KR102882704B1 - Method for Single-Base Genome Editing Using CRISPR/Cas9 System and Uses Thereof - Google Patents

Abstract

The present invention relates to a genome editing method based on the CRISPR/Cas9 system and its use. The CRISPR system using oligonucleotide-induced mutation and 5'-truncated sgRNA according to the present invention achieves a significant genome editing effect on target DNA. Therefore, the CRISPR system of the present invention is expected to be used in a wide range of fields, such as a composition for gene correction using gene scissors, genome-level screening, development of a composition for treating various diseases including cancer, diagnosis or imaging of diseases, and development of transgenic plants and animals.

Description

Method for Single-Base Genome Editing Using CRISPR/Cas9 System and Uses Thereof

The present invention relates to a method for single-base editing of a genome based on mismatch intolerance of the CRISPR/Cas9 system and its use.

The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated) system is an adaptive immune system of microorganisms that, after being infected with foreign DNA such as bacteriophage, cuts out part of the DNA sequence and stores it in its own genome when a surviving microorganism is infected. When it is reinfected with foreign DNA with a similar sequence, it recognizes it and induces double-strand breaks to protect itself. As the CRISPR/Cas system evolved as an immune system to respond to bacteriophage that constantly evolves through mutations, it has mismatch tolerance that causes double-strand breaks in the target DNA even if the target DNA sequence and the target recognition sequence of the sgRNA do not completely match. Due to this, in eukaryotic systems with high genome complexity, the off-target effect, in which unwanted sites are recognized as targets and double-strand breaks occur, has become a problem.

The CRISPR/Cas system is modular in function, consisting of a guide RNA that recognizes target DNA and a Cas protein with nuclease activity that induces double-strand breaks upon approaching the target via the guide RNA. For the CRISPR/Cas system to induce double-strand breaks at the target site, base pairing between the sgRNA and the target DNA must occur. Therefore, the design of the sgRNA is the most crucial element for the CRISPR/Cas system to be utilized as a genome-editing tool. The top priority for the CRISPR/Cas system as a genome-editing tool is to enhance its specificity for the target DNA, and optimizing the design of the sgRNA is considered the easiest way to achieve this.

Since many CRISPR/Cas systems are widely used for genome editing not only in microorganisms but also in eukaryotes, the accuracy of inducing double-strand breaks only at the target site while not causing double-strand breaks at other sites is important. Therefore, research has been conducted to increase the specificity of CRISPR/Cas systems for target DNA to reduce the tolerance of mismatches in CRISPR/Cas systems.

However, there is a problem that most of them are based on the introduction of random mutations by NHEJ, resulting in a somewhat low level of sophistication.

Therefore, there is a need to develop a CRISPR/Cas9 gene scissors that can easily and efficiently edit the genome of a target, including microorganisms, with a simple design and universal usability that complements the sophistication of conventional genome editing methods.

US 2020/0071730 A1 Republic of Korea 10-2020-0177636

Fu YF, Sander JD, Reyon D, Cascio VM, Joung JK. 2014. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32: 279-284. Kim H, Lee WJ, Oh Y, Kang SH, Hur JK, Lee H, Song W, Lim KS, Park YH, Song BS et al. 2020. Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide. Nucleic Acids Res 48: 8601-8616. Li P, Kleinstiver BP, Leon MY, Prew MS, Navarro-Gomez D, Greenwald SH, Pierce EA, Joung JK, Liu Q. 2018. Allele-Specific CRISPR-Cas9 Genome Editing of the Single-Base P23H Mutation for Rhodopsin-Associated Dominant Retinitis Pigmentosa. CRISPR J 1: 55-64. Park HM, Liu H, Wu J, Chong A, Mackley V, Fellmann C, Rao A, Jiang F, Chu H, Murthy N et al. 2018. Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo. Nat Commun 9:3313. Zhang X, Xu L, Fan R, Gao Q, Song Y, Lyu Cell Discov 4: 36.

