rich in uridine (U) but also the DNA sequence encoding the same, and this is appropriately interpreted depending on the context. The present inventors have experimentally elucidated the structure and effect of the U-rich tail sequence in detail, which will be described in more detail below with specific implementation examples.

For example, a U-rich tail sequence can be represented as Ux. Wherein x can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In one embodiment, x can be an integer within two numerical ranges selected in the immediately preceding sentence. For example, x can be an integer between 1 and 6. In another example, x can be an integer between 1 and 20. In one embodiment, x can be an integer greater than or equal to 20.

Also, in one embodiment, the U-rich tail sequence can be represented as (UaN)nUb, where N is one of adenosine (A), uridine (U), cytidine (C), and guanosine (G). Wherein, a is an integer between 1 and 5, and n is an integer greater than or equal to 0. In one embodiment, n can be an integer between 0 and 2. In one embodiment, b can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, b can be an integer within two numerical ranges selected in the immediately preceding sentence. For example, b can be an integer between 1 and 6.

In one embodiment, the U-rich tail sequence can be represented as (UaV)nUb, where V is one of adenosine (A), cytidine (C), and guanosine (G). Wherein, a is an integer from 1 to 4, and n is an integer greater than or equal to 0. In one embodiment, n can be 1 or 2. In one embodiment, b can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In one embodiment, b can be an integer within two numerical ranges selected in the immediately preceding sentence. For example, b can be an integer from 1 to 6. In another example, b can be an integer from 1 to 20. As an example implementation, b can be an integer greater than or equal to 20.

In addition, the U-rich tail sequence may be a combined form of a sequence represented by Ux and a sequence represented by (UaV)n. In one embodiment, the U-rich tail sequence may be represented by U)n1-V1-(U)n2-V2-Ux. At this time, V1 and V2 are each one of adenine (A), cytidine (C), and guanine (G). At this time, n1 and n2 may each be an integer between 1 and 4. At this time, x may be an integer between 1 and 20.

Additionally, the length of the U-rich tail sequence may be 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt. In one embodiment, the length of the U-rich tail sequence may be 20 nt or longer.

In one embodiment, the sequence of the U-rich tail is 5'-U-3', 5'-UU-3', 5'-UUU-3', 5'-UUUU-3', 5'-UUUUU-3', 5'-UUUUU-3', 5'-UUUUUU-3', 5'-UUURUUU-3' (SEQ ID NO: 67), 5'-UUURUUUUU-3' (SEQ ID NO: 68), 5'-UUUURU-3' (SEQ ID NO: 69), 5'-UUUURUU-3' (SEQ ID NO: 70), 5'-UUUURUUU-3' (SEQ ID NO: 71), 5'-UUUURUUUU-3' (SEQ ID NO: 72), 5'-UUUURUUUUU-3' (SEQ ID NO: 73), or It may be 5'-UUUURUUUUU-3' (SEQ ID NO: 74). Here, R may be A or G. Preferably, the sequence of the U-rich tail may be one in which R is A in any one of the base sequences of SEQ ID NOs: 67 to 74, and may include or consist of any one of the base sequences of SEQ ID NOs: 75 to 82.

In addition, the sequence of the U-rich tail may be a sequence of any one of SEQ ID NOs: 67 to 74, wherein R is G, and may include or consist of any one of SEQ ID NOs: 83 to 90. Most preferably, the sequence of the U-rich tail may be 5'-UUUUAUUUU-3' (SEQ ID NO: 80), 5'-UUUUAUUUUUUU-3' (SEQ ID NO: 82), 5'-UUUUGUUUUUU-3' (SEQ ID NO: 90) or 5'-UUUUUUU-3' (SEQ ID NO: 91).

In one embodiment, the U-rich tail sequence can comprise or consist of any one nucleotide sequence selected from the group consisting of SEQ ID NO: 67 to SEQ ID NO: 91.

(6) Additional sequence

The engineered tracrRNA of the present invention may optionally further comprise an additional sequence. The additional sequence may be located at the 3'-terminus of the engineered tracrRNA. The additional sequence may be located at the 3'-terminus of the fourth region. In addition, the additional sequence may also be located at the 5'-terminus of the engineered tracrRNA. For example, the additional sequence may be located at the 5'-terminus of the first region.

The additional sequence can be from 1 to 40 nucleotides. In one embodiment, the additional sequence can be any nucleotide sequence or any randomly arranged nucleotide sequence. For example, the additional sequence can be the sequence 5'-AUAAAGGUGA-3' (SEQ ID NO: 97).

Additionally, the additional sequence may be a known nucleotide sequence. For example, the additional sequence may be a hammerhead ribozyme nucleotide sequence. Here, the hammerhead ribozyme nucleotide sequence may be a 5'-CUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC-3' (SEQ ID NO: 98) sequence or a 5'-CUGCUCGAAUGAGCAAAGCAGGAGUGCCUGAGUAGUC-3' (SEQ ID NO: 99) sequence. The above examples are merely illustrative and are not limiting.

(7) Chemical modification

In some embodiments, the engineered tracrRNA or engineered crRNA may optionally have at least one or more nucleotides with a chemical modification. The chemical modification may be a modification of various covalent bonds that may occur in the bases and/or sugars of the nucleotides.

For example, the chemical modification may be methylation, halogenation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS) or 2'-O-methyl 3'thioPACE (MSP). The above examples are merely illustrative and are not limiting.

When used in an ultra-small gene editing system comprising the augment RNA and Cas12f1 mutant protein complex of the present invention, the effect of significantly improving cleavage or editing of a target nucleic acid or target gene within a cell is exhibited compared to when using a guide RNA found in nature.

Above all, the engineered guide RNA may have the following effects: optimization of the length showing high efficiency and reduction of the guide RNA synthesis cost accordingly, securing additional space or capacity when inserted into a viral vector, normal expression of tracrRNA, increase in the expression of operable guide RNA, increase in the stability of the guide RNA, increase in the stability of the guide RNA and Cas12f1 variant protein complex, induction of formation of a highly efficient guide RNA and Cas12f1 variant protein complex, increase in the cleavage efficiency of the target nucleic acid by the ultra-small gene editing system including the guide RNA and Cas12f1 variant protein complex, and increase in the editing efficiency of the target nucleic acid by the system. Accordingly, the use of the engineered guide RNA described above for the Cas12f1 variant protein or a homolog protein thereof can overcome the limitations of the above-mentioned conventional technology and can cleave or edit genes with high efficiency in cells.

In addition, since the engineered guide RNA has a short length compared to guide RNAs found in nature, it has a high potential for application in the field of gene editing technology. Using the engineered guide RNA, the size of the ultra-small gene editing system containing the guide RNA and Cas12f1 variant protein complex is very small, and the editing efficiency is excellent, so that it can be utilized in various gene editing technologies.

4. Composition for gene editing

According to another aspect of the present invention, a gene editing composition comprising the gene editing system described above is provided. In addition, a gene editing composition comprising the vector system described below or both the gene editing system and the vector system is provided.

In one embodiment, the composition for gene editing can comprise a small endonuclease comprising a Cas12f1 variant protein or a homolog protein thereof, or a nucleic acid encoding the endonuclease; and a guide RNA or a nucleic acid encoding the guide RNA. For the "Cas12f1 variant protein", "homolog protein" and "guide RNA", see above.

It is obvious that the gene editing composition of the present invention may additionally contain, in addition to each component of the ultra-small gene editing system according to the present invention, an appropriate material necessary for the gene editing purpose.

III. Nucleic acids encoding components of a miniature gene editing system

Since each component of the ultra-small gene editing system provided in the present invention is intended to be expressed within a cell, according to one aspect of the present invention, a nucleic acid or polynucleotide encoding each component of the ultra-small gene editing system is provided.

Specifically, the nucleic acid or polynucleotide comprises a nucleic acid sequence encoding a gene editing protein and/or guide RNA included in the ultra-small gene editing system to be expressed. At this time, the sequence of the nucleic acid or polynucleotide may include not only a nucleic acid sequence encoding a wild-type gene editing protein and a wild-type guide RNA, but also a nucleic acid sequence encoding an engineered augment RNA and/or a codon-optimized gene editing protein, a nucleic acid sequence encoding an engineered gene editing protein, or a nucleic acid sequence encoding a gene editing protein with lost or reduced DNA double-strand cleavage activity.

In the present invention, the nucleic acid or polynucleotide may include a sequence configured to express a Cas12f1 variant protein or a homolog protein thereof, which is an ultra-small gene editing protein. Here, the Cas12f1 variant protein or a homolog protein thereof may be a protein having an activity of cleaving a double-stranded or single-stranded DNA.

In one embodiment, the nucleic acid or polynucleotide may comprise a sequence configured to express a Cas12f1 variant protein. Here, the wild-type Cas12f1 variant protein may be a protein consisting of an amino acid sequence of SEQ ID NO: 1. In addition, an analog of the Cas12f1 variant protein according to the present invention may be a protein having 1 to 28 amino acids added to the N-terminus of a Cas12f1 protein consisting of an amino acid sequence of SEQ ID NO: 5.

Additionally, the nucleic acid or polynucleotide may comprise a sequence encoding a Cas12f1 variant protein or a homolog protein thereof. Preferably, the nucleic acid or polynucleotide may comprise a human codon optimized nucleic acid sequence encoding a Cas12f1 variant protein or a homolog protein thereof. Here, the Cas12f1 variant protein may be a protein consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4, and the homolog protein may be a protein consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 141 to 232.

Additionally, the nucleic acid or polynucleotide may comprise a sequence encoding a modified Cas12f1 variant protein or a Cas12f1 variant fusion protein. In one embodiment, the nucleic acid or polynucleotide may comprise a sequence configured to express a Cas12f1 variant protein that is modified to cleave only one strand of a duplex of a target nucleic acid. In one example, the modified Cas12f1 variant protein may be modified to be able to cleave only one strand of a duplex of a target nucleic acid and perform base editing or prime editing on the strand that is not cut. Alternatively, the nucleic acid or polynucleotide may comprise a sequence encoding a Cas12f1 variant protein that is modified to be able to perform base editing or prime editing or gene expression regulation function on the target nucleic acid.

In addition, the nucleic acid or polynucleotide may be configured to express an augmented guide RNA engineered to have optimal targeting efficiency for the Cas12f1 variant, or may comprise a sequence configured to express one or more different engineered guide RNAs. For example, the augmented RNA sequence may comprise a scaffold sequence, a spacer sequence, and a U-rich tail sequence. Specifically, the augmented RNA sequence may comprise a modified tracrRNA sequence and/or a modified crRNA sequence, and may comprise a U-rich tail sequence therein. For example, the U-rich tail sequence may be expressed as (UaN)nUb, wherein N is one of adenosine (A), uridine (U), cytidine (C), and guanosine (G). Here, a is an integer greater than or equal to 1 and less than or equal to 4, n is an integer greater than or equal to 0, 1, and 2, and b is an integer greater than or equal to 1 and less than or equal to 10. In another embodiment, the U-rich tail sequence can be represented as (UaV)nUb, where a, n, and b are integers, a can be 1 or more and 4 or less, n can be 0 or more, and b can be 1 or more and 10 or less.

IV. Design of vector for expression of ultra-small gene-editing system

In order to use the ultra-small gene editing system provided by the present invention for gene editing, a method can be used in which a vector including a sequence encoding each component of the gene editing system is introduced into a target cell, and each component of the gene editing system is expressed in the target cell.

In addition, in order to achieve excellent targeting efficiency in the ultra-small gene editing system of the present invention for editing a target nucleic acid or a target gene, it is preferable that each component of the guide RNA and the Cas12f1 variant protein complex be operably linked and included as a single vector. Here, an effector protein may be linked to the nucleic acid-decomposing protein or the guide molecule as needed to form a fused protein.

For example, the fused protein may include an orthogonal RNA-binding protein or an adaptor protein present in a bacteriophage coat protein. Here, the coat protein may include MS2, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ΦCb5, ΦCb8r, ΦCb12r, ΦCb23r, 7s, and PRR1. Additionally, the fused protein may be delivered via one or more lipid nanoparticles.

In one embodiment, the Cas12f1 variant protein or a homolog protein thereof, which is a component of the ultra-small gene editing system of the present invention, can be delivered to a cell as one or more guide RNAs or one or more mRNA molecules encoding the same. In this case, the RNA molecules can be delivered via one or more lipid nanoparticles.

Additionally, the components of the micro-gene editing system of the present invention may be in the form of one or more DNA molecules. Here, the one or more DNA molecules may include one or more regulatory elements operable to express a gene editing protein or a guide molecule. Optionally, the one or more regulatory elements may include an inducible promoter.

The DNA molecules constituting the above ultra-small gene editing system can be delivered into a cell by being contained in one or more adeno-associated virus (AAV) vectors. Preferably, the DNA molecules can be delivered into a cell by being contained in a single adeno-associated virus (AAV) vector.

More specifically, components of a vector that allows the ultra-small gene editing system of the present invention to be expressed in a cell include:

1. A nucleic acid structure encoding a component of a miniature gene editing system.

Since the purpose of the above vector is to express each component of the ultra-small gene editing system of the present invention within a cell, the sequence of the vector must essentially include at least one of the nucleic acid sequences encoding each component of the ultra-small gene editing system.

In one embodiment, the vector system can comprise a first nucleic acid construct operably linked to a nucleotide sequence encoding a small endonuclease comprising a Cas12f1 variant protein or a homolog protein thereof; and a second nucleic acid construct operably linked to a nucleotide sequence encoding a guide RNA. Wherein, the first nucleic acid construct and the second nucleic acid construct can be located on the same vector of the vector system or on different/separate vectors. Herein, the linkage can be directly linked or via a linker.

In one embodiment, the nucleic acid construct can comprise a nucleic acid encoding an engineered guide RNA. The engineered guide RNA can comprise an engineered tracrRNA and/or an engineered crRNA. The engineered guide RNA can have the same composition as the engineered guide RNA embodiments described above.

Specifically, the guide RNA may include/be comprised of a nucleic acid sequence encoding an augment RNA having a modification in MS1 (SEQ ID NO: 44), an augment RNA having a modification in MS1/MS2 (SEQ ID NO: 45), an augment RNA Cas12f1_ge3.0 having a modification in MS1/MS2/MS3 (SEQ ID NO: 46), an augment RNA Cas12f1_ge4.0 having a modification in MS2/MS3/MS4 (SEQ ID NO: 47), and/or an augment RNA Cas12f1_ge4.1 having a modification in MS2/MS3/MS4/MS5 (SEQ ID NO: 48). In addition, the guide RNA may include/be comprised of a nucleic acid encoding an augment RNA in which the MS2 modification of the present invention is added to each of an engineered augment RNA selected from any one of SEQ ID NOs: 105 to 137 or an engineered augment RNA consisting of the base sequences of SEQ ID NOs: 125 to 137.

In addition, in the nucleic acid structure, the novel ultra-small gene editing protein Cas12f1 variant protein is a protein comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, and the nucleic acid structure may comprise a nucleic acid encoding the protein or a codon-optimized nucleic acid of the protein. Preferably, the ultra-small gene editing protein may be a gene editing protein characterized by comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, and the codon-optimized nucleic acid encoding the same may be a human codon-optimized nucleic acid consisting of any one nucleotide sequence selected from SEQ ID NO: 101 to SEQ ID NO: 104.

The novel ultra-small gene editing protein of the above nucleic acid structure, Cas12f1 variant protein or a homolog protein thereof, may have a peptide of a certain length added thereto. The peptide may comprise or consist of a nucleic acid sequence encoding any one amino acid sequence selected from the group consisting of SEQ ID NO: 49 to SEQ ID NO: 51.

In addition, the nucleic acid structure may include at least one nuclear localization signal (NLS) or nuclear export signal (NES) sequence at the N-terminus or C-terminus. The NLS sequence refers to a peptide of a certain length or its sequence that acts as a kind of "tag" by attaching to a protein, etc., which is a target of transport, when transporting a substance outside the cell nucleus into the nucleus by nuclear transport. The NES sequence refers to a peptide of a certain length or its sequence that acts as a kind of "tag" by attaching to a protein, which is a target of transport, when transporting a substance inside the cell nucleus into the nucleus by nuclear transport.

The above NLS sequence may be a nucleotide sequence of SEQ ID NO: 52 or SEQ ID NO: 53, or may comprise or consist of a nucleic acid sequence encoding any one amino acid sequence selected from SEQ ID NO: 54 to SEQ ID NO: 57.

The sequence of the above vector includes a nucleic acid sequence encoding a guide RNA and/or a gene editing protein included in the above ultra-small gene editing system to be expressed. For information regarding the nucleic acid sequence, refer to the contents described in “III. Nucleic Acids Encoding Components of the Ultra-small Gene Editing System.”

The vector may be configured to express two or more different engineered guide RNAs. In one embodiment, the vector may be configured to express a first augment RNA and a second augment RNA. In one embodiment, the first augment RNA sequence may comprise a first scaffold sequence, a first spacer sequence, and a first U-rich tail sequence, and the second augment RNA sequence may comprise a second scaffold sequence, a second spacer sequence, and a second U-rich tail sequence.

Additionally, the vector may contain, in addition to the components of the aforementioned miniature gene editing system, a nucleic acid sequence encoding additional expression elements that a person skilled in the art desires to express as needed.

For example, the additional expression element may be a tag. Specifically, the additional expression element may be a herbicide resistance gene such as glyphosate, glufosinate ammonium or phosphinothricin, an antibiotic resistance gene such as ampicillin, kanamycin, G418, Bleomycin, hygromycin or chloramphenicol.

2. Regulating and/or controlling components

In order to express the above vector in a cell, it must include one or more regulatory and/or control elements. Specifically, the regulatory and/or control elements may include, but are not limited to, a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an Internal Ribosome Entry Site (IRES), a splice acceptor, a 2A sequence and/or a replication origin. Here, the replication origin may be, but is not limited to, an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin and/or a BBV replication origin.

3. Promoter

In order to express the nucleic acid sequence encoding the hypercompact TaRGET system of the present invention contained in the above vector in a cell, a promoter sequence must be operably linked to the sequence encoding each component so that an RNA transcription factor can be activated in the cell. The promoter sequence can be designed differently depending on the corresponding RNA transcription factor or expression environment, and is not limited as long as it can appropriately express the components of the hypercompact TaRGET system in a cell.

For example, the promoter sequence may be a promoter that promotes transcription of RNA polymerase RNA Pol I, Pol II or Pol III. Specifically, the promoter may be one of the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), the rous sarcoma virus (RSV) promoter, the human U6 small nuclear promoter (U6), the enhanced U6 promoter, the 7SK promoter (7SK) and the human H1 promoter (H1).

4. End signal

When the above vector sequence includes a promoter sequence, transcription of a sequence operably linked to the promoter is induced by an RNA transcription factor, and a termination signal that induces termination of transcription of the RNA transcription factor may be included. The termination signal may vary depending on the type of the promoter sequence. Specifically, when the promoter is a U6 or H1 promoter, the promoter recognizes a TTTTT (T5) or TTTTTT (T6) sequence, which is a continuous sequence of thymidine (T), as a termination signal.

The sequence of the engineered guide RNA provided in the present invention includes a U-rich tail sequence at its 3'-terminus. Accordingly, the sequence encoding the engineered guide RNA includes a T-rich sequence corresponding to the U-rich tail sequence at its 3'-terminus. As described above, some promoter sequences recognize a thymidine (T) sequence, for example, a sequence in which five or more thymidines (T) are connected in sequence, as a termination signal, and therefore, in some cases, the T-rich sequence may be recognized as a termination signal.

In other words, when the vector sequence provided herein includes a sequence encoding an engineered guide RNA, a sequence encoding a U-rich tail sequence included in the augment RNA sequence can be used as a termination signal.

In one embodiment, when the vector sequence comprises a U6 or H1 promoter sequence and a sequence encoding an engineered guide RNA operably linked thereto, a portion of the sequence encoding a U-rich tail sequence included in the augment RNA sequence can be recognized as a termination signal. In this case, the U-rich tail sequence comprises a sequence in which five or more uridines (U) are consecutively linked.

5. Additional expression factors

The above vector may be configured to express additional components, such as NLS, NES and/or tag proteins, as required.

In one embodiment, the additional component can be expressed independently of the Cas12f1 variant protein, a homolog of the Cas12f1 variant protein, and/or an engineered guide RNA (gRNA) for the Cas12f1 variant.

In another embodiment, the additional component can be expressed directly or via a linker with the Cas12f1 variant protein, a homolog of the Cas12f1 variant protein, and/or an engineered guide RNA (gRNA) for the Cas12f1 variant.

For example, a nucleic acid construct encoding a component of an ultra-small gene editing system according to the present invention may be a nucleic acid construct characterized in that it includes at least one nuclear localization sequence (NLS) sequence at the N-terminus or the C-terminus. The NLS sequence may be a nucleic acid construct characterized in that it includes/consists of a base sequence encoding any one amino acid sequence selected from SEQ ID NO: 54 to SEQ ID NO: 57. Here, the additional component may be a component that is generally expressed when it is desired to express an ultra-small gene editing system, and reference may be made to known technologies widely recognized by those skilled in the art.

In addition, the present invention provides, as an embodiment, a nucleic acid included in a vector or the like for expressing an engineered guide RNA (gRNA) according to the present invention or a nucleic acid encoding the same and/or a component of a micro-gene editing system. Here, the nucleic acid may be DNA or RNA existing in nature, and may be a modified nucleic acid in which a part or all of the nucleic acid has been chemically modified. For example, the nucleic acid may be one in which one or more nucleotides have been chemically modified. In this case, the chemical modification may include all modifications of nucleic acids known to those skilled in the art.

6. Types and forms of expression vectors

The vector according to the present invention may be a viral vector. More specifically, the viral vector may be at least one selected from the group consisting of a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus. In one embodiment, the viral vector may be an adeno-associated virus vector.

In addition, the vector according to the present invention may be a non-viral vector. More specifically, the non-viral vector may be at least one selected from the group consisting of a plasmid, a phage, naked DNA, a DNA complex, and mRNA. In one embodiment, the plasmid may be selected from the group consisting of pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19. In one embodiment, the phage may be M13, and the vector may be a PCR amplicon.

The vector according to the present invention can be designed in the form of a linear or circular vector. If the vector is a linear vector, RNA transcription is terminated at the 3'-end even if the linear vector sequence does not separately include a termination signal. However, if the vector is a circular vector, RNA transcription is not terminated if the circular vector sequence does not separately include a termination signal. Therefore, when using a circular vector as the vector, a termination signal corresponding to a transcription factor associated with each promoter sequence must be included in order to express the intended target.

V. Gene editing method using engineered guide RNA

The present invention provides a method for editing or targeting a target nucleic acid or target gene within a target cell or within a test tube using an engineered guide RNA (augment RNA) that has optimal target editing activity for a Cas12f1 mutant protein or a homolog protein thereof. The gene editing method may be a method for cleaving a nucleic acid at a target site. The target gene or target nucleic acid comprises a target sequence, and the target nucleic acid may be single-stranded DNA, double-stranded DNA, and/or RNA.

According to one aspect of the present invention, a gene editing method is provided, comprising a step of contacting a gene editing system of the present invention, a vector system of the present invention, or a gene editing composition of the present invention with a target gene or a target nucleic acid.

In one embodiment, the gene editing method comprises delivering an engineered guide RNA (augment RNA) for a Cas12f1 variant and a Cas12f1 variant protein or a homolog protein thereof, or a nucleic acid encoding each of them, into a target nucleic acid or a target cell containing the target gene. As a result, a guide RNA and Cas12f1 variant protein complex comprising the engineered guide RNA is injected into the target cell, or formation of the guide RNA and Cas12f1 variant protein complex is induced, and the target gene is edited by the guide RNA and Cas12f1 variant protein complex. The gene editing comprises nucleic acid cleavage of a double-stranded DNA, a single-stranded DNA, or a hybrid duplex of DNA and RNA having a target sequence in the target gene or target nucleic acid. Here, the Cas12f1 variant protein may be a wild-type Cas12f1 variant protein, an engineered Cas12f1 variant protein, a modified Cas12f1 variant protein, or a homolog protein of the Cas12f1 variant.

In one embodiment, the gene editing method may comprise delivering into a target cell a nucleic acid encoding a Cas12f1 variant protein or a homolog protein thereof; and an engineered guide RNA (augment RNA) or a nucleic acid encoding the same, wherein the engineered guide RNA (augment RNA) sequence comprises a sequence of a modified scaffold region, a spacer sequence, and a U-rich tail sequence. Here, the sequence of the modified scaffold region may have the same characteristics and structure as those described in the aforementioned sections “3. 3. Engineered Guide RNA for Cas12f1 Variant Protein” and “(2) Scaffold Region.”

For example, the engineered tracrRNA may comprise/consist of any one base sequence selected from SEQ ID NO: 29 to SEQ ID NO: 32, and the engineered crRNA may comprise/consist of the base sequence of SEQ ID NO: 33.

As another example, the engineered tracrRNA may comprise/consist of any one base sequence selected from SEQ ID NO: 39 to SEQ ID NO: 42, and the engineered crRNA may comprise/consist of the base sequence of SEQ ID NO: 43.

In one embodiment, the engineered guide RNA may comprise/consist of any one base sequence selected from SEQ ID NO: 44 to SEQ ID NO: 48.

In addition, the spacer sequence can complementarily bind to a target gene or a target nucleic acid included in the target cell, and the U-rich tail sequence can be expressed as (UaV)nUb. Here, a, n, and b are integers, a is 1 or more and 4 or less, n is 0 or more, and b is 1 or more and 10 or less. In another embodiment, the U-rich tail sequence can be expressed as (UaN)nUb. Here, N is one of adenosine (A), uridine (U), cytidine (C), and guanosine (G). Here, a is an integer of 1 or more and 4 or less, n is an integer of 0, 1, and 2, and b is an integer of 1 or more and 10 or less. For example, the sequence of the U-rich tail is 5'-U-3', 5'-UU-3', 5'-UUU-3', 5'-UUUU-3', 5'-UUUUU-3', 5'-UUUUUU-3', 5'-UUURUUU-3' (SEQ ID NO: 67), 5'-UUURUUUUU-3' (SEQ ID NO: 68), 5'-UUUURU-3' (SEQ ID NO: 69), 5'-UUUURUU-3' (SEQ ID NO: 70), 5'-UUUURUUU-3' (SEQ ID NO: 71), 5'-UUUURUUUU-3' (SEQ ID NO: 72), 5'-UUUURUUUUU-3' (SEQ ID NO: 73), or It can be 5'-UUUURUUUUU-3' (SEQ ID NO: 74). Here, R can be A or G. In this case, the sequence of the U-rich tail can be any one of SEQ ID NO: 75 to SEQ ID NO: 90. Preferably, the sequence of the U-rich tail can be 5'-UUUUAUUUU-3' (SEQ ID NO: 80) or 5'-UUUUGUUUU-3' (SEQ ID NO: 88).

Below, the steps of a gene editing method using engineered guide RNA (augment RNA) are described.

1. Determination of target sequence, spacer sequence, and target of gene editing

The target of gene editing using the hypercompact TaRGET system of the present invention may be a nucleic acid in a test tube or a nucleic acid in a prokaryotic cell or a eukaryotic cell. More specifically, the eukaryotic cell may be, but is not limited to, yeast, an insect cell, a plant cell, an animal cell, and/or a human cell.

The target nucleic acid, target gene or target sequence can be determined in consideration of the purpose of gene editing, the environment of the target of editing, the PAM sequence recognized by the Cas12f1 variant protein or its homolog protein, and/or other variables. Here, if a target sequence having an appropriate length or a PAM sequence recognized by the Cas12f1 variant protein or its homolog protein can be determined within the target nucleic acid or target gene, the method can be performed without particular limitation by utilizing known techniques.

Once the target sequence is determined, a spacer sequence in the corresponding guide RNA is designed. The spacer sequence is designed as a sequence that can bind to the target sequence.

In one embodiment, the spacer sequence is designed as a sequence capable of complementarily binding to the target nucleic acid or target gene. Specifically, the spacer sequence can be designed as a sequence complementary to a target sequence included in the target strand sequence of the target nucleic acid or target gene.

In addition, the spacer sequence may be designed as an RNA sequence corresponding to the DNA sequence of the protospacer included in the non-target strand sequence of the target nucleic acid. Specifically, the spacer sequence may be designed as a sequence having the same base sequence as the protospacer sequence, and in which each of the thymidines (T) included in the base sequence is substituted with uridine (U).

In one embodiment, the spacer sequence may be a complementary sequence having at least 60% sequence identity with the target sequence. Preferably, the spacer sequence may be a complementary sequence having from 60% to 90% sequence identity with the target sequence. More preferably, the spacer sequence may be a complementary sequence having from 90% to 100% sequence identity with the target sequence.

In addition, the spacer sequence according to the present invention may be a complementary sequence having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches with the target sequence. In one embodiment, the spacer sequence may have 1 to 5 mismatches with the target sequence. In addition, the spacer sequence may have 6 to 10 mismatches with the target sequence.

2. Intracellular delivery of each component of the hypercompact TaRGET system

The base correction and gene editing method provided in this specification utilizes the fact that the hypercompact TaRGET system according to the present invention has the activity of recognizing and editing a target sequence for a target nucleic acid or target gene.

The gene editing method provided herein presupposes that a miniature gene editing system or vector comprising an engineered guide RNA (gRNA) for a Cas12f1 variant is brought into contact with a target nucleic acid or a target sequence site of a target gene within a target cell.

Accordingly, the gene editing method of the present invention includes effectively delivering the ultra-small gene editing system within a target cell. Preferably, the method includes contacting or inducing contact of the nucleic acid structure of the ultra-small gene and/or each component of the ultra-small gene editing system comprising the same with a target nucleic acid or a target sequence site of a target gene within the target cell.

In one embodiment, the gene editing method may comprise delivering a Cas12f1 variant protein or a homolog thereof, or a nucleic acid encoding it; and an engineered guide RNA (augment RNA) or a nucleic acid encoding it into a target cell. In addition, the gene editing method may comprise delivering a Cas12f1 variant protein or a homolog thereof; and an engineered guide RNA (augment RNA) into a target cell. In another embodiment, the gene editing method may comprise delivering a Cas12f1 variant protein or a homolog thereof; and a nucleic acid encoding an engineered guide RNA (augment RNA) into a target cell. In yet another embodiment, the gene editing method may comprise delivering a nucleic acid encoding a Cas12f1 variant protein or a homolog thereof; and an engineered guide RNA (augment RNA) into a target cell. In yet another embodiment, the gene editing method may comprise delivering a nucleic acid encoding a Cas12f1 variant protein or a homolog thereof; and delivering a nucleic acid encoding an engineered guide RNA (augment RNA) into a target cell.

The Cas12f1 variant protein or a homolog protein thereof, or a nucleic acid encoding the same; and an engineered guide RNA (augment RNA) or a nucleic acid encoding the same can be delivered into a target cell in various delivery forms using various delivery methods. Here, the induction is not particularly limited as long as it is a method by which the ultra-small gene editing system or ultra-small gene editing nucleic acid structure including the engineered guide RNA (augment RNA) for the Cas12f1 variant comes into contact with the target nucleic acid within the cell.

(1) Transmission form

The delivery form of the ultra-small gene editing nucleic acid construct for the method of the present invention and the ultra-small gene editing system (Hypercompact TaRGET system) comprising the same is not particularly limited as long as it can deliver the Cas12f1 mutant protein or a homolog protein thereof, or a nucleic acid encoding the same; and the engineered guide RNA (augment RNA) or a nucleic acid encoding the same into a cell in an appropriate delivery form.

As a delivery form of the ultra-small gene editing nucleic acid structure and the ultra-small gene editing system comprising the same for the method of the present invention, a ribonucleoprotein particle (RNP) in which a guide RNA engineered for a Cas12f1 variant and a Cas12f1 variant protein are bound can be used.

In one embodiment, the gene editing method may comprise injecting into a target cell a complex of guide RNA and Cas12f1 variant protein, wherein the guide RNA and Cas12f1 variant protein are engineered for the Cas12f1 variant.

In another delivery form, a nonviral vector may be used comprising a Cas12f1 variant protein or a homologous protein thereof, or a nucleic acid encoding the same; and an engineered guide RNA or a nucleic acid encoding the same.

In one embodiment, the gene editing method can comprise introducing into a target cell a nonviral vector comprising a nucleic acid sequence encoding a Cas12f1 variant protein and a nucleic acid sequence encoding a guide RNA engineered for the Cas12f1 variant. Specifically, the nonviral vector can be, but is not limited to, a plasmid, naked DNA, a DNA complex, mRNA, or a linear PCR amplicon.

In another embodiment, the gene editing method can comprise introducing into a target cell a first nonviral vector comprising a nucleic acid sequence encoding a Cas12f1 variant protein and a second nonviral vector comprising a nucleic acid sequence encoding a guide RNA engineered for the Cas12f1 variant. Specifically, the first nonviral vector and the second nonviral vector can each be one selected from the group consisting of, but not limited to, a plasmid, a naked DNA, a DNA complex, an mRNA, and a linear PCR amplicon.

Another delivery form may utilize a virus comprising a nucleic acid sequence encoding a Cas12f1 variant protein and a nucleic acid sequence encoding a guide RNA engineered for the Cas12f1 variant.

In one embodiment, the gene editing method may comprise introducing into a target cell a virus comprising a nucleic acid sequence encoding a Cas12f1 variant protein and a nucleic acid sequence encoding a guide RNA engineered for the Cas12f1 variant. Specifically, the virus may be one selected from the group consisting of a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus, but is not limited thereto. Preferably, the virus may be an adeno-associated virus.

As another example, the gene editing method may comprise introducing into a target cell a first virus comprising a nucleic acid sequence encoding a Cas12f1 variant protein or a homolog protein thereof and a second virus comprising a nucleic acid sequence encoding a guide RNA engineered for the Cas12f1 variant. Specifically, the first viral vector and the second viral vector may each be selected from the group consisting of, but not limited to, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus.

Additionally, the delivery form may be a method of delivering a Cas12f1 variant protein or a homolog protein thereof, or a nucleic acid encoding the same; and a guide RNA engineered for the Cas12f1 variant, or a nucleic acid encoding the same, using nanoparticles.

In one embodiment, the delivery method may be to deliver the Cas12f1 variant protein or a nucleic acid encoding it, the first engineered guide RNA for the Cas12f1 variant or a nucleic acid encoding it, and/or the second engineered guide RNA for the Cas12f1 variant or a nucleic acid encoding it using nanoparticles.

Here, the delivery method may be, but is not limited to, cationic liposome method, lithium acetate-DMSO, lipid-mediated transfection, calcium phosphate precipitation, lipofection, electroporation, gene gun, sonoporation, magnetofection, and/or transient cell compression or squeezing, PEI (Polyethyleneimine)-mediated transfection, DEAE-dextran-mediated transfection or nanoparticle-mediated nucleic acid delivery.

In addition, the delivery form of the method for editing a target gene by delivering into a cell a Cas12f1 variant protein or a homolog protein thereof, or a nucleic acid encoding the same; and a guide RNA engineered for the Cas12f1 variant or a nucleic acid encoding the same in the present invention can be performed by combining the aforementioned delivery forms.

In one embodiment, the gene editing method can comprise delivering the Cas12f1 variant protein or a homolog protein thereof, or a nucleic acid encoding it, in a first delivery form, and delivering the guide RNA engineered for the Cas12f1 variant or a nucleic acid encoding it in a second delivery form. In this case, the first delivery form and the second delivery form can each be any one of the delivery forms described above.

In one embodiment, the delivery form of the gene editing method is not particularly limited as long as it is a delivery form that can deliver the ultra-small gene editing nucleic acid structure according to the present invention to be delivered in one vector or a component of the ultra-small gene editing system including the same to an environment where a target nucleic acid or target gene to be edited exists.

(2) Order of delivery

The above gene editing method comprises delivering into a cell a Cas12f1 variant protein or a homologous protein thereof, or a nucleic acid encoding the same; and a guide RNA engineered for the Cas12f1 variant, or a nucleic acid encoding the same, wherein the constructs can be delivered into the cell simultaneously, but can be delivered sequentially with a time difference.

In one embodiment, the gene editing method can comprise simultaneously delivering a Cas12f1 variant protein or a homolog protein thereof, or a nucleic acid encoding the same; and a guide RNA engineered for the Cas12f1 variant, or a nucleic acid encoding the same.

In another embodiment, the gene editing method can comprise delivering into a cell a Cas12f1 variant protein or a homologous protein thereof, or a nucleic acid encoding the same, followed by, at a time interval, delivering into the cell a guide RNA engineered for the Cas12f1 variant, or a nucleic acid encoding the same.

In another embodiment, the gene editing method can comprise delivering into a cell a guide RNA engineered for a Cas12f1 variant or a nucleic acid encoding the same, followed by timed delivery into the cell of the Cas12f1 variant protein or a homolog protein thereof, or a nucleic acid encoding the same.

In addition, the gene editing method provided by the present invention may include delivering into a target cell a Cas12f1 variant protein or a homolog protein thereof, or a nucleic acid encoding the same; and a guide RNA engineered for two or more Cas12f1 variants, or a nucleic acid encoding the same.

Through the above method, two or more guide RNA and Cas12f1 variant protein complexes targeting different sequences can be injected into a target cell, or two or more guide RNA and Cas12f1 variant protein complexes can be formed within the target cell. As a result, two or more target genes or target nucleic acids contained within the cell can be edited.

In one embodiment, the gene editing method comprises delivering a Cas12f1 variant protein or a nucleic acid encoding it, a first engineered guide RNA for the Cas12f1 variant or a nucleic acid encoding it, and a second engineered guide RNA for the Cas12f1 variant or a nucleic acid encoding it into a target cell comprising a target gene or a target nucleic acid. In this case, each of the components can be delivered into the cell using one or more of the aforementioned delivery forms and delivery methods. Here, two or more components can be delivered into the cell simultaneously or sequentially with a time difference.

Additionally, in one embodiment, the hypercompact TaRGET system comprises an engineered guide RNA that maximizes its targeting activity or gene editing activity for a Cas12f1 variant protein or a homolog protein thereof.

This may be any one or more of those described in the above-mentioned “3. Engineered guide RNA for Cas12f1 variant protein (augment RNA)” and “(2) Scaffold region” sections. Preferably, the guide RNA may be an augment RNA having a modification in MS1 (SEQ ID NO: 44), an augment RNA having a modification in MS1/MS2 (SEQ ID NO: 45), an augment RNA Cas12f1_ge3.0 having a modification in MS1/MS2/MS3 (SEQ ID NO: 46), an augment RNA Cas12f1_ge4.0 having a modification in MS2/MS3/MS4 (SEQ ID NO: 47), and/or an augment RNA Cas12f1_ge4.1 having a modification in MS2/MS3/MS4/MS5 (SEQ ID NO: 48). These are merely examples and are not limiting.

In addition, it is obvious that the gene editing composition of the present invention may additionally contain appropriate substances necessary for gene editing purposes in addition to each component of the ultracompact TaRGET system according to the present invention.

The present invention also provides a method of editing a nucleic acid, comprising the step of contacting a target sequence with the ultra-small gene editing system according to the present invention or the composition comprising the same. Here, the nucleic acid editing may be nucleic acid cleavage. As a result, nucleic acid editing of an indel occurs, in which any base in the target nucleic acid or target gene is deleted or added.

In one embodiment, the gene editing method may comprise delivering an ultra-small gene editing system in the form of a ribonucleoprotein particle, wherein the ultra-small gene editing protein according to the present invention, the Cas12f1 variant protein or a homolog protein thereof is bound to a guide RNA engineered for a Cas12f1 variant, into a eukaryotic cell. In this case, the delivery may be performed using electroporation or lipofection.

In another embodiment, the gene editing method can comprise delivering into a cell comprising a target nucleic acid or target gene using an adeno-associated virus (AAV) vector, preferably comprising both a nucleic acid sequence encoding a guide RNA engineered for a Cas12f1 variant and a nucleic acid sequence encoding a Cas12f1 variant protein or a homologous protein thereof.

Hereinafter, the invention provided by the present specification will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are intended only to illustrate the content disclosed by the present specification, and that the scope of the content disclosed by the present specification is not limited by these examples.

Example

Example 1. Fabrication of components of a hypercompact TaRGET system

Example 1.1. Gene editing protein and human codon-optimized nucleic acid encoding same

The present invention relates to a protein constituting a Hypercompact TaRGET (Tiny nuclease-augment RNA-based Genome Editing Technology) system, which is an ultra-small gene editing system, and comprises a Cas12f1 variant protein or a homolog protein thereof. Preferably, the Cas12f1 variant protein comprises a protein consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence in which 1 to 28 amino acids are deleted or substituted from the N-terminus based on the sequence (except for a Cas12f1 protein consisting of the amino acid sequence of SEQ ID NO: 5), and as a representative example of a Cas12f1 variant protein (engineered Cas12f variant protein) consisting of an amino acid sequence in which 1 to 28 amino acids are deleted or substituted from the N-terminus based on the amino acid sequence of SEQ ID NO: 1, a Cas12f1 variant v1 protein (SEQ ID NO: 2) comprising N-terminal 26aa of CasX at the N-terminus of Cas12f1, a Cas12f1 variant v2 protein (SEQ ID NO: 3) comprising a 28aa random sequence, or a Cas12f1 variant v3 protein (SEQ ID NO: 4) comprising a 26aa random sequence is provided. In addition, the Cas12f1 variant protein includes a protein consisting of an amino acid sequence having 1 to 600 amino acids added to the N-terminus or C-terminus of a Cas12f1 variant comprising or consisting of the amino acid sequence of SEQ ID NO: 1. Here, the 1 to 600 amino acids added to the N-terminus or C-terminus may include or consist of the amino acid sequence of SEQ ID NO: 233 or SEQ ID NO: 234, and one or more NLS sequences may be further included between the added sequence and the Cas12f1 variant protein. In addition, a homolog of the Cas12f1 variant protein may be a protein comprising or consisting of any one amino acid sequence selected from SEQ ID NO: 141 to SEQ ID NO: 232.

The present invention also provides a human codon-optimized gene for a Cas12f1 mutant protein using a codon optimization program to construct a hypercompact TaRGET system expressed in human cells and a hypercompact nucleic acid construct for nucleic acid cleavage.

A nucleic acid construct including a human codon-optimized Cas12f1 variant gene (SEQ ID NO: 101) was produced by adding NLS sequences, 5'-CCAAAGAAGAAGCGGAAGGTC-3' (SEQ ID NO: 52) and 5'-AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG-3' (SEQ ID NO: 53), to the 5'-terminal and 3'-terminal of the codon-optimized gene, respectively, and synthesizing a gene in which a linker 5'-GGTATCCACGGAGTCCCAGCAGCC-3' (SEQ ID NO: 100) was connected between the 5'-terminal NLS sequence and the start codon of the Cas12f1 variant.

Table 1 below shows the human codon-optimized Cas12f1 variant base sequence encoding the Cas12f1 variant protein produced above and the amino acid sequence of the Cas12f1 variant protein. In addition, Table 2 shows the base sequences of human codon-optimized nucleic acids encoding Cas12f1 variant v1 to v3 proteins, respectively. These were used as nucleic acids encoding the gene editing proteins constituting the ultra-small gene editing system according to the present invention.

Label Sequence (5' to 3') SEQ ID
NO:
Cas12f1 mutant protein

MGEKSSRRRRNGKSGAWTAAITSCVGGKMAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIDVGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP

1

Human codon-optimized nucleic acid encoding Cas12f1 mutant protein

ATGGGGGAGAAAAGTTCCCGCCGCCGACGGAATGGAAAAAGCGGTGCGTGGACTGCTGCTATAACAAGCTGTGTTGGGGGTAAGATGGCCAAGAACACAATTACAAAGACACTGAAGCTGAGGATCGTGAGACCATACAACAGCGCTGAGGTCGAGAAGATTGTGGCTGATGAAAAGAACAACAGGGAAAAGATCGCCCTCGAGAAGAACAAGGATAAGGTGAAGGAGGCCTGCTCTAAGCACCTGAAAGTGGCCGCCTACTGCACCACACAGGTGGAGAGGAACGCCTGTCTGTTTTGTAAAGCTCGGAAGCTGGATGATAAGTTTTACCAGAAGCTGCGGGGCCAGTTCCCCGATGCCGTCTTTTGGCAGGAGATTAGCGAGATCTTCAGACAGCTGCAGAAGCAGGCCGCCGAGATCTACAACCAGAGCCTGATCGAGCTCTACTACGAGATCTTCATCAAGGGCAAGGGCATTGCCAACGCCTCCTCCGTGGAGCACTACCTGAGCGACGTGTGCTACACAAGAGCCGCCGAGCTCTTTAAGAACGCCGCTATCGCTTCCGGGCTGAGGAGCAAGATTAAGAGTAACTTCCGGCTCAAGGAGCTGAAGAACATGAAGAGCGGCCTGCCCACTACAAAGAGCGACAACTTCCCAATTCCACTGGTGAAGCAGAAGGGGGGCCAGTACACAGGGTTCGAGATTTCCAACCACAACAGCGACTTTATTATTAAGATCCCCTTTGGCAGGTGGCAGGTCAAGAAGGAGATTGACAAGTACAGGCCCTGGGAGAAGTTTGATTTCGAGCAGGTGCAGAAGAGCCCCAAGCCTATTTCCCTGCTGCTGTCCACACAGCGGCGGAAGAGGAACAAGGGGTGGTCTAAGGATGAGGGGACCGAGGCCGAGATTAAGAAAGTGATGAACGGCGACTACCAGACAAGCTACATCGAGGTCAAGCGGGGCAGTAAGATTGGCGAGAAGAGCGCCTGGATGCTGAACCTGAGCATTGACGTGCCAAAGATTGATAAGGGCGTGGATCCCAGCATCATCGGAGGGATCGATGTGGGGGTCAAGAGCCCCCTCGTGTGCGCCATCAACAACGCCTTCAGCAGGTACAGCATCTCCGATAACGACCTGTTCCACTTTAACAAGAAGATGTTCGCCCGGCGGAGGATTTTGCTCAAGAAGAACCGGCACAAGCGGGCCGGACACGGGGCCAAGAACAAGCTCAAGCCCATCACTATCCTGACCGAGAAGAGCGAGAGGTTCAGGAAGAAGCTCATCGAGAGATGGGCCTGCGAGATCGCCGATTTCTTTATTAAGAACAAGGTCGGAACAGTGCAGATGGAGAACCTCGAGAGCATGAAGAGGAAGGAGGATTCCTACTTCAACATTCGGCTGAGGGGGTTCTGGCCCTACGCTGAGATGCAGAACAAGATTGAGTTTAAGCTGAAGCAGTACGGGATTGAGATCCGGAAGGTGGCCCCCAACAACACCAGCAAGACCTGCAGCAAGTGCGGGCACCTCAACAACTACTTCAACTTCGAGTACCGGAAGAAGAACAAGTTCCCACACTTCAAGTGCGAGAAGTGCAACTTTAAGGAGAACGCCGATTACAACGCCGCCCTGAACATCAGCAACCCTAAGCTGAAGAGCACTAAGGAGGAGCCCAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG

101

Label Sequence (5' to 3') SEQ ID
NO:
Human codon-optimized nucleic acid encoding Cas12f1 variant v1 protein

ATGGAAAAGAGAATCAACAAGATCAGGAAGAAGCTGAGCGCCGACAACGCCACCAAGCCTGTGTCAGGAGTGGCCCCCATGGCCAAGAACACAATTACAAAGACACTGAAGCTGAGGATCGTGAGACCATACAACAGCGCTGAGGTCGAGAAGATTGTGGCTGATGAAAAGAACAACAGGGAAAAGATCGCCCTCGAGAAGAACAAGGATAAGGTGAAGGAGGCCTGCTCTAAGCACCTGAAAGTGGCCGCCTACTGCACCACACAGGTGGAGAGGAACGCCTGTCTGTTTTGTAAAGCTCGGAAGCTGGATGATAAGTTTTACCAGAAGCTGCGGGGCCAGTTCCCCGATGCCGTCTTTTGGCAGGAGATTAGCGAGATCTTCAGACAGCTGCAGAAGCAGGCCGCCGAGATCTACAACCAGAGCCTGATCGAGCTCTACTACGAGATCTTCATCAAGGGCAAGGGCATTGCCAACGCCTCCTCCGTGGAGCACTACCTGAGCGACGTGTGCTACACAAGAGCCGCCGAGCTCTTTAAGAACGCCGCTATCGCTTCCGGGCTGAGGAGCAAGATTAAGAGTAACTTCCGGCTCAAGGAGCTGAAGAACATGAAGAGCGGCCTGCCCACTACAAAGAGCGACAACTTCCCAATTCCACTGGTGAAGCAGAAGGGGGGCCAGTACACAGGGTTCGAGATTTCCAACCACAACAGCGACTTTATTATTAAGATCCCCTTTGGCAGGTGGCAGGTCAAGAAGGAGATTGACAAGTACAGGCCCTGGGAGAAGTTTGATTTCGAGCAGGTGCAGAAGAGCCCCAAGCCTATTTCCCTGCTGCTGTCCACACAGCGGCGGAAGAGGAACAAGGGGTGGTCTAAGGATGAGGGGACCGAGGCCGAGATTAAGAAAGTGATGAACGGCGACTACCAGACAAGCTACATCGAGGTCAAGCGGGGCAGTAAGATTGGCGAGAAGAGCGCCTGGATGCTGAACCTGAGCATTGACGTGCCAAAGATTGATAAGGGCGTGGATCCCAGCATCATCGGAGGGATCGATGTGGGGGTCAAGAGCCCCCTCGTGTGCGCCATCAACAACGCCTTCAGCAGGTACAGCATCTCCGATAACGACCTGTTCCACTTTAACAAGAAGATGTTCGCCCGGCGGAGGATTTTGCTCAAGAAGAACCGGCACAAGCGGGCCGGACACGGGGCCAAGAACAAGCTCAAGCCCATCACTATCCTGACCGAGAAGAGCGAGAGGTTCAGGAAGAAGCTCATCGAGAGATGGGCCTGCGAGATCGCCGATTTCTTTATTAAGAACAAGGTCGGAACAGTGCAGATGGAGAACCTCGAGAGCATGAAGAGGAAGGAGGATTCCTACTTCAACATTCGGCTGAGGGGGTTCTGGCCCTACGCTGAGATGCAGAACAAGATTGAGTTTAAGCTGAAGCAGTACGGGATTGAGATCCGGAAGGTGGCCCCCAACAACACCAGCAAGACCTGCAGCAAGTGCGGGCACCTCAACAACTACTTCAACTTCGAGTACCGGAAGAAGAACAAGTTCCCACACTTCAAGTGCGAGAAGTGCAACTTTAAGGAGAACGCCGATTACAACGCCGCCCTGAACATCAGCAACCCTAAGCTGAAGAGCACTAAGGAGGAGCCC

102

Human codon-optimized nucleic acid encoding Cas12f1 variant v2 protein

ATGGCTGGCGGACCAGGCGCAGGTAGTGCTGCGCCAGTTTCTTCAACTTCCTCCCTGCCCCTGGCTGCGCTTAACATGCGCGTGATGGCCAAGAACACAATTACAAAGACACTGAAGCTGAGGATCGTGAGACCATACAACAGCGCTGAGGTCGAGAAGATTGTGGCTGATGAAAAGAACAACAGGGAAAAGATCGCCCTCGAGAAGAACAAGGATAAGGTGAAGGAGGCCTGCTCTAAGCACCTGAAAGTGGCCGCCTACTGCACCACACAGGTGGAGAGGAACGCCTGTCTGTTTTGTAAAGCTCGGAAGCTGGATGATAAGTTTTACCAGAAGCTGCGGGGCCAGTTCCCCGATGCCGTCTTTTGGCAGGAGATTAGCGAGATCTTCAGACAGCTGCAGAAGCAGGCCGCCGAGATCTACAACCAGAGCCTGATCGAGCTCTACTACGAGATCTTCATCAAGGGCAAGGGCATTGCCAACGCCTCCTCCGTGGAGCACTACCTGAGCGACGTGTGCTACACAAGAGCCGCCGAGCTCTTTAAGAACGCCGCTATCGCTTCCGGGCTGAGGAGCAAGATTAAGAGTAACTTCCGGCTCAAGGAGCTGAAGAACATGAAGAGCGGCCTGCCCACTACAAAGAGCGACAACTTCCCAATTCCACTGGTGAAGCAGAAGGGGGGCCAGTACACAGGGTTCGAGATTTCCAACCACAACAGCGACTTTATTATTAAGATCCCCTTTGGCAGGTGGCAGGTCAAGAAGGAGATTGACAAGTACAGGCCCTGGGAGAAGTTTGATTTCGAGCAGGTGCAGAAGAGCCCCAAGCCTATTTCCCTGCTGCTGTCCACACAGCGGCGGAAGAGGAACAAGGGGTGGTCTAAGGATGAGGGGACCGAGGCCGAGATTAAGAAAGTGATGAACGGCGACTACCAGACAAGCTACATCGAGGTCAAGCGGGGCAGTAAGATTGGCGAGAAGAGCGCCTGGATGCTGAACCTGAGCATTGACGTGCCAAAGATTGATAAGGGCGTGGATCCCAGCATCATCGGAGGGATCGATGTGGGGGTCAAGAGCCCCCTCGTGTGCGCCATCAACAACGCCTTCAGCAGGTACAGCATCTCCGATAACGACCTGTTCCACTTTAACAAGAAGATGTTCGCCCGGCGGAGGATTTTGCTCAAGAAGAACCGGCACAAGCGGGCCGGACACGGGGCCAAGAACAAGCTCAAGCCCATCACTATCCTGACCGAGAAGAGCGAGAGGTTCAGGAAGAAGCTCATCGAGAGATGGGCCTGCGAGATCGCCGATTTCTTTATTAAGAACAAGGTCGGAACAGTGCAGATGGAGAACCTCGAGAGCATGAAGAGGAAGGAGGATTCCTACTTCAACATTCGGCTGAGGGGGTTCTGGCCCTACGCTGAGATGCAGAACAAGATTGAGTTTAAGCTGAAGCAGTACGGGATTGAGATCCGGAAGGTGGCCCCCAACAACACCAGCAAGACCTGCAGCAAGTGCGGGCACCTCAACAACTACTTCAACTTCGAGTACCGGAAGAAGAACAAGTTCCCACACTTCAAGTGCGAGAAGTGCAACTTTAAGGAGAACGCCGATTACAACGCCGCCCTGAACATCAGCAACCCTAAGCTGAAGAGCACTAAGGAGGAGCCC

103

Human codon-optimized nucleic acid encoding Cas12f1 variant v3 protein

ATGGCTGGCGGACCAGGCGCAGGTAGTGCTGCGCCAGTTTCTTCAACTTCCTCCCTGCCCCTGGCTGCGCTTAACATGATGGCCAAGAACACAATTACAAAGACACTGAAGCTGAGGATCGTGAGACCATACAACAGCGCTGAGGTCGAGAAGATTGTGGCTGATGAAAAGAACAACAGGGAAAAGATCGCCCTCGAGAAGAACAAGGATAAGGTGAAGGAGGCCTGCTCTAAGCACCTGAAAGTGGCCGCCTACTGCACCACACAGGTGGAGAGGAACGCCTGTCTGTTTTGTAAAGCTCGGAAGCTGGATGATAAGTTTTACCAGAAGCTGCGGGGCCAGTTCCCCGATGCCGTCTTTTGGCAGGAGATTAGCGAGATCTTCAGACAGCTGCAGAAGCAGGCCGCCGAGATCTACAACCAGAGCCTGATCGAGCTCTACTACGAGATCTTCATCAAGGGCAAGGGCATTGCCAACGCCTCCTCCGTGGAGCACTACCTGAGCGACGTGTGCTACACAAGAGCCGCCGAGCTCTTTAAGAACGCCGCTATCGCTTCCGGGCTGAGGAGCAAGATTAAGAGTAACTTCCGGCTCAAGGAGCTGAAGAACATGAAGAGCGGCCTGCCCACTACAAAGAGCGACAACTTCCCAATTCCACTGGTGAAGCAGAAGGGGGGCCAGTACACAGGGTTCGAGATTTCCAACCACAACAGCGACTTTATTATTAAGATCCCCTTTGGCAGGTGGCAGGTCAAGAAGGAGATTGACAAGTACAGGCCCTGGGAGAAGTTTGATTTCGAGCAGGTGCAGAAGAGCCCCAAGCCTATTTCCCTGCTGCTGTCCACACAGCGGCGGAAGAGGAACAAGGGGTGGTCTAAGGATGAGGGGACCGAGGCCGAGATTAAGAAAGTGATGAACGGCGACTACCAGACAAGCTACATCGAGGTCAAGCGGGGCAGTAAGATTGGCGAGAAGAGCGCCTGGATGCTGAACCTGAGCATTGACGTGCCAAAGATTGATAAGGGCGTGGATCCCAGCATCATCGGAGGGATCGATGTGGGGGTCAAGAGCCCCCTCGTGTGCGCCATCAACAACGCCTTCAGCAGGTACAGCATCTCCGATAACGACCTGTTCCACTTTAACAAGAAGATGTTCGCCCGGCGGAGGATTTTGCTCAAGAAGAACCGGCACAAGCGGGCCGGACACGGGGCCAAGAACAAGCTCAAGCCCATCACTATCCTGACCGAGAAGAGCGAGAGGTTCAGGAAGAAGCTCATCGAGAGATGGGCCTGCGAGATCGCCGATTTCTTTATTAAGAACAAGGTCGGAACAGTGCAGATGGAGAACCTCGAGAGCATGAAGAGGAAGGAGGATTCCTACTTCAACATTCGGCTGAGGGGGTTCTGGCCCTACGCTGAGATGCAGAACAAGATTGAGTTTAAGCTGAAGCAGTACGGGATTGAGATCCGGAAGGTGGCCCCCAACAACACCAGCAAGACCTGCAGCAAGTGCGGGCACCTCAACAACTACTTCAACTTCGAGTACCGGAAGAAGAACAAGTTCCCACACTTCAAGTGCGAGAAGTGCAACTTTAAGGAGAACGCCGATTACAACGCCGCCCTGAACATCAGCAACCCTAAGCTGAAGAGCACTAAGGAGGAGCCC

104

The above-mentioned ultra-small gene editing nucleic acid construct was manufactured by the following method. The nucleic acid construct used in the present invention includes the gene sequence of a human codon-optimized Cas12f1 variant (including an engineered variant). PCR amplification was performed using the gene sequence as a template, and cloning was performed in accordance with the desired cloning sequence into a vector having a promoter capable of expression in a eukaryotic cell system and a poly(A) signal sequence by the Gibson assembly method. After cloning, the sequence of the obtained recombinant plasmid vector was finally confirmed through the Sanger sequencing method.

Example 1.2. Expression and purification of gene editing proteins

The gene produced in Example 1.1 above was expressed and the protein was purified.

First, the nucleic acid construct was cloned into the pMAL-c2 plasmid vector and transformed into BL21 (DE3) E. coli cells. The transformed E. coli colonies were grown in LB broth at 37°C until the optical density reached 0.7. The transformed E. coli cells were cultured overnight at 18°C in the presence of 0.1 mM isopropylthio-β-D-galactoside. Then, the cultured cells were collected by centrifugation at 3,500 g for 30 minutes, and the collected cells were resuspended in 20 mM Tris-HCl (pH 7.6), 500 mM NaCl, 5 mM β-mercaptoethanol, and 5% glycerol. The cells were lysed in a lysis buffer and then disrupted by sonication. The sample containing the disrupted cells was centrifuged at 15,000 g for 30 min, and the supernatant obtained was filtered through a 0.45 ㎛ syringe filter (Millipore), and the filtered supernatant was loaded onto a Ni ²⁺ -affinity column using an FPLC purification system (KTA Purifier, GE Healthcare). The bound fractions were eluted with a gradient of 80-400 mM imidazole, 20 mM Tris-HCl (pH 7.5).

The eluted protein was cleaved by treating with TEV protease for 16 h. The cleaved protein was purified on a Heparin column with a linear gradient of 0.15-1.6 M NaCl. The recombinant Cas12f1 mutant protein purified on the Heparin column was dialyzed against a solution of 20 mM Tris pH 7.6, 150 mM NaCl, 5 mM β-mercaptoethanol, and 5% glycerol. The dialyzed protein was purified by passing it through an MBP column, and then repurified on a monoS column (GE Healthcare) or EnrichS with a linear gradient of 0.5-1.2 M NaCl.

The above purified proteins were collected and dialyzed against a solution of 20 mM Tris pH 7.6, 150 mM NaCl, 5 mM β-mercaptoethanol, and 5% glycerol to purify the ultra-small gene editing protein (small endonuclease) used in the present invention. The concentration of the produced ultra-small gene editing protein was quantified using the Bradford assay using bovine serum albumin (BSA) as a standard and measured electrophoretically on a coomassie blue-stained SDS-PAGE gel.

Example 1.3. Engineered guide RNA for Cas12f1 variants or homologs thereof

In this example, as a component of an ultra-small gene editing system for nucleic acid or gene editing, an engineered guide RNA (augment RNA) that has high-efficiency targeting activity and gene editing activity for a Cas12f1 mutant protein or a homolog thereof was produced. The activity of an ultra-small gene editing system for nucleic acid or gene editing is important for the endonuclease activity of the Cas12f1 mutant protein or a homolog thereof constituting the system, but it is estimated that a large difference in the activity will occur depending on the degree to which the gene editing protein binds to a target nucleic acid or a target gene region. Accordingly, an engineered augment RNA for a Cas12f1 mutant was produced as follows.

The augment RNA for the Cas12f1 mutant protein or its homolog protein is a guide RNA found in nature that adds a new structure or deletes or modifies part of its structure, and may include a new structure, a U-rich tail, at the 3'-terminus. Specifically, the augment RNA is characterized by including an engineered tracrRNA sequence including a modified first to fourth regions of the scaffold, an engineered crRNA sequence including a modified fifth to sixth regions of the scaffold, and/or a U-rich tail sequence which is a modified seventh region (see FIGS. 2A and 2B ).

The fourth region and the fifth region are complementary binding sites and include modification site 1 (MS1) and modification site 4 (MS4), and the seventh region, the U-rich tail sequence, corresponds to modification site 2 (MS2). The first region is modification site 3 (MS3), and the second region includes modification site 5 (MS5).

Figure 1 illustrates in detail the "modification site (MS) MS1 to MS5", which is a site where modification is performed in a naturally existing guide RNA to produce a wild-type guide RNA for a Cas12f1 mutant and a highly efficient augment RNA for the Cas12f1 mutant protein provided by the present invention and its homologues. Figures 2a and 2b illustrate exemplary structures of various modification sites for producing an engineered single guide RNA (augment RNA) of the present invention. Figure 2a illustrates a modification site of a canonical sgRNA for a Cas12f1 mutant, and Figure 2b illustrates a modification site of a mature form sgRNA for an engineered Cas12f1 mutant.

In addition, the present invention produced engineered guide RNAs for Cas12f1 variants (Cas12f1 variant augment RNAs) according to “3. Engineered guide RNAs for ultra-small gene editing systems” and “7. Modifications for making single guide RNA” in the aforementioned “II. High-efficiency miniature genome editing system/composition” section. However, these augment RNAs are only representative examples of the engineered guide RNAs used in the present invention, and the Cas12f1 variant augment RNAs of the present invention are not limited to the exemplified sequences.

The specific base sequences of the exemplary Cas12f1 variant augment RNA are detailed in Tables 3 and 4 below. Here, the 5'-NNNNNNNNNNNNNNNNNNNN-3' portion serves as a spacer sequence and may be composed of a base sequence of 15 to 50 bases.

sgRNA Sequence (5' to 3') SEQ ID
NO: Canonical
sgRNA CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAGacgaaUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 6 MS1/MS2/MS3
(Cas12f1_ge3.0) ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAGUGCUCCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAGAGCAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNUUUUAUUUUUU 46 MS2/MS3/MS4
(Cas12f1_ge4.0) ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAgaaaGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNNUUUUAUUUUUU 47 MS2/MS3/MS4/MS5
(Cas12f1_ge4.1) ACCGCUUCACUUAGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAgaaaGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNNNNUUUUAUUUUUU 48

sgRNA Sequence (5' to 3') SEQ ID
NO: Canonical
sgRNA CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAGacgaaUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 6 MS1 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 44 MS1/MS2 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNUUUUAUUUU 45 MS1/MS3-1 GAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAGAGCAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 105 MS1/MS3-2 UGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAGUGCUCCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAGAGCAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN 106 MS1/MS3-3 ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAGUGCUCCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAGAGCAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 107 MS1/MS4 ^* -1 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCCAAUUCgaaaGAACCCGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 108 MS1/MS4 ^* -2 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCgaaaGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 109 MS1/MS4 ^* -3 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUCA GUGCU gaaa AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN 110 MS1/MS5-1 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUuuagAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN 111 MS1/MS5-2 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCuuagGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 112 MS1/MS5-3 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN 113 MS1/MS2/MS3
(Cas12f1_ge3.0) ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAGUGCUCCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAGAGCAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNUUUUAUUUUUU 46 MS1/MS2/MS4 ^* -2 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCgaaaGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNUUUUUAUUUU 114 MS1/MS3-3/MS4 ^* -2 ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCgaaaGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 115 MS1/MS2/MS5-3 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNUUUUAUUUU 116 MS1/MS3-3/MS5-3 ACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 117 MS1/MS4 ^* -2/MS5-3 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCgaaaGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 118 MS1/MS2/MS3-3/
MS4 ^* -2 ACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCgaaaGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNNUUUUUAUUUU 119 MS1/MS2/MS3-3/
MS5-3 ACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNNUUUUAUUUU 120 MS1/MS2/MS4 ^* -2/
MS5-3 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCgaaaGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNNUUUUUAUUUU 121 MS1/MS3-3/MS4 ^* -2/
MS5-3 ACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCgaaaGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 122 MS1/MS2/MS3-3/
MS4 ^* -2/MS5-3 ACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA GUGCU CCUCUCgaaaGAAUAG AGCAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNNUUUUAUUUU 123

In addition, we attempted to confirm the difference in target nucleic acid or target gene cleavage or editing activity of the ultra-small gene editing system by the MS1, which is a modified portion in the Cas12f1 mutant augment RNA. To this end, we produced an sgRNA in which MS1 was removed from the canonical sgRNA (SEQ ID NO: 6), and named it "mature form sgRNA". The mature form sgRNA may be composed of a base sequence of SEQ ID NO: 124, and one or more pairs of base sequences forming a complementary pair may be additionally removed. Table 5 shows exemplary Cas12f1 mutant mature form sgRNAs and their specific base sequences.

sgRNA Sequence (5' to 3') SEQ ID
NO: Mature form sgRNA CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA UUU gaaa GAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN 124 MS3-1 GAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA UUU gaaa GAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 125 MS3-2 UGGAGAACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA UUU gaaa GAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 126 MS3-3 ACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA UUU gaaa GAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 127 MS4-1 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA U gaaa A UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 128 MS4-2 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCgaaaGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN 129 MS4-3 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAgaaaGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 130 MS5-1 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUuuagAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAUUCA UUU gaaa GAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 131 MS5-2 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUuuagAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA UUU gaaa GAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 132 MS5-3 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA UUU gaaa GAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 133 MS3-3/MS4-3 ACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAgaaaGGAAUGCAACNNNNNNNNNNNNNNNNNNNN 134 MS3-3/MS5-3 ACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA UUU gaaa GAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN 135 MS4-3/MS5-3 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAgaaaGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 136 MS3-3/MS4-3/
MS5-3 ACCGCUUCACCAAuuagUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAgaaaGGAAUGCAACNNNNNNNNNNNNNNNNNNNNNN 137

The above-mentioned exemplified Cas12f1 mutant augment RNA was prepared by the following method. First, the engineered guide RNA was chemically synthesized from a pre-designed guide RNA to prepare the same, and then a PCR amplicon containing the synthesized guide RNA sequence and a T7 promoter sequence was prepared. U-rich tail ligation to the 3'-terminus of the engineered Cas12f1 mutant guide RNA was performed using Pfu PCR Master Mix5 (Biofact) in the presence of a sequence-modified primer and a Cas12f1 mutant guide RNA plasmid vector. The PCR amplicon was purified using the HiGene ^TM Gel & PCR Purification System (Biofact).

Modification of the second, fourth and fifth regions of the engineered scaffold regions of the engineered Cas12f1 mutant guide RNA was performed by cloning synthetic oligonucleotides (Macrogen) that delivered the modified sequences into the linearized guide RNA encoding vector using Apo I and Bam HI restriction enzymes.

In addition, the modification of the first region among the engineered scaffold regions of the engineered Cas12f1 mutant guide RNA was performed by PCR amplification of the canonical or engineered template plasmid vector using a forward primer targeting the 5'-terminal portion of tracrRNA and a reverse primer targeting the U6 promoter region. The PCR amplification was performed by Q5 Hot Start high-fidelity DNA polymerase (NEB), and the PCR products were ligated using KLD Enzyme Mix (NEB). The ligated PCR products were transformed into DH5α E. coli cells. Mutagenesis was confirmed by Sanger sequencing analysis.

The engineered plasmid vectors were purified using the NucleoBond® Xtra Midi EF kit (MN). One microgram of the purified plasmid was used as a template for mRNA synthesis using T7 RNA polymerase (NEB) and NTPs (Jena Bioscience). The engineered guide RNAs for the above-mentioned Cas12f1 variants were purified using the Monarch® RNA cleanup kit (NEB), aliquoted into cryogenic vials, and stored in liquid nitrogen.

Next, amplicons of canonical guide RNA and engineered guide RNA were prepared. To this end, the canonical guide RNA template DNA plasmid and the augment RNA template DNA plasmid were subjected to PCR amplification using a U6-complementary forward primer and a protospacer sequence-complementary reverse primer using KAPA HiFi HotStart DNA polymerase (Roche) or Pfu DNA polymerase (Biofact).

The above PCR amplification results were purified using the Higene ^TM Gel & PCR purification system (Biofact) to obtain canonical guide RNA and augment RNA amplicons.

Using the above PCR amplicon as a template, in vitro transcription was performed using NEB T7 polymerase. The in vitro transcription result was treated with DNase I (NEB), and then purified using Monarch RNA Cleanup Kit (NEB), to obtain guide RNA. Thereafter, a plasmid vector containing a pre-designed guide RNA sequence and a T7 promoter sequence was produced according to the T-blunt plasmid (Biofact) cloning method.

After the guide RNA sequence including the T7 promoter sequence in the above vector was double cut and purified, in vitro transcription was performed on the resultant using T7 polymerase (NEB). After treating the resultant of the above in vitro transcription with DNase I (NEB), the guide RNA was obtained by purifying it using the Monarch RNA Cleanup Kit (NEB).

Example 1.4. Preparation of ribonucleoprotein particles (RNPs)

The miniature genome editing system can be a ribonucleoprotein (RNP) formed by the interaction between an engineered guide RNA (augment RNA) and one Cas12f1 variant protein, or an RNP formed by the interaction between an engineered guide RNA and two Cas12f1 variant proteins.

To this end, the ultra-small gene editing protein (small endonuclease) purified in Example 1.2 and the engineered guide RNA prepared in Example 1.3 were incubated together at concentrations of 300 nM and 900 nM for 10 minutes at room temperature to prepare ribonucleoprotein particles (RNPs).

Example 2. Design and production of plasmid vector

A Cas12f1 variant, a tiny gene editing protein, was human codon-optimized for expression in human cells, and oligonucleotides of the codon-optimized Cas12f1 variant gene were constructed.

In addition, an oligonucleotide including the base sequence of the above-mentioned Cas12f1 mutant gene and a nuclear localization signal (NLS) sequence and a linker sequence at each of the 5'-terminal and 3'-terminal was synthesized (Bionics), thereby synthesizing a polynucleotide of a human codon-optimized Cas12f1 mutant nucleic acid construct for cleavage of the target nucleic acid or target gene of the present invention. The polynucleotide of the codon-optimized Cas12f1 mutant nucleic acid construct was operably linked to a plasmid including a sequence encoding eGFP linked to a chicken β-actin (CBA) promoter and a self-cleavage T2A peptide (2A), and cloned.

In addition, template DNA for the canonical guide RNA used in this experiment was synthesized (Twist Bioscience) and cloned into the pTwist Amp plasmid vector. Template DNA for the engineered guide RNA was produced using the enzyme cloning technique and cloned into the pTwist Amp plasmid.

Using the above plasmid as a template, a U6-complementary forward primer and a protospacer sequence-complementary reverse primer were used to prepare amplicons of the canonical guide RNA or engineered guide RNA. If necessary, the prepared amplicons were cloned into a T-blunt plasmid (Biofact) for replication.

Additionally, oligonucleotides encoding engineered tracrRNA and engineered crRNA were cleaved with restriction enzymes BamHI and HindIII (NEB) and cloned into the pSilencer 2.0 vector (ThermoFisher Scientific) to produce engineered dual guide RNA.

Template DNAs encoding "Cas12f1_ge3.0", "Cas12f1_ge4.0", and "Cas12f1_ge4.1", which are Cas12f1 variant augment RNAs showing high efficiency for Cas12f1 variants, were synthesized and cloned into the pTwist Amp plasmid vector (Twist Bioscience). If necessary, the vector was used as a template for amplification of the guide RNA encoding sequence using a U6-complementary forward primer and a protospacer-complementary reverse primer.

Vectors expressing components of the miniature genome editing system were prepared by cloning a polynucleotide encoding an engineered Cas12f1 variant augment RNA into a vector comprising the human codon-optimized Cas12f1 variant gene or a nucleic acid construct comprising the same using Gibson assembly.

Specifically, as a vector expressing the above-described ultra-small gene editing system, an adeno-associated virus inverted terminal repeat (AAV) plasmid vector (AAV vector) was prepared, in which 1) a sequence encoding eGFP linked to a chicken β-actin (CBA) promoter and a self-cleavage T2A peptide (2A), 2) a polynucleotide of a human codon-optimized nucleic acid construct encoding a Cas12f1 variant protein or a homolog protein thereof, and 3) a Cas12f1 variant augment RNA according to the present invention are operably linked.

Here, the transcription of the nucleic acid construct encoding the Cas12f1 variant protein or its homolog protein and the guide RNA were promoted by the chicken β and U6 promoters, respectively. In addition, the AAV plasmid vector (AAV vector) can be appropriately changed depending on the purpose of gene editing or modification, such as the number of eGFP, Cas12f1 variant augment RNA, and/or the addition of an effector protein.

To mass-produce the above AAV vector, the AAV vector and helper plasmid were transduced into HEK 293T cells. The transduced HEK293 T cells were cultured in DMEM medium containing 2% FBS. Recombinant pseudotyped AAV vector stocks were generated using PEI coprecipitation with PEIpro (Polyplus-transfection) and triple-transfection with plasmids at the same molar ratio. After 72 h of incubation, the cells were lysed, and the AAV vectors were purified from the lysate by iodixanol (Sigma-Aldrich) step gradient ultra-centrifugation.

Example 3. Cell Transfection

HEK 293T (ATCC CRL-11268), HeLa (ATCC CLL-2), U-2 OS (ATCC HTB-96), and K-562 (ATCC CCL-243) cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, and 0.1 mM nonessential amino acids at 37°C under 5% CO ₂ conditions.

For cell transfection of the target nucleic acid or the nucleic acid construct for target gene cleavage produced in Example 2, the vector containing it, or DNA encoding engineered guide RNA, 1.0 × 10 ⁵ HEK 293T cells were seeded 1 day before transfection. Cell transfection was performed by electroporation or lipofection. In the case of electroporation, 2-5 ㎍ each of the nucleic acid construct, the plasmid vector containing it, or DNA encoding engineered guide RNA was transfected into 4 × 10 ⁵ HEK-293 T cells using the Neon transfection system (Invitrogen). Electroporation was performed under the conditions of 1300 V, 10 mA, and 3 pulses. For lipofection, 6-15 μl FuGene reagent (Promega) was mixed with 2-5 μg of plasmid vector encoding Cas12f1 mutant protein and 1.5-5 μg of PCR amplicon. The mixture (300 μl) was added to 1.5 ml DMEM medium on which 1 × 10 ⁶ cells were plated 1 day before transfection. The cells were cultured in the presence of the mixture for 1 to 10 days and then collected. Genomic DNA of the cells was isolated manually using the PureHelix ^TM genomic DNA preparation kit (NanoHelix) or using the Maxwell RSC Cultured cells DNA Kit (Promega).

For cell transfection of the AAV vector containing the nucleic acid sequence encoding the gene editing protein constructed in Example 2, human HEK293T cells were infected with the AAV vector at different multiplicities of infection (MOI) of 1, 5, 10, 50 and 100 as determined by quantitative PCR. The transfected HEK293T cells were cultured in DMEM medium containing 2% FBS. Cells were harvested for isolation of genomic DNA at different time points, for example, on day 1, day 3, day 5 and day 7.

In addition, ribonucleoprotein particles (RNPs) manufactured according to Example 1.4 were transfected into cells using electroporation, or 1 day after transfection using the lipofection method, engineered guide RNAs manufactured according to Example 1.3 were transfected into cells using electroporation.

Example 4. Results Analysis

Example 4.1. Analysis of nucleic acid cleavage efficiency

To analyze the target nucleic acid or target gene cleavage efficiency of the miniature genome editing system, the region including the protospacer among the genomic DNA isolated from HEK 293T cells was subjected to PCR using target-specific primers in the presence of KAPA HiFi HotStart DNA polymerase (Roche). The amplification method followed the manufacturer's instructions. The PCR amplicon resulting from the amplification, which contained Illumina TruSeq HT dual indexes, was subjected to 150-bp pair-end sequencing using Illumina iSeq 100.

Indel frequency was calculated using MAUND, which is available at https://github.com/ibscge/maund.

PCR products were obtained using BioFACT ^TM Lamp Pfu DNA polymerase. The PCR products (100-300 μg) were reacted with 10 units of T7E1 enzyme (NEB) in a 25 μg reaction mixture at 37°C for 30 min. 20 μl of the reaction mixture was directly loaded onto a 10% acrylamide gel, and the cleaved PCR products were run in a TBE buffer system. The gel image was stained with ethidium bromide solution and digitized using a Printgraph 2 M gel imaging system (Atto). The digitized results were analyzed to evaluate the gene editing efficiency.

Example 4.2. Analysis of intracellular nucleic acid cleavage activity

Analysis of the cleavage activity of the ultra-small gene editing system for the target nucleic acid or target site of the target gene in cells was performed as follows.

The adeno-associated virus (AAV) vector produced in Example 2 was transfected into HEK 293T cells. After 3, 5, and 7 days, genomic DNA was obtained from the transfected HEK 293T cells and purified using a Genomic DNA prep kit (QIAGEN, catalog #: 69504). The target nucleic acid or the target region of the target gene was amplified from the purified product by PCR, and the final PCR product was analyzed using targeted deep sequencing. The target region was amplified using the KAPA HiFi HotStart PCR kit (KAPA Biosystem, #: KK2501) for library generation. The library was sequenced using MiniSeq of the TruSeq HT Dual Index system (Illumina).

Example 4.3. Statistical Analysis

Statistical significance was verified by two-tailed Student's t-test using Sigma Plot software (ver. 14.0). A p-value less than 0.05 was considered statistically significant, and the p-value is shown in each figure. The error bars of all data were plotted using Sigmaplot and represent the standard deviation of each data. The sample size was not determined in advance based on statistical methods. Each experiment was repeated three times, and the average of each value was used for analysis.

Example 5. Confirmation of nucleic acid cleavage by an ultra-small gene editing system

Example 5.1. Confirmation of Indel Activity of Cas12f1 Variants for Target Sequence

Whether a hypercompact TaRGET system has an activity of cleaving a target nucleic acid or a target sequence of a gene in a cell and the target sequence cleavage activity of the hypercompact TaRGET system according to the type of engineered guide RNA were investigated. Indels (deletion and insertion; indel) may occur due to nucleic acid cleavage in a target nucleic acid or a target gene. The indels are generated by non-homologous end joining (NHEJ), in which two compatible ends formed by cleavage of a double-stranded DNA frequently contact each other to repair or repair a double-stranded break in DNA, resulting in partial insertion and/or deletion (insertion) of a nucleic acid sequence at a NHEJ repair site. As a result, nucleic acid editing in which one or more bases are deleted and/or added in a target gene or a target nucleic acid may occur due to the target nucleic acid cleavage of the gene editing system.

In order to verify whether the ultra-small gene editing system fabricated in this example exhibits effective nucleic acid cleavage activity, three human endogenous DNA target sites containing a "PAM sequence" recognized as a cleavage site by the Cas12f1 mutant protein or its homolog protein were identified. The target sequences (Target-1 to Target-3) used in the experiment are shown in Table 6 below.

Target name Target sequence (5' to 3') SEQ ID NO: Target-1 [TTTG]CACACACACAGTGGGCTACC 138 Target-2 [TTTG]CATCCCCAGGACACACACAC 139 Target-3 [TTTA]AGAACACATACCCCTGGGCC 140

First, we investigated the indel efficiency of the target sequence by representative engineered guide RNAs (augment RNAs) having one or more modifications among the modification sites MS1 to MS5 in the canonical sgRNA, namely MS1/MS2/MS3 augment RNA (Cas12f1_ge3.0), MS2/MS3/MS4 augment RNA (Cas12f1_ge4.0), and MS2/MS3/MS4/MS5 augment RNA (Cas12f1_ge4.1).

As a result, as shown in FIGS. 3a to 3c, for all of the target sequences Target-1 to Target-3, each of the engineered sgRNAs, i.e., MS1/MS2/MS3 augment RNA (Cas12f1_ge3.0), MS2/MS23/MS4 augment RNA (Cas12f1_ge4.0), and MS2/MS3/MS4/MS5 augment RNA (Cas12f1_ge4.1), enabled the Cas12f1 mutant protein or the Cas12f1 mutant v1 to v3 proteins to cleave the target nucleic acid with an efficiency of 90% or higher, showing similar indel efficiencies.

In contrast, the non-engineered canonical sgRNA did not induce any indel effect for the Cas12f1 mutant proteins and Cas12f1 mutants v1 to v3 proteins to cleave the target nucleic acids (Target-1 to Target-3) (Figs. 3a to 3c). Moreover, in the case of the Cas12f1 mutants (Cas12f1 variant-extension) in which the amino acid sequence of SEQ ID NO: 233 at the N-terminus of the Cas12f1 mutant protein (wtTnpB) or the amino acid sequence of SEQ ID NO: 234 at the C-terminus of the Cas12f1 mutant protein was linked to the NLS sequence (SEQ ID NO: 54; PKKKRKV), the Cas12f1 mutants also showed indel efficiency for cleaving Target-1 and Target-2 together with the engineered guide RNA [MS2/MS3/MS4/MS5 augment RNA (Cas12f1_ge4.1)] (Fig. 3d).

These results imply that the engineered guide RNA (augment RNA) is a highly efficient guide RNA that enables the Cas12f1 mutant protein to cleave the target nucleic acid compared to the canonical sgRNA, and that the ultra-compact TaRGET system of the present invention including the same is a novel gene editing system that exhibits excellent nucleic acid editing activity.

Example 5.2. Comparison of Indel Efficiency in Cells with Existing Gene Editing Proteins

Next, the indel efficiency of the ultra-small gene editing system including Cas12f1 mutant proteins, which are ultra-small gene editing proteins; and MS2/MS3/MS4/MS5 augment RNAs (Cas12f1_ge4.1) engineered to have the shortest length, was compared with that of representative gene editing systems known to have excellent indel activity.

To this end, the CRISPR/SpCas9 system, the CRISPR/AsCas12a system, the CRISPR/Cas12f1 system, and the representative Cas12f1 mutant system, Cas12f1 mutant v1 system, and Cas12f1 mutant v2 system of the ultra-small gene editing system (Hypercompact TaRGET system) of the present invention were transfected into HEK 293T cells, respectively, and then the indel efficiency at the endogenous locus of 5'-[TTTA]AGAACACATACCCCTGGGCC-3' (Target-3, SEQ ID NO: 140) was confirmed through deep sequencing analysis.

As a result, the CRISPR/SpCas9 system showed an indel efficiency of about 10%, while the Cas12f1 mutant system, the Cas12f1 mutant v1 system, and the Cas12f1 mutant v2 system showed indel efficiencies of 45%, 55%, and 38%, respectively (Fig. 4). This means that the ultra-compact genome editing system (Hypercompact TaRGET system) including the ultra-compact genome editing protein Cas12f1 mutant according to the present invention has the advantage of expanding the range of applications for various genome editing due to its small size as described above, and has been confirmed to have significantly increased target nucleic acid or target gene cleavage efficiency compared to the most widely studied and currently used CRISPR/Cas system in terms of cleavage of target nucleic acids.

Example 5.3. Analysis of Indel activity according to combination of augment RNA and Cas12f1 variants

Example 5.3.1. Comparison of Indel Activity According to Augment RNA

In this example, we aimed to confirm that the engineered guide RNA (augment RNA) induces superior target nucleic acid cleavage activity for Cas12f1 mutant proteins (including mutants v1 to v3) compared to the canonical sgRNA. To this end, each of the modification sites MS1 to MS5 in the canonical sgRNA was further subdivided into three sections. By combining them to have one or more modifications, various engineered augment RNAs were produced as exemplified in Example 1.3. The indel activity of the engineered augment RNAs produced above was tested.

As a result, as shown in FIGS. 5A, 5B, and 6, the hypercompact TaRGET system including the canonical sgRNA (full length) and the Cas12f1 mutant protein did not cause cleavage of the target strand, but the engineered augment RNA used in the test affected the indel efficiency of the Cas12f1 mutant protein for the target nucleic acid depending on its base sequence and target sequence.

Specifically, in the gene editing system (Hypercompact TaRGET system) containing the Cas12f1 mutant protein for the target sequence 5'-[TTTG]CACACACACAGTGGGCTACC-3' (Target-1, SEQ ID NO: 138), MS1/MS2/MS3 augment RNA, MS1/MS2/MS4 ^* -2 augment RNA, MS1/MS3-3/MS4 ^* -2 augment RNA, and MS1/MS2/MS3-3/MS4 ^* -2 augment RNA showed high indel efficiencies of about 50% to 65%, and MS1/MS3-3 augment RNA, MS1/MS2/MS5-3 augment RNA, MS1/MS2/MS3-3/MS5-3 augment RNA, MS1/MS2/MS4 ^* -2/MS5-3 augment RNA, and MS1/MS2/MS3-3/MS4 ^* -2/MS5-3 augment RNA showed indel efficiencies of about 30% to 65%. It showed an indel efficiency of 40% (Fig. 5a).

In a gene editing system (Hypercompact TaRGET system) containing Cas12f1 mutant protein for the target sequence 5'-[TTTG]CATCCCCAGGACACACACAC-3' (Target-2, SEQ ID NO: 139), MS1/MS2/MS3 augment RNA, MS1/MS2/MS3-3/MS4 ^* -2 augment RNA, MS1/MS2/MS3-3/MS5-3 augment RNA, and MS1/MS2/MS3-3/MS4 ^* -2/MS5-3 augment RNA showed an indel efficiency of about 35% to 45%, and MS1/MS2/MS4 ^* -2 augment RNA, MS1/MS3-3/MS4 ^* -2 augment RNA, MS1/MS2/MS5-3 augment RNA, MS1/MS3-3/MS5-3 augment RNA, MS1/MS4 ^* -2/MS5-3 augment RNA, MS1/MS2/MS4 ^* -2/MS5-3 augment RNA and MS1/MS3-3/MS4 ^* -2/MS5-3 augment RNA showed indel efficiencies of approximately 15% to 20% (Fig. 5b).

Example 5.3.2. Comparison of Indel Activity of Cas12f1 Variants According to High-Efficiency Augment RNA

The indel efficiency for the target nucleic acid of the ultra-small gene editing system comprising the MS1/MS2/MS3 augment RNA, MS1/MS2/MS4 ^* -2 augment RNA, MS1/MS3-3/MS4 ^* -2 augment RNA or MS1/MS2/MS3-3/MS4 ^* -2 augment RNA showing high-efficiency indel effect and the Cas12f1 mutant protein was confirmed.

As a result, Cas12f1 mutant v2 and Cas12f1 mutant v3 showed excellent indel efficiencies of about 45% to about 65%, similar to the case of Cas12f1 mutant proteins (Fig. 6). However, Cas12f1 mutant v1 showed a somewhat lower indel efficiency of about 15%. However, compared to the almost no indel activity by canonical sgRNA, all of the engineered MS1/MS2/MS3 augment RNA, MS1/MS2/MS4 ^* -2 augment RNA, MS1/MS3-3/MS4 ^* -2 augment RNA, and MS1/MS2/MS3-3/MS4 ^* -2 augment RNA of the present invention can be said to have significantly increased the indel efficiency of the gene scissors system including Cas12f1 mutant v1 (Fig. 6).

Example 5.3.3. Comparison of Indel Activity of Cas12f1 Variants According to Augment RNA Based on Mature Form sgRNA

Next, to obtain a highly efficient engineered single guide RNA (augment RNA) for the Cas12f1 mutant protein, the mature form sgRNA, 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCuuagGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA UUU gaaa GAA UGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN-3' (SEQ ID NO: 124) and the augment RNA having a partial modification of the nucleic acid sequence in the mature form sgRNA were prepared (Table 5), and the indel efficiency of the ultra-compact TaRGET system of the present invention was measured.

As a result, most of the engineered augment RNAs produced showed improved indel efficiency than canonical sgRNA, and in particular, MS3-3/MS4-3 augment RNA (SEQ ID NO: 134) showed indel efficiency of about 40% and about 20% in target sequences Target-1 and Target-2, respectively (Figs. 7a and 7c).

In addition, in the case of augment RNA having an MS2 modification of U ₄ AU ₄ added to the 3'-terminus of each of the augment RNAs (SEQ ID NOs: 125 to 137, Table 5) having partial modifications in the nucleic acid sequence in the Mature form sgRNA, the indel efficiency was higher than that of the Mature form sgRNA (Figs. 7b and 7d). Even when the MS2 modification of U ₄ AU ₄ was added to the 3'-terminus of MS3-3/MS4-3 augment RNA (SEQ ID NO: 134), the indel efficiencies were about 60% and about 50% in the target sequences Target-1 and Target-2, respectively, confirming that the addition of the MS2 modification to the 3'-terminus significantly increases the indel efficiency (Figs. 7b and 7d).

Among the engineered augment RNAs having additional modifications in the above Mature form sgRNA, the MS3-3 augment RNA (SEQ ID NO: 127), MS3-3/MS4-3 augment RNA (SEQ ID NO: 134), or MS3-3/MS4-3/MS5-3 augment RNA (SEQ ID NO: 137) showing high indel effects and the indel efficiency of an ultra-compact gene editing system (Hypercompact TaRGET system) containing Cas12f1 mutant protein were confirmed for the target nucleic acid.

As a result, as can be confirmed in FIG. 8, all of the Cas12f1 mutant v1 to v3 proteins exhibited excellent indel efficiency, similar to the case of the Cas12f1 mutant protein. In addition, the engineered augment RNA in which the MS2 modification U4AU4 was added to the 3'-terminus of the MS3-3 augment RNA (SEQ ID NO: 127), MS3-3/MS4-3 augment RNA (SEQ ID NO: 134), or _MS3-3 / _MS4-3 /MS5-3 augment RNA (SEQ ID NO: 137) also exhibited significantly increased indel efficiency for the Cas12f1 mutant v1, Cas12f1 mutant v2, and Cas12f1 mutant v3 proteins, similar to the Cas12f1 mutant proteins (FIG. 8).

In summary of the above results, it is concluded that the ultra-small gene editing system comprising the Cas12f1 variant protein of the present invention (including Cas12f variant v1 to v3 proteins) and homolog proteins of the Cas12f1 variant protein exhibiting the same biological activity as these; and augment RNA has an increased cleavage activity of a target nucleic acid or a target gene by a modification in which at least one base sequence is deleted or substituted or by a modification in which a U-rich tail is added to the 3'-end of the canonical guide RNA, compared to the case in which the nucleic acid cleavage activity is almost absent when the canonical guide RNA is included.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

[Sequence List Free Text]

Sequence information for skipped sequences (sequences having fewer than 10 defined nucleic acids; SEQ ID NOs: 15, 18, 21, 22, 23, 60, 67, 69, 70, 71, 72, 75, 77, 78, 79, 80, 83, 85, 86, 87, 88, and 91) in the electronic sequence listing file attached to this specification is provided below.

Sequence number: 15

Sequence length: 13

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: The fourth region of tracrRNA for Cas12f1 variant gRNA

order:

caaauucann nnn

Sequence number: 18

Sequence length: 13

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: The fourth region of tracrRNA for Cas12f1 variant gRNA

order:

caaauucann ncn

Sequence number: 21

Sequence length: 12

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: The fifth region of crRNA for Cas12f1 variant gRNA

order:

nnnnnugaag ga

Sequence number: 22

Sequence length: 12

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: The fifth region of crRNA for Cas12f1 variant gRNA

order:

nbnnnugaag ga

Sequence number: 23

Sequence length: 7

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: The sixth region of crRNA for Cas12f1 variant gRNA

order:

augcaac

Sequence number: 60

Sequence length: 12

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: The fifth region of crRNA for Cas12f1 variant gRNA

order:

nbnnnugaag ga

Sequence number: 67

Sequence length: 7

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuruuu

Sequence number: 69

Sequence length: 6

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuuru

Sequence number: 70

Sequence length: 7

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuuruu

Sequence number: 71

Sequence length: 8

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuuruuu

Sequence number: 72

Sequence length: 9

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

Sequence:

uuuuruuuu

Sequence number: 75

Sequence length: 7

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuauuu

Sequence number: 77

Sequence length: 6

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuuau

Sequence number: 78

Sequence length: 7

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuuuu

Sequence number: 79

Sequence length: 8

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuuuuu

Sequence number: 80

Sequence length: 9

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuuuuuu

Sequence number: 83

Sequence length: 7

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuguuu

Sequence number: 85

Sequence length: 6

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuugu

Sequence number: 86

Sequence length: 7

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuuguu

Sequence number: 87

Sequence length: 8

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuuguuu

Sequence number: 88

Sequence length: 9

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuuguuuu

Sequence number: 91

Sequence length: 6

Sequence type: RNA

Sequence Origin: Artificial Sequence

Sequence name: U-rich tail sequence

order:

uuuuuu

Attach electronic file of sequence list

Claims (42)

A system for editing a nucleic acid comprising a target sequence in a cell, comprising an endonuclease comprising a Cas12f1 variant protein represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a nucleic acid encoding the endonuclease; and one or more guide RNAs or a nucleic acid encoding the guide RNAs. In the first paragraph,
The above Cas12f1 mutant protein consists of the amino acid sequence of sequence number 1.
System. In the first paragraph,
The above Cas12f1 mutant protein is part of a fusion protein.
System. In the first paragraph,
The above cell is a eukaryotic cell
System. In the first paragraph,
The above editing includes double-strand cleavage of the target nucleic acid.
System. In the first paragraph,
The system comprises two or more guide RNAs, wherein the two or more guide RNAs target the same or different target sequences.
System. In the first paragraph,
The above guide RNA is sgRNA.
System. In Article 7,
The above guide RNA contains a U-rich tail sequence of 5'-(UaN)dUe-3', 5'-UaVUaVUe-3' or 5'-UaVUaVUaVUe-3' at the 3' end,
N is A, C, G or U respectively,
V is independently A, C or G,
a is an integer between 0 and 5,
d is an integer between 0 and 4,
e is an integer between 0 and 10
System. In Article 8,
The above U-rich tail sequence comprises any one nucleotide sequence selected from the group consisting of SEQ ID NO: 67 to SEQ ID NO: 91.
System. In the first paragraph,
The above guide RNA does not contain more than five consecutive uridine sequences.
System. In the first paragraph,
The above guide RNA comprises a tracrRNA comprising a first region, a second region, a third region and a fourth region in the 5'-3' direction, a crRNA comprising a fifth region and a sixth region, a spacer sequence and a seventh region,
The first region comprises a nucleotide sequence of SEQ ID NO: 7 or a sequence in which one or more nucleotides are sequentially deleted from the 5' end of SEQ ID NO: 7, or the first region is absent,
The second region comprises a nucleotide sequence of SEQ ID NO: 9 or a part thereof, wherein the 5'-UUAG-3' sequence in the nucleotide sequence of SEQ ID NO: 9 is substituted or not substituted with a 5'-GAAA-3' sequence, and the part of the sequence is a sequence in which one or more pairs of nucleotide sequences forming a stem in a stem-loop structure formed by the nucleotide sequence of SEQ ID NO: 9 are deleted.
The third region comprises a nucleotide sequence of sequence number 13 or a sequence having at least 70% sequence identity to the sequence,
The fourth region comprises a nucleotide sequence of SEQ ID NO: 14 or a part of the sequence, wherein the part of the sequence comprises a sequence of 5'-CAAAUUCANNNNN-3' (SEQ ID NO: 15) in the sequence of SEQ ID NO: 14, but a part of the sequence at the 3' end is deleted,
The above fifth region comprises a nucleotide sequence of SEQ ID NO: 20 or a part of the sequence, wherein the part of the sequence comprises the sequence 5'-NNNNNUGAAGGA-3' (SEQ ID NO: 21) among the base sequences of SEQ ID NO: 20, but a part of the sequence at the 5' end is deleted,
The above sixth region comprises a 5'-AUGCAAC-3' (SEQ ID NO: 23) sequence or a sequence having 70% or more sequence identity to the 5'-AUGCAAC-3' sequence,
The above spacer sequence comprises a sequence of 10 to 50 nucleotides that hybridizes to the target sequence,
The seventh region is absent or comprises a U-rich tail sequence of 5'-(UaN)dUe-3', 5'-UaVUaVUe-3' or 5'-UaVUaVUaVUe-3', wherein in the sequence, N is each A, C, G or U, V is each independently A, C or G, a is an integer from 0 to 5, d is an integer from 0 to 4 and e is an integer from 0 to 10.
System. In Article 11,
The first region is 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 8), 5'-UGGAGAA-3' or
5'-A-3'
System. In Article 11,
The second region comprises a nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, wherein the 5'-UUAG-3' sequence in the nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 is or is not replaced with a 5'-GAAA-3' sequence.
System. In Article 11,
The above fourth region and the above fifth region are connected by a linker.
System. In the first paragraph,
The above guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NOs: 44 to 48, SEQ ID NOs: 105 to 123, SEQ ID NO: 124, and SEQ ID NOs: 125 to 137.
System. In the first paragraph,
The above guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 46 to 48.
System. In the first paragraph,
The above Cas12f1 mutant protein comprises an amino acid sequence of SEQ ID NO: 1, and the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 46 to 48.
System. In the first paragraph,
The above Cas12f1 mutant protein and the above guide RNA form a ribonucleoprotein particle.
System. In the first paragraph,
The above system is a composition
System. A vector system comprising a first nucleic acid construct operably linked to a nucleotide sequence encoding an endonuclease comprising a Cas12f1 variant protein represented by SEQ ID NO: 1 or SEQ ID NO: 2; and a second nucleic acid construct operably linked to a nucleotide sequence encoding a guide RNA, wherein the first nucleic acid construct and the second nucleic acid construct are contained in the same vector or in different vectors. In Article 20,
The above vector further comprises a promoter for a nucleotide sequence encoding the guide RNA.
Vector system. In Article 21,
The above promoter is U6 promoter, H1 promoter or 7SK promoter.
Vector system. In Article 20,
The first nucleic acid construct comprises a codon-optimized nucleic acid encoding the Cas12f1 variant protein.
Vector system. In Article 20,
The first or second nucleic acid construct comprises at least one nuclear localization signal (NLS) or nuclear export signal (NES) sequence at the N-terminus or C-terminus.
Vector system. In Article 20,
The above vector may be mRNA, plasmid, linear PCR amplicon or viral vector.
Vector system. In Article 25,
The above viral vector is a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a poxvirus vector or a herpes simplex virus vector.
Vector system. In Article 20,
The above guide RNA contains a U-rich tail sequence of 5'-(UaN)dUe-3', 5'-UaVUaVUe-3' or 5'-UaVUaVUaVUe-3' at the 3' end,
N is A, C, G or U respectively,
V is independently A, C or G,
a is an integer between 0 and 5,
d is an integer between 0 and 4,
e is an integer between 0 and 10
Vector system. In Article 27,
The above U-rich tail sequence comprises any one nucleotide sequence selected from the group consisting of SEQ ID NO: 67 to SEQ ID NO: 91.
Vector system. In Article 20,
The above guide RNA does not contain five or more consecutive uridine sequences.
Vector system. In Article 20,
The above guide RNA comprises a tracrRNA comprising a first region, a second region, a third region and a fourth region in the 5'-3' direction, a crRNA comprising a fifth region and a sixth region, a spacer sequence and a seventh region,
The first region comprises a nucleotide sequence of SEQ ID NO: 7 or a sequence in which one or more nucleotides are sequentially deleted from the 5' end of SEQ ID NO: 7, or the first region is absent,
The second region comprises a nucleotide sequence of SEQ ID NO: 9 or a part thereof, wherein the 5'-UUAG-3' sequence in the nucleotide sequence of SEQ ID NO: 9 is substituted or not substituted with a 5'-GAAA-3' sequence, and the part of the sequence is a sequence in which one or more pairs of nucleotide sequences forming a stem in a stem-loop structure formed by the nucleotide sequence of SEQ ID NO: 9 are deleted.
The third region comprises a nucleotide sequence of sequence number 13 or a sequence having at least 70% sequence identity to the sequence,
The fourth region above comprises a nucleotide sequence of SEQ ID NO: 14 or a part of the sequence, wherein the part of the sequence comprises a sequence of 5'-CAAAUUCANNNNN-3' (SEQ ID NO: 15) in the sequence of SEQ ID NO: 14, but a part of the sequence at the 3' end is deleted,
The above fifth region comprises a nucleotide sequence of SEQ ID NO: 20 or a part of the sequence, wherein the part of the sequence comprises the sequence 5'-NNNNNUGAAGGA-3' (SEQ ID NO: 21) among the base sequences of SEQ ID NO: 20, but a part of the sequence at the 5' end is deleted,
The above sixth region comprises a 5'-AUGCAAC-3' (SEQ ID NO: 23) sequence or a sequence having 70% or more sequence identity to the 5'-AUGCAAC-3' sequence,
The above spacer sequence comprises a sequence of 10 to 50 nucleotides that hybridizes with a target sequence in a cell,
The seventh region is absent or comprises a U-rich tail sequence of 5'-(UaN)dUe-3', 5'-UaVUaVUe-3' or 5'-UaVUaVUaVUe-3', wherein in the sequence, N is each A, C, G or U, V is each independently A, C or G, a is an integer from 0 to 5, d is an integer from 0 to 4 and e is an integer from 0 to 10.
Vector system. In Article 30,
The first region is 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 8), 5'-UGGAGAA-3' or
5'-A-3'
Vector system. In Article 30,
The second region comprises a nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, wherein the 5'-UUAG-3' sequence in the nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 is or is not replaced with a 5'-GAAA-3' sequence.
Vector system. In Article 30,
The above fourth region and the above fifth region are connected by a linker.
Vector system. In Article 20,
The above guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NOs: 44 to 48, SEQ ID NOs: 105 to 123, SEQ ID NO: 124, and SEQ ID NOs: 125 to 137.
Vector system. In Article 20,
The above guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 46 to 48.
Vector system. In Article 20,
The above Cas12f1 mutant protein comprises an amino acid sequence of SEQ ID NO: 1, and the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 46 to 48.
Vector system. In Article 20,
The above vector system further comprises a third nucleic acid construct operably linked to a nucleotide sequence encoding a guide RNA, wherein the guide RNAs of the third nucleic acid construct and the second nucleic acid construct target the same or different target sequences in a cell, and the first nucleic acid construct to the third nucleic acid construct are contained in the same vector or in different vectors.
Vector system. An in vitro gene editing method comprising the step of contacting a system according to any one of claims 1 to 19 or a vector system according to any one of claims 20 to 37 with a target gene or a target nucleic acid. In Article 38,
The above gene editing is the cleavage of double-stranded DNA, single-stranded DNA, or hybrid double-stranded nucleic acid of DNA and RNA containing a target sequence within a target gene or target nucleic acid.
A gene editing method performed in a test tube. In Article 38,
The above method is performed in a prokaryotic or eukaryotic cell in which a target nucleic acid or target gene exists.
A gene editing method performed in a test tube. In Article 40,
The above eukaryotic cell is a yeast, insect cell, plant cell, non-human animal cell or human cell.
A gene editing method performed in a test tube. In Article 38,
The above gene editing system or the vector system is packaged into a virus selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus and herpes simplex virus and delivered into a prokaryotic cell or a eukaryotic cell.
A gene editing method performed in a test tube.