WO2025007951A1 - Sacas9 sgrna targeting ttr and modification mode thereof - Google Patents

Abstract

The present invention provides an saCas9 sgRNA targeting TTR and a modification mode thereof. The modified saCas9 sgRNA comprises a guide RNA having a length of 20 to 22 nucleotides and an sgRNA scaffold having a length of 77 nucleotides, and the nucleotide sequence of the saCas9 sgRNA comprises at least one chemical modification; and the saCas9 sgRNA targeting TTR comprises a guide RNA at the 5' end, and the sequence of the guide RNA is at least 20, at least 21, or all 22 consecutive nucleotides of a nucleotide sequence as shown in any one of SEQ ID NOs: 1-9. The saCas9 sgRNA provided by the present invention can significantly improve the editing activity and reduce the off-target effects.

Description

A saCas9 sgRNA targeting TTR and its modification method

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application No. 202310828316.9 filed on July 6, 2023, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention belongs to the field of gene editing, and more specifically, relates to a variety of saCas9 nuclease-specific sgRNA chemical modification methods and targeted TTR gene sequences.

Background Art

CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) nucleases have achieved effective gene editing in a variety of species, different cells, and animals. By recognizing specific PAM sequences and artificially designed sgRNA (guide RNA, gRNA), Cas9 specifically binds to different locations on the genome, causing double-strand breaks (DSB) in DNA, and activating endogenous DNA repair mechanisms to produce insertions or deletions at the gaps, thereby achieving the purpose of gene knockout or knock-in.

Long-term expression of Cas9 in cells can significantly increase its off-target efficiency, and delivery in the form of RNA to achieve transient expression can significantly reduce off-target and improve safety.

Compared to the widely used SpCas9, SaCas9 is smaller in size but has comparable cleavage activity, and the development of the SaCas9KKH variant has expanded the PAM recognition sequence of SaCas9 from "NNGRRT" to "NNNRRT", increasing the application flexibility of SaCas9. However, the cleavage activity of SaCas9 and its sgRNA in cells based on transient delivery in the form of modified RNA has not been studied.

Cationic lipid nanoparticles are used to deliver Cas9 mRNA and chemically modified sgRNA into cells or animals and humans, which can achieve effective gene knockout and achieve the purpose of gene therapy. Severely modified sgRNA is also more stable than sgRNA modified only at the head and tail, and has a higher knockout efficiency. However, some positions are not suitable for excessive modification, otherwise it will affect the enzyme activity and ultimately affect the cutting efficiency. Therefore, the gene Severe modification of sgRNA based on structure is the best strategy, but the existing crystal structure of SaCas9 has some omissions, and the structural-based sgRNA modification strategy remains to be studied.

Familial amyloidosis (ATTR) is caused by mutations in the gene TTR that encodes the thyroxine transporter protein. TTR is mainly synthesized in the liver, with a small amount produced in the choroid plexus and retina, and is responsible for transporting retinol and thyroxine throughout the body. TTR usually circulates in the blood as a soluble tetrameric protein. TTR mutations lead to misfolding and deposition, resulting in amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), etc. It is estimated that approximately 50,000 patients worldwide may be affected by FAP and FAC. A series of treatments for ATTR have been studied, but no approved drugs can stop disease progression and improve quality of life.

Summary of the invention

The object of the present invention is to provide a sgRNA suitable for saCas9 nuclease that can significantly improve editing activity and reduce off-target efficiency.

In a first aspect, the present invention provides a modified saCas9 sgRNA comprising a guide RNA of 20 to 22 nucleotides in length and a sgRNA backbone of 77 nucleotides in length, and the nucleotide sequence of the sgRNA comprises at least one chemical modification.

As used herein, the term "saCas9" refers to Staphylococcus aureus Cas9.

As used herein, the term "sgRNA" is a single-stranded guide RNA molecule that includes a guide RNA sequence complementary to a target DNA and a sgRNA backbone.

As used herein, the term "saCas9 sgRNA" (hereinafter also referred to as "sgRNA") refers to an sgRNA suitable for use in combination with saCas9 nuclease.

As used herein, the term “guide RNA” refers to an RNA fragment that is complementary to the target DNA and is located at the 5’ end of the saCas9 sgRNA.