Under the circumstances described above, the present inventors have made extensive research efforts to develop a method capable of precisely editing a target genome at the single base level based on the CRISPR/Cas9 system. Accordingly, the present inventors introduced site-directed mutagenesis into the CRISPR/Cas9 system, which includes a 5'-truncated sgRNA that deletes the 5'-terminal nucleotide of the target DNA, with an oligonucleotide containing a base sequence having a single base mismatch in the target DNA. As a result, when two nucleotides at the 5'-terminus of the guide 5'-truncated sgRNA having homology to the target DNA were deleted (removed), the CRISPR/Cas9 system exhibited mismatch intolerance that distinguishes single base mismatches, thereby significantly improving the efficiency and accuracy of single base editing (proofreading) of the E. coli genome, thereby completing the present invention.

Accordingly, one object of the present invention is to provide a genome editing method based on the CRISPR/Cas9 system.

In addition, another object of the present invention is to provide a composition for genome editing based on the CRISPR/Cas9 system.

In addition, another object of the present invention is to provide a method for increasing genome editing efficiency based on the CRISPR/Cas9 system.

In addition, another object of the present invention is to provide a method for producing a subject in which target DNA is edited based on the CRISPR/Cas9 system.

In addition, another object of the present invention is to provide a subject having edited target DNA, manufactured by a method for manufacturing a subject having edited target DNA based on a CRISPR/Cas9 system.

Other objects and advantages of the present invention will become more apparent from the detailed description of the invention and the claims below.

The terminology used herein is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

Additionally, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

The terms "nucleic acid sequence", "nucleotide sequence" and "polynucleotide sequence" as used herein mean oligonucleotides or polynucleotides, and fragments or portions thereof, and DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded, and represent the sense or antisense strand.

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, the present invention provides a genome editing method based on a CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) system, comprising a donor nucleic acid molecule complementarily binding to a target DNA and a guide RNA (guide RNA, gRNA), and comprising a step of deleting 1 to 3 nucleotides from the 5'-end of the guide RNA comprising a nucleotide sequence complementary to the target DNA.

As used herein, the terms "edit," "editing," or "edited" refer to a method of altering the nucleic acid sequence of a polynucleotide (e.g., a wild-type naturally occurring nucleic acid sequence or a mutated naturally occurring sequence) by selectively deleting a specific genomic target or incorporating a new specific sequence using an exogenously supplied DNA template. Such specific genomic targets may include, but are not limited to, a chromosomal region, mitochondrial DNA, a gene, a promoter, an open reading frame, or any nucleic acid sequence.

As used herein, the term “genome editing” means editing, restoring, correcting, losing and/or altering gene function by deletion, insertion, substitution, etc. of a nucleic acid molecule by Cas9 cleavage at a target site of a target DNA, unless otherwise specified.

The terms "delete", "deleted", "deleting" or "deletion" as used herein may be defined as a change in a nucleotide or amino acid sequence, respectively, whereby one or more nucleotides or amino acid residues are absent (removed) or rendered absent.

Preferably, in the present invention, a guide RNA having 1 to 3 nucleotides deleted (5'-truncated) from the 5'-end of a guide RNA comprising a nucleotide sequence complementary to a target DNA comprises a region consisting of 17 to 19 consecutive nucleotides complementary to the target DNA, and such a truncated guide RNA is characterized in that it rather enhances the editing effect of the CRISPR system.

As long as the purpose of the present invention can be achieved, the number of deleted nucleotides is not limited, but preferably, the number of deleted nucleotides is 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, even more preferably 1 to 2, and most preferably 2.

Additionally, the deleted nucleotides on the guide RNA may be positioned consecutively or discontinuously.

According to the present invention, the position of the nucleotide to be deleted on the guide RNA of the present invention, i.e., the 5'-truncated guide RNA, may be located at a site immediately adjacent to the 5'-end of the guide RNA, which is spaced apart by 1 to 3 nucleotides.

The term "immediately adjacent" as used herein to refer to a deleted nucleotide position on a 5'-truncated guide RNA means that it is located at a site juxtaposed in the 5'-end direction on the guide RNA containing a nucleotide sequence complementary to the target DNA, i.e., spaced apart by one nucleotide.

The term "guide RNA" refers to an RNA that is specific to target DNA, can form a complex with a Cas protein, and brings the Cas protein to the target DNA.

The above guide RNA may be a dual RNA (dualRNA) comprising a crRNA (CRISPR RNA) and a tracrRNA (transactivating crRNA) that hybridizes with the target DNA, or a single-stranded guide RNA (sgRNA) comprising portions of the crRNA and tracrRNA and hybridizing with the target DNA, and in one embodiment of the present invention, the guide RNA is sgRNA.

Any guide RNA can be used in the present invention as long as the guide RNA comprises essential portions of crRNA and tracrRNA and a portion complementary to the target.

The above crRNA can hybridize with target DNA.

Guide RNA can be delivered to cells or organisms in the form of RNA or DNA encoding the guide RNA. Furthermore, the guide RNA may be in the form of isolated RNA, RNA contained in a viral vector, or encoded within a vector. Preferably, the vector may be, but is not limited to, a viral vector, a plasmid vector, or an Agrobacterium vector.

The term "5'-truncated guide RNA" used in the present invention when referring to genome editing based on the CRISPR/Cas9 system refers to an sgRNA comprising a crRNA in which 1 to 3 nucleotides are deleted from the 5'-end of a guide RNA comprising a nucleotide sequence complementary to a target DNA, and is used interchangeably with 5'-truncated sgRNA in the present specification.

The term "donor nucleic acid molecule" or "donor nucleic acid sequence" used in the present invention when referring to CRISPR/Cas9 system-based genome editing refers to a natural or modified polynucleotide, RNA-DNA chimera, or DNA fragment, or PCR-amplified ssDNA or dsDNA fragment or analog thereof, which contains the desired nucleotide sequence to be inserted into the target DNA.

These donor nucleic acid molecules may be in any form, such as single-stranded and double-stranded, as long as they can induce a modification on the target DNA to achieve the purpose of the present invention.

The modification on the target DNA may include a substitution of one or more nucleotides at any desired position, an insertion of one or more nucleotides, a deletion of one or more nucleotides, a knockout, a knockin, a substitution with a homologous, endogenous or heterologous nucleic acid sequence, or a combination thereof.

In the present invention, preferably, the modification on the target DNA is a point mutation introduced (induced) by substitution of one or more nucleotides in the wild-type DNA sequence, and the introduction of such point mutation is, for example, by an oligonucleotide.

The term "hybridization" as used herein refers to the formation of a double-stranded nucleic acid from complementary single-stranded nucleic acids. Hybridization may occur when the complementarity between the two nucleic acid strands is perfect (a perfect match), or may occur even when some bases mismatch.

As used herein, the term “Cas protein” refers to an essential protein element in the CRISPR/Cas system, which forms an active endonuclease or nickase when complexed with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).

Information on Cas genes and proteins is available from, but not limited to, GenBank at the National Center for Biotechnology Information (NCBI). CRISPR-associated (CRISPR) genes encoding Cas proteins are often associated with CRISPR repeat-spacer arrays. CRISPR-Cas systems are classified into two classes and six types, among which the Type ± system, which involves the Cas9 protein and crRNA and tracrRNA, is the most well-known.

The target DNA comprises a nucleotide sequence complementary to the crRNA or sgRNA and a protospacer-adjacent motif (PAM).

The above oligonucleotide comprises a protospacer-adjacent motif (PAM) of the target DNA.

The above PAM is a 5'-NGG-3' trinucleotide.

Conventional mutagenic oligonucleotides, when inserted into cells, only introduced mutations at low yields during DNA replication. Even when using the conventional CRISPR/Cas9 system, single-nucleotide point mutations were difficult to introduce due to the mismatch tolerance of CRISPR/Cas9.

Accordingly, in order to solve the above-described problem, the present inventors introduced site-directed mutagenesis into a CRISPR/Cas9 system including a 5'-truncated sgRNA that deleted the nucleotide at the 5'-end of the target DNA, using an oligonucleotide including a base sequence having a single base mismatch in the target DNA.

As a result, it was found that when two nucleotides at the 5'-end of the guide 5'-truncated sgRNA having homology to the target DNA are deleted, the CRISPR/Cas9 system exhibits mismatch intolerance that distinguishes single base mismatches, thereby achieving a significant improvement in the efficiency and accuracy of single base editing (correction) of the genome.

Therefore, the method of the present invention demonstrates that the genome of a target subject can be precisely edited at the single base level by introducing a single base mutation.

In addition, according to another aspect of the present invention, the present invention provides a composition for genome editing based on the CRISPR/Cas9 system, comprising a donor nucleic acid molecule complementarily binding to a target DNA and a guide RNA, wherein the guide RNA comprising a nucleotide sequence complementary to the target DNA has 1 to 3 nucleotides deleted from its 5'-end.

According to one embodiment of the present invention, the composition of the present invention comprises a structure capable of expressing a guide RNA that recognizes a target gene in a CRISPR-Cas9 system but has 1 to 3 nucleotides deleted from a selected target DNA sequence, wherein the guide RNA and donor DNA (e.g., oligonucleotide) are simultaneously delivered into a cell, and when the target DNA is cleavage by the Cas9 protein, the donor DNA containing a mutant sequence instead of the target DNA is included in the genome through the donor DNA, thereby increasing the efficiency of substitution mutation of the target DNA.

Since the composition of the present invention utilizes the method of the present invention described above, redundant descriptions are omitted to avoid excessive complexity of the present specification.

In addition, according to another aspect of the present invention, the present invention provides a method for increasing genome editing efficiency based on a CRISPR/Cas9 system, comprising a donor nucleic acid molecule and a guide RNA that complementarily bind to a target DNA, and comprising a step of deleting 1 to 3 nucleotides from the 5'-end of a guide RNA comprising a nucleotide sequence complementary to the target DNA.

Since the method of the present invention utilizes the above-described method, description of duplicate content is omitted to avoid excessive complexity of this specification.

In addition, according to another aspect of the present invention, the present invention provides a method for producing a subject whose target DNA is edited based on a CRISPR/Cas9 system, comprising the following steps:

(a) a step of producing a donor nucleic acid molecule that complementarily binds to a target DNA and induces a modification on the target DNA;

(b) a step of producing a guide RNA having 1 to 3 nucleotides deleted from the 5'-end of the guide RNA including a nucleotide sequence complementary to the target DNA;

(c) A step of injecting the donor nucleic acid molecule of step (a) and the guide RNA of step (b) into a subject to be edited to edit the target DNA of the subject.

In addition, the CRISPR/Cas9 system of the present invention can use any selection marker known in the art as long as it can achieve the purpose of the present invention.

The subject of the present invention is not limited to a plasmid, a virus, a prokaryotic cell, an isolated eukaryotic cell, or a eukaryotic organism other than a human, as long as the method of the present invention can be applied thereto.

The eukaryotic cell may be a cell of yeast, fungus, plant, insect, amphibian, mammal, etc., and may be, for example, but is not limited to, cells cultured in vitro, transplanted cells, primary cell cultures, in vivo cells, and cells of mammals including humans, which are commonly used in the art.

Any nucleic acid encoding the Cas9 protein or the Cas9 protein may be used as long as it can achieve the purpose of the present invention, but is preferably derived from the genus Streptococcus .

According to another aspect of the present invention, the present invention provides a subject having edited target DNA, produced by the method for producing a subject having edited target DNA described above.

The present invention relates to a method for editing and repairing the genome of a target target at the single base level, and has the effect of providing a method for producing a strain that optimizes the production ability of useful substances, etc. by correctly repairing the genome of a target target, for example, a microbial strain, in which a mutation has occurred in the target gene, or by inducing a change in a codon, etc.

Since the CRISPR system using oligonucleotide-induced mutation and 5'-truncated sgRNA according to the present invention achieved a significant genome editing effect on the target DNA, the CRISPR system of the present invention is expected to be used in a wide range of fields, such as gene correction compositions using gene scissors, genome-level screening, development of therapeutic agents for various diseases including cancer, compositions for disease diagnosis or imaging, and development of transgenic plants and animals.

Figure 1 is a graph showing the colony forming units after negative selection of CRISPR/Cas9 using mismatch or 5'-terminal deletion guide RNAs of various lengths (Figure 1A) and a conceptual diagram of mismatch intolerance for the production of experimental groups (1) to (4) above (Figure 1B).
Figure 2 shows the experimental method for confirming single-base editing efficiency for each galK and xylB gene through 5'-truncated sgRNA/Cas9 negative selection using mismatch intolerance (Figure 2A) and the results thereof as a colony forming unit graph (Figure 2B).
Figure 3 is a graph showing the base editing efficiency at various locations in the galK (Figure 3A) and xylB (Figure 3B) genes to verify the single base editing ability of CRISPR/Cas9 using 5'-truncated sgRNA.
Figures 4A and 4B are graphs showing the change in single or double base insertion/deletion efficiency and the operation of the gene scissors according to the difference in the 5'-end removal nucleotide length of sgRNA in negative selection using 5'-truncated sgRNA/Cas9.
Figure 5 is a graph showing the single-base editing efficiency and colony forming unit for each of the galK and xylB genes by negative selection with 5'-truncated sgRNA in CRISPR/Cas9-NG (Figure 5A) and a schematic diagram of the experimental group (Figure 5B).

The following examples are provided solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

Example 1. Verification of mismatch tolerance of 5’-truncated sgRNA in CRISPR/Cas9.

The present inventors used the CRISPR/Cas9 system based on the HK1059 strain (a mutant E. coli strain in which the cas9 gene is inserted into the genome, E. coli MG1655 araBAD ::P _BAD - cas9 -KmR) invented in PCT/KR2021/004155 and the lambda-red beta expression plasmid pHK463 and sgRNA plasmid to assist recombination by oligonucleotides in the following examples.

After spreading the HK1059 strain on LB solid medium, a single colony grown was inoculated into 200 ml of LB liquid medium, cultured at 37°C until OD _{600 nm} became 0.4, centrifuged at 3500 rpm for 20 minutes, and washed twice with 40 ml of 10% glycerol to produce electrocompetent cells.

Plasmids expressing guide RNAs with 1 to 3 mismatch sequences, 1 to 3 5'-terminal nucleotide deletions, and 1 mismatch and 1 to 3 5'-terminal nucleotide deletions in the sgRNA homologous to the target DNA were plated on LB solid medium added to HK1059 at a concentration of 1 ml ^-1 . If the CRISPR/Cas9 system operates normally, the number of colonies formed on the LB solid medium decreases, which indirectly confirmed whether CRISPR/Cas9 operates by sgRNA plasmids with mismatches or 5'-terminal nucleotide deletions.

As a result, as shown in Fig. 1, the CRISPR/Cas9 system worked only when there were 1 to 2 mismatch sequences with the target DNA, 1 to 2 5'-terminal nucleotide deletions, or 1 mismatch and 1 5'-terminal nucleotide deletion within the sgRNA, but it was confirmed that the number of surviving colonies increased when there were 3 or more mismatches or 5'-terminal nucleotide deletions, or when there were 1 mismatch and 2 or more 5'-terminal nucleotide deletions.

Accordingly, the inventors of the present invention exploited the intolerance of mismatches of CRISPR/Cas9 to precisely edit the genome at the single base level. A system has been established. More specifically, the present inventors first designed a guide RNA such that nucleotides are removed from the 5'-end of the target gene sequence to which the guide RNA complementarily binds. Accordingly, when a mutation is introduced into the target DNA sequence of the gene scissors by an oligonucleotide, the sequence in which a single base has been edited due to mismatch intolerance is not recognized as the target sequence by the gene scissors, allowing cells to survive, whereas the target in which no mutation is induced is double-stranded DNA cut due to the mismatch tolerance of the gene scissors, and the cell does not survive (negative selection), so that the target genome can be effectively edited at the single base level as well as selected.

Example 2. Confirmation of single-base editing capability of 5’-truncated sgRNA through negative selection.

To determine whether single-base editing of a 5'-truncated sgRNA is possible through negative selection, the present inventors designed an experiment as shown in Fig. 2A.

Briefly, it is as follows: The lambda-red beta expression plasmid pHK463 to assist recombination by oligonucleotides was inserted into the HK1059 strain of Example 1, and a single colony formed after plating was inoculated into 200 ml of LB liquid medium and cultured at 30°C until the OD _{600 nm} became 0.4. L-arabinose was added at a concentration of 1 mM and cultured for an additional 3 hours to overexpress lambda-red beta protein and Cas9 protein. After centrifugation at 3500 rpm for 20 minutes and washing with 40 ml of 10% glycerol twice, the cells were manufactured into electrocompetent cells.

Mutagenic oligonucleotides were designed to substitute a single nucleotide at base 504 or 510 of the galK gene (NCBI accession no. 945358) and bases 643 and 652 of the xylB gene (NCBI accession no. 948133). The guide RNA-expressing plasmid and oligonucleotides were electroporated into HK1059 cells, plated on MacConkey plate selective medium supplemented with 5 g/L galactose, and cultured at 37°C.

When a mutation is introduced into the galK or xylB gene by oligonucleotides, each gene is not expressed normally, so galactose or xylose metabolism is impossible, resulting in the formation of white colonies on the MacConkey selective medium. Conversely, if no mutation occurs and galactose or xylose is metabolized, the pH of the MacConkey medium is lowered to below 6.8 by the metabolites, forming red colonies. The color ratio of colonies formed on the solid medium [white colonies / (white colonies + red colonies)] is calculated by CRISPR/Cas9. The editing efficiency of the system was calculated.

As a result, as shown in Fig. 2B, the editing efficiency increased to the maximum as the number of 5'-terminal nucleotide deletions in sgRNA increased to 2, and the single base editing at position 504 of the galK gene was introduced with an editing efficiency of 80%, the single base editing at position 510 was introduced with an editing efficiency of 83%, the single base editing at position 643 of the xylB gene was introduced with an editing efficiency of 82%, and the single base editing at position 652 was introduced with an editing efficiency of 60%. However, when the number of 5'-terminal nucleotide deletions in sgRNA was 3 or more, the CRISPR/Cas9 system did not function properly, and the number of surviving colonies increased significantly to the level of 10 ⁸ CFU/㎍ DNA.

Example 3. Verification of single-base editing efficiency of 5'-truncated sgRNA through random candidate sequence analysis.

To determine whether 5'-truncated sgRNAs can effectively edit various bases in addition to editing bases 504 and 510 of galK or 643 and 652 of xylB , the inventors constructed oligonucleotides to substitute single point mutations at various positions within the target DNA sequence (N ₂₀ ) of Cas9 with three bases other than itself (A→G/T/C, T→G/A/C, G→A/T/C, or C→G/A/T).

Briefly, it is as follows: After electroporation of a plasmid expressing three different guide RNAs, each having a guide RNA matching the target sequence per target site and a mismatch sequence to the right of the point mutation position, and a point mutagenic oligonucleotide (10 or 8 targets X 3 point mutagenic oligonucleotides = 30 or 24 electroporations), the cells were plated on LB medium and cultured at 37°C. Four colonies were randomly selected from the formed colonies and Sanger sequence analysis was performed to confirm whether mutations were introduced.

As a result, as shown in Fig. 3A, in galK, point mutations were successfully induced in 63% (19/30) of 30 possible point mutation cases (10 targets X 3 point mutagenic oligonucleotides). In addition, as shown in Fig. 3B, in xylB, point mutations were successfully induced in 62% (15/24) of 24 possible point mutation cases (8 targets X 3 point mutagenic oligonucleotides).

Meanwhile, point mutations are a phenomenon in which a single base pair changes the sequence, and there are Transition and Transversion. Transition is a phenomenon in which a Py base changes to a Py base, or a Pu base changes to a Pu base, and Transversion is a phenomenon in which a Py base changes to a Pu base, or a Pu base changes to a Py base (Py = pyrimidine = C or T, Pu = purine = G or A).

When the results of 19 electroporations of galK and 15 electroporations of xylB (/192 electroporations) that successfully introduced mutations were divided according to the mutation types (64 Transitions + 128 Transversions), Transitions were 50% (5/10) and 50% (4/8), respectively, and Transversions were 70% (14/20) and 68% (11/16), respectively, showing that Transversions were more dominant.

This demonstrates that the optimal condition for increasing the efficiency of point mutagenesis is to reversely utilize the mismatch intolerance of CRISPR/Cas9 exhibited by the 5’-truncated sgRNA of the present invention.

Example 4. Confirmation of single nucleotide insertion/deletion efficiency using CRISPR/Cas9 5’-truncated sgRNA.

The present inventors confirmed that 5’-truncated sgRNA can be applied not only to improve single-base editing efficiency but also to insertion or deletion of single nucleotides, and can be utilized for correction of frameshift mutations.

Briefly, if base 504 or 510 of the galK gene is deleted or a single nucleotide is inserted at position 504 or 510, a frame shift in the galK gene occurs, creating a stop codon in the 600s, leading to premature translation termination and preventing normal synthesis of the GalK protein. Strains with a single nucleotide deletion or insertion are unable to metabolize galactose normally and form white colonies on MacConkey medium. Therefore, the efficiency of single nucleotide deletion or insertion can be assessed by the color change of the colonies formed on MacConkey medium.

The color change of colonies formed on MacConkey medium was observed by inserting an oligonucleotide that deleted nucleotides 504 or 504-505 of the galK gene, which is located 5' from the PAM sequence, or inserted Cytosine at position 504 or Cytosine and Thymine at positions 504-505, and an sgRNA expression plasmid having a deletion of 0-3 nucleotides at the 5' end of the sgRNA.

As a result, as shown in Fig. 4, in all cases where the 5'-terminal nucleotide was not deleted or only one nucleotide was deleted, an editing efficiency of 8% or less was observed. On the other hand, when two nucleotides were deleted from the 5'-terminus of sgRNA, the deletion efficiency of nucleotide 504 was 74%, the single nucleotide insertion efficiency at position 504 was 78% (Fig. 4A), the deletion efficiency of nucleotides 504-505 was 48%, and the insertion efficiency of two nucleotides at positions 504-505 was 47%, showing that in all cases, the highest editing efficiency was observed when two nucleotides at the 5'-terminus of sgRNA were deleted (Fig. 4B).

When sgRNA had three or more deleted nucleotides at the 5'-end, the CFU increased to more than 10 ⁷ /㎍ DNA regardless of the position of nucleotide deletion or insertion. This result showed the same trend as in Examples 1 and 2, showing that when using 5'-truncated sgRNA in the CRISPR/Cas9 system, deletion of two nucleotides is most effective for both single-base editing, insertion, or deletion.

Example 5. Confirmation of single-base editing capability of 5’-truncated sgRNA in CRISPR/Cas9-NG.

We constructed a mutant E. coli strain HK1159 ( E. coli MG1655 araBAD ::P _BAD - cas9-NG -KmR) with the cas9-NG gene inserted into its genome through lambda-red recombination. Similarly, the lambda-red beta expression plasmid pHK463 was inserted to facilitate oligonucleotide-mediated recombination, and a single colony formed after plating was inoculated into 200 ml of LB liquid medium and cultured at 30°C until the OD _{600 nm} reached 0.4. 1 mM L-arabinose was added and cultured for an additional 3 hours to overexpress lambda-red beta and Cas9 proteins. After centrifugation at 3500 rpm for 20 minutes and washing twice with 40 ml of 10% glycerol, the cells were prepared as electrocompetent cells.

When a mutation was introduced into the galK or xylB gene by oligonucleotide in the HK1159 strain using the same method as in Example 2, as shown in FIG. 5, in CRISPR/Cas9-NG, the editing efficiency increased to the maximum as the number of 5'-terminal nucleotide deletions in sgRNA increased to 3, and the single base editing at position 504 of the galK gene was introduced with an editing efficiency of 85%, the single base editing at position 510 was introduced with an editing efficiency of 78%, the single base editing at position 643 of the xylB gene was introduced with an editing efficiency of 74%, and the single base editing at position 652 was introduced with an editing efficiency of 66%. When the number of 5'-terminal nucleotide deletions was 3, the number of surviving colonies slightly increased to the level of 10 ⁶ CFU/㎍ DNA. The differences in the number of different 5'-terminal nucleotide deletions and the number of viable colonies suggest that Cas9 and Cas9-NG operate through different mechanisms within cells due to structural differences in their proteins. When the number of 5'-terminal nucleotide deletions in sgRNA is four or more, the CRISPR/Cas9-NG system does not function properly, resulting in a significant increase in the number of viable colonies to 10 ⁸ CFU/μg DNA, as with Cas9.

Therefore, the present invention not only improves the efficiency of introducing point mutations with a simple design method compared to the conventional CRISPR/Cas9 system, but also achieves a precise gene editing effect at the actual single base unit that works in both Cas9 and Cas9-NG, so that it can be used in various ways, such as to correctly repair the genome of microbial strains in which a mutation (e.g., point mutation) has occurred in the target gene, or to induce changes in codons, etc., thereby creating strains with optimized production ability of useful substances by having codons and metabolic pathways optimized for material production, and is very useful for industrial applications related to the production of useful products.

While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (11)

Contains a donor nucleic acid molecule and a guide RNA (gRNA) that complementarily bind to the target DNA,
The donor nucleic acid molecule is one that induces a modification on the target DNA,
A genome editing method based on the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) system, wherein the above modification comprises a substitution of one nucleotide, an insertion of one nucleotide, a deletion of one nucleotide, or a combination thereof.
A genome editing method based on the CRISPR/Cas9 system, comprising a step of deleting two nucleotides from the 5'-end of a guide RNA comprising a nucleotide sequence complementary to the target DNA.
In the first paragraph,
The above guide RNA is characterized in that it comprises a region consisting of 18 consecutive nucleotides complementary to the target DNA.
Genome editing method based on the CRISPR/Cas9 system.
In the first paragraph,
The guide RNA is characterized in that it is a dual RNA (dualRNA) including a crRNA (CRISPR RNA) and a tracrRNA (transactivating crRNA) that hybridizes with the target DNA, or a single-stranded guide RNA (sgRNA) that includes portions of the crRNA and tracrRNA and hybridizes with the target DNA.
Genome editing method based on the CRISPR/Cas9 system.
In the third paragraph,
The target DNA is characterized in that it comprises a nucleotide sequence complementary to the crRNA or sgRNA and a protospacer-adjacent motif (PAM).
Genome editing method based on the CRISPR/Cas9 system.
In the first paragraph,
The donor nucleic acid molecule is characterized in that it is in a single-stranded or double-stranded form.
Genome editing method based on the CRISPR/Cas9 system.
delete delete A composition for genome editing based on the CRISPR/Cas9 system, comprising a donor nucleic acid molecule and a guide RNA that complementarily bind to target DNA,
The guide RNA comprising a nucleotide sequence complementary to the target DNA has two nucleotides deleted from its 5'-end,
The donor nucleic acid molecule is one that induces a modification on the target DNA,
wherein the above modification comprises a substitution of one nucleotide, an insertion of one nucleotide, a deletion of one nucleotide, or a combination thereof.
A composition for genome editing based on the CRISPR/Cas9 system.
Comprising a donor nucleic acid molecule and a guide RNA that complementarily bind to the target DNA,
The donor nucleic acid molecule is one that induces a modification on the target DNA,
A method for increasing genome editing efficiency based on the CRISPR/Cas9 system, characterized in that the above modification comprises a substitution of one nucleotide, an insertion of one nucleotide, a deletion of one nucleotide, or a combination thereof.
A method for increasing genome editing efficiency based on the CRISPR/Cas9 system, comprising a step of deleting two nucleotides from the 5'-end of a guide RNA comprising a nucleotide sequence complementary to the target DNA.
(a) a step of producing a donor nucleic acid molecule that complementarily binds to a target DNA and induces a modification on the target DNA;
(b) a step of producing a guide RNA having two nucleotides deleted from the 5'-end of the guide RNA including a nucleotide sequence complementary to the target DNA;
(c) a method for producing a subject whose target DNA has been edited based on the CRISPR/Cas9 system, comprising the step of injecting the donor nucleic acid molecule of step (a) and the guide RNA of step (b) into the subject to be edited to edit the target DNA of the subject;
The above modification includes a substitution of one nucleotide, an insertion of one nucleotide, a deletion of one nucleotide, or a combination thereof,
A method for producing a target DNA-edited object based on the CRISPR/Cas9 system, characterized in that the target object is a prokaryotic cell, an isolated eukaryotic cell, or a eukaryotic organism other than a human.
A target DNA-edited subject manufactured by the method for manufacturing a target DNA-edited subject of Article 10,
A subject whose target DNA has been edited, characterized in that the subject is a prokaryotic cell, an isolated eukaryotic cell, or a eukaryotic organism other than a human.