As used herein, the term "sgRNA backbone" is a chimera of a repeat sequence of CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) in the CRISPR targeting recognition system, wherein crRNA contains a guide RNA in addition to the repeat sequence and can target different target DNAs, while tracrRNA is a fixed sequence in sgRNA, contains a stem-loop structure, and can bind to the Cas9 protein.

More specifically, the sgRNA backbone in this article comprises a repeat sequence/inverted repeat sequence region, a stem loop 1 region, a linker region, a stem loop 2 region and a 3’ terminal region in the 5’ to 3’ direction, wherein the repeat sequence/inverted repeat sequence region includes a 14nt repeat sequence (repeat), a 4nt tetraloop (tetraloop) and a 16nt inverted repeat sequence (anti-repeat), which are respectively located at the 1-14nt position, 15-18nt position and 19-34nt position of the 5’ end of the sgRNA backbone, and the repeat sequence and the inverted repeat sequence are complementary to each other to form a stem loop, which binds to Cas9; the stem loop 1 region (stem loop1) is 14nt in total and is located at the 35-48nt position of the 5’ end of the sgRNA backbone; the stem loop 2 region (stem loop2) is 24nt in total and is located at the 54-77nt position of the 5’ end of the sgRNA backbone; the 5nt linker region (linker) connecting the stem loop 1 and the stem loop 2 is located at the 49-53nt position of the 5’ end of the sgRNA backbone. Furthermore, the 3’ end of the saCas9 sgRNA of the present invention is a variable sequence. More specifically, the saCas9 sgRNA may optionally include a nucleotide sequence UUUU at the 3’ end.

According to the above description of the structural composition of the saCas9 sgRNA of the present invention, based on the different lengths of the guide RNA (which can be 20-22nt) and whether the nucleotide sequence UUUU is contained at the 3' end, the length of the saCas9 sgRNA of the present invention can be between 97-103nt (e.g., 97nt, 98nt, 99nt, 100nt, 101nt, 102nt or 103nt).

In one embodiment of the present invention, the at least one chemical modification may be selected from at least one of 2'-O-methyl modification, 2'-F modification, 2'-MOE modification and modification of thiophosphate bond.

As used herein, the term "chemical modification" refers to the substitution of a certain atom or group on a nucleotide molecule with another atom or group.

As used herein, the term "2'-O-methyl modification" refers to the replacement of the hydroxyl group on the carbon 2 of the pentose in the nucleotide molecule by -O-methyl (or "methoxy", "-OMe", etc.). Further, in this article, the terms "mA", "mC", "mU", and "mG" are used to represent 2'-O-methyl modified nucleotides.

As used herein, the term "2'-F" refers to a hydroxyl group on the carbon 2 of a pentose in a nucleotide molecule being replaced by F (or "fluorine", "fluorine atom", etc.). Further, herein, the terms "fA", "fC", "fU", and "fG" are used to represent 2'-F modified nucleotides.

As used herein, the term "2'-MOE" refers to a nucleotide molecule in which the hydroxyl group on the carbon 2 of the pentose is replaced by a MOE group (or "methoxyethyl", "-(CH ₂ ) ₂ OCH ₃ ", etc.). Further, herein, the terms "moeA", "moeC", "moeU", "moeG" are used to represent 2'-O-MOE modified nucleotides.

As used herein, the term "modification of phosphorothioate bonds (or PS modification)" refers to the replacement of "P=O" in the phosphodiester bonds connecting nucleotide molecules by "P=S". Further, in this article, the terms "A*", "C*", "U*", "G*" are used to represent nucleotides whose 3' ends are modified by phosphorothioate bonds. When the hydroxyl group on the carbon No. 2 of the pentose in the nucleotide molecule and the phosphodiester bond at the 3' end are modified at the same time, a combination of two labels is used to represent the nucleotide, such as "mA*", "mC*", "mU*", "mG*", "fA*", "fC*", "fU*", "fG*", etc.

In one embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present on at least 1, at least 2, at least 3 or at least 4 nucleotides of the 7 nucleotides at the 5' end and/or the 3' end of the saCas9 sgRNA. For example, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present on 1, 2, 3, 4, 5, 6 or all 7 nucleotides of the 7 nucleotides at the 5' end of the saCas9 sgRNA, and/or on 1, 2, 3, 4, 5, 6 or all 7 nucleotides of the 7 nucleotides at the 3' end of the saCas9 sgRNA, wherein the number and position of modifications at the two ends are not mutually affected. In a preferred embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present on at least 3 or at least 4 nucleotides of the 5' end and/or the 3' end of the saCas9 sgRNA.

In one embodiment of the present invention, the 2’-O-methyl modification, 2’-F modification or 2’-MOE modification may be present in at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides at positions -14 to -9, -3, -1, 1, 3 to 9, 11 to 27, 29 to 33, 36 to 39, 41, 42, 44 to 47, 49 to 77 of the saCas9 sgRNA.

According to the present invention, the numbering method of nucleotide positions can be referred to Figure 2. Taking the structure of Figure 2 as an example, the first nucleotide at the 5' end of the sgRNA backbone is used as the starting point and is numbered 1, 2, 3...77, 78, 79, 80, 81 toward the 3' end; and the first nucleotide at the 3' end of the guide RNA is used as the starting point and is numbered -1, -2, -3, -4, -5...-18, -19, -20, -21 toward the 5' end.

In a more preferred embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present in at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of the nucleotides at positions 11-22 and 62-69 of the saCas9 sgRNA. In a more preferred embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present in all nucleotides at positions 11-22 of the saCas9 sgRNA. In a more preferred embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present in at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of the nucleotides at positions 11-22 and 62-69 of the saCas9 sgRNA. In a more preferred embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present on all nucleotides at positions 11-22 and 62-69 of the saCas9 sgRNA.

In one embodiment of the present invention, the modification of the phosphorothioate bond may be present between at least 2, at least 3, at least 4 or at least 5 nucleotides of the 7 nucleotides at the 5' end and/or the 3' end of the saCas9sgRNA. For example, the modification of the phosphorothioate bond may be present between 2, 3, 4, 5, 6 or all 7 nucleotides of the 7 nucleotides at the 5' end of the saCas9sgRNA; and/or the modification of the phosphorothioate bond may be present between 2, 3, 4, 5, 6 or all 7 nucleotides of the 7 nucleotides at the 3' end of the saCas9 sgRNA, wherein the number and position of modifications at the two ends are not mutually affected. In a preferred embodiment of the present invention, the modification of the phosphorothioate bond may be present between at least 4 or at least 5 nucleotides of the 5' end and/or the 3' end of the saCas9 sgRNA.

In a preferred embodiment of the present invention, the chemical modification in the present invention is any one of Mod001-Mod0013.

In a second aspect, the present invention also provides a saCas9 sgRNA targeting TTR, which comprises a guide RNA at the 5’ end, the sequence of the guide RNA being at least 20, at least 21, or all 22 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-9.

As used herein, the term "TTR" refers to transthyretin.

In one embodiment of the present invention, the sequence of the guide RNA may be at least 20, at least 21, or all 22 consecutive nucleotides at the 3' end of the nucleotide sequence as shown in any one of SEQ ID NOs: 1-9. More preferably, the sequence of the guide RNA may be 21 consecutive nucleotides at the 3' end of the nucleotide sequence as shown in any one of SEQ ID NOs: 1-9. For a clearer explanation, taking SEQ ID NO: 1 as an example, the 21 consecutive nucleotides at the 3' end of the nucleotide sequence shown in SEQ ID NO: 1 are GUGUCUGAGGCUGGCCCUACG.

In one embodiment of the present invention, the saCas9 sgRNA may also include a sgRNA backbone of 77 nucleotides in length.

In one embodiment of the present invention, the nucleotide sequence of the saCas9 sgRNA may include In a preferred embodiment of the present invention, the at least one chemical modification is selected from at least one of 2'-O-methyl modification, 2'-F modification, 2'-MOE modification and modification of phosphorothioate bond.

In one embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present on at least 1, at least 2, at least 3 or at least 4 nucleotides of the 7 nucleotides at the 5' end and/or the 3' end of the saCas9 sgRNA. For example, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present on 1, 2, 3, 4, 5, 6 or all 7 nucleotides of the 7 nucleotides at the 5' end of the saCas9 sgRNA, and/or on 1, 2, 3, 4, 5, 6 or all 7 nucleotides of the 7 nucleotides at the 3' end of the saCas9 sgRNA, wherein the number and position of modifications at the two ends are not mutually affected. In a preferred embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present on at least 3 or at least 4 nucleotides of the 5' end and/or the 3' end of the saCas9 sgRNA.

In a more preferred embodiment of the present invention, the 2’-O-methyl modification, 2’-F modification or 2’-MOE modification may be present in at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides at positions -14 to -9, -3, -1, 1, 3 to 9, 11 to 27, 29 to 33, 36 to 39, 41, 42, 44 to 47, 49 to 77 of the saCas9 sgRNA.

In a more preferred embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present on at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of the nucleotides at positions 11-22 and 62-69 of the saCas9 sgRNA. In a more preferred embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present on all nucleotides at positions 11-22 of the saCas9 sgRNA. In a more preferred embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present on all nucleotides at positions 62-69 of the saCas9 sgRNA. In a more preferred embodiment of the present invention, the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification may be present on all nucleotides at positions 11-22 and 62-69 of the saCas9 sgRNA.

In one embodiment of the present invention, the modification of the phosphorothioate bond may be present between at least 2, at least 3, at least 4 or at least 5 nucleotides of the 7 nucleotides at the 5' end and/or the 3' end of the saCas9sgRNA. For example, the modification of the phosphorothioate bond may be present between 2, 3, 4, 5, 6 or all 7 nucleotides of the 7 nucleotides at the 5' end of the saCas9sgRNA; and/or the modification of the phosphorothioate bond may be present in the 7 nucleotides at the 3' end of the saCas9 sgRNA. In a preferred embodiment of the present invention, the modification of the phosphorothioate bond may be present between at least 4 or at least 5 nucleotides at the 5' end and/or 3' end of the saCas9 sgRNA.

In a more preferred embodiment of the present invention, the nucleotide sequence of the saCas9 sgRNA is shown in any one of SEQ ID NO: 12-25 and 27-29.

In a third aspect, the present invention also provides a composition comprising the above-mentioned saCas9 sgRNA, and saCas9 nuclease or mRNA encoding saCas9 nuclease.

In one embodiment of the present invention, the composition may be, for example, a pharmaceutical composition.

In a fourth aspect, the present invention also provides a kit comprising the saCas9 sgRNA or the composition described above, and optionally instructions for use.

In the fifth aspect, the present invention also provides the use of the above-mentioned saCas9 sgRNA, the above-mentioned composition, or the above-mentioned kit in modifying target DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

Figure 1 is a schematic diagram showing the design and screening of guide RNA targeting the human TTR gene, A: guide RNA targeting the human TTR gene designed by CRISPOR and the corresponding exon location, B: saCas9-sgRNA plasmid was transfected into HEK293 cells, and the editing efficiency was detected after 3 days, where CR003353 is the spCas9 TTR guide RNA used by Intellia therapeutics as a positive control.

Figure 2 shows the effective editing ability of saCas9 mRNA and the head-tail modified sgRNA of the present invention in cells. AB: The design of the head-tail modified form of sgRNA includes selecting a 21nt length guide RNA, Four U bases were added to the 3' end, as well as 2'-OMe modification and PS modification of the first and last three bases; C: Editing efficiency of TTR-sg12, TTR-sg20 and VEGFA-sg8 with first and last modifications in cells; Natural sgRNA is a natural unmodified sgRNA; the mass ratio of saCas9 mRNA to sgRNA is 5:1 or 5:5, respectively.

Figure 3 shows the editing efficiency when the length of the guide RNA in the head-tail modified sgRNA is 21 nt. A: sgRNA design containing guide RNAs of different lengths; B: The editing efficiency is highest when the length of the guide RNA is 21 nt. The mass ratio of saCas9 mRNA to sgRNA is 5:1 or 5:5, respectively.

Figure 4 shows the design of the heavily modified form of sgRNA of saCas9 of the present invention. The bases marked with a single underline are 2'-OMe modified, the bases marked with a double underline are 2'-F modified, and the bases marked with an asterisk are PS modified.

Figure 5 shows the editing efficiency of heavily modified sgRNA in cells, where sgRNAs with different modified forms were co-transfected with saCas9 mRNA at different mass ratios into the human Huh7 cell line, and genomic DNA was extracted 3 days later to detect the editing efficiency. Natural sgRNA is a natural unmodified sgRNA. Mod001 is a 102nt head-tail modified sgRNA, and G000480 is a heavily modified spCas9 TTR sgRNA used by Intellia therapeutics.

Figure 6 shows the application of sgRNA modification forms in other sgRNA sequences, where TTR-sg1sgRNA was modified in the forms of Mod001, Mod011 and Mod013, and co-transfected with saCas9 mRNA into human Huh7 cell line, and genomic DNA was extracted 3 days later to detect editing efficiency. Unmodified natural sgRNA was used as a control.

DETAILED DESCRIPTION

The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

Hereinafter, the present invention will be described in more detail by way of examples.

Example

Example 1: Materials and Methods

A. Synthetic sgRNA

The guide sequences targeting endogenous genes such as VEGFA and EMX1 are reported sequences (Ran, Cong et al. 2015; Chu et al., 2019). The guide RNA targeting the TTR gene provided in the present invention was designed by CRISPOR. The guide RNA connected to the AAV vector was synthesized by Jinweizhi, and the forward and reverse strand oligonucleotides were annealed and connected to the linearized saCas9 vector (addgene, NO.61591) after BsaI digestion. The modified sgRNA was synthesized by Suzhou Beixin Company.

B.saCas9-sgRNA plasmid transfection

When HEK293 cells reached 90% confluency, fresh medium was replaced for transfection. 1.5 μg of saCas9-sgRNA plasmid was transfected into each 6-well plate by lipofectamine 3000 (Thermo Fisher Scientific). Genomic DNA was collected after 3 days of culture to detect on-target and off-target efficiencies.

C.saCas9 mRNA in vitro transcription (IVT)

Wild-type saCas9 and spCas9 were connected to the TOPO vector, respectively. Using the TOPO vector as a template, T7-AG-F and BZ_Tail_2 as primers, PCR amplification was performed to obtain T7-cas9-polyA fragments of corresponding sizes. The PCR products after gel recovery were used as templates for IVT. The corresponding reaction system was:

Incubate at 37°C for 4-6 hours, add DNase I at a final concentration of 0.3U/μl, mix well and incubate at 37°C for 15 minutes to digest the DNA template. Then, the transcribed RNA was purified and recovered using magnetic beads (Novozyme). The nucleotide sequences of the transcribed saCas9 mRNA and spCas9 mRNA are shown in SEQ ID NO: 52 and SEQ ID NO: 53, respectively.

D. Cell transfection with saCas9 mRNA and sgRNA

When Huh7 cells reached 90% confluency, fresh medium was replaced for transfection. Each 24-well plate was transfected with 500 ng saCas9 mRNA and 500 ng or 100 ng sgRNA by RNAimax (Thermo Fisher Scientific), with a weight ratio of (5:5 or 5:1). Genomic DNA was extracted 3 days after transfection to detect editing efficiency.

E. Editing efficiency detection (Indels analysis)

Primers were designed using Primer 3.0 to specifically amplify a 500 bp fragment upstream and downstream of the sgRNA target sequence. The purified and recovered amplified fragments were subjected to Sanger sequencing, and the sequencing peak graph was analyzed for editing efficiency using TIDE online software (https://tide.nki.nl).

Example 2: Screening of guide RNA targeting TTR

The four exon sequences in the human TTR gene were input into the CRISPOR software (http://crispor.tefor.net) respectively, and then nine 20nt guide RNA sequences were designed according to the NNGRRT PAM of saCas9, wherein the 22nt guide RNA sequences shown in SEQ ID NO: 1-9 are shown in Table 1, which are based on the above 20nt guide RNA sequences, and according to the base pairing with the exon, two nucleotides are added to the 5' end of the 20nt guide RNA sequence to form a nucleotide structure. The guide RNA sequences used in the examples are all continuous nucleotides in the 22nt nucleotide sequences shown in SEQ ID NO: 1-9.

Table 1

The above 9 20nt guide RNAs were connected to the saCas9 vector and transfected into HEK293 cells. Indels were detected 3 days later. The results showed that the cleavage activity of sg1, sg12 and sg20 guide RNAs was comparable to that of the reported spCas9 guide RNAs. The results are shown in Figure 1.

Example 3: saCas9 single-stranded sgRNA head-to-tail modification pattern

In this example, a 21nt guide RNA sequence is used as an example, and 4 Us are added to the 3' end of the sgRNA to prevent the modification of the sgRNA end from affecting the binding of the saCas9 protein to the sgRNA. Then, the three phosphodiester bonds at the head and tail of the obtained 102nt sgRNA are PS modified to obtain the nucleotide structure shown in Figure 2 (A), and further, the three bases at the head and tail are modified with 2'-OMe to obtain the nucleotide structure shown in Figure 2 (B). Subsequently, it is transfected into the human huh7 cell line simultaneously with 100ng or 500ng of saCas9 mRNA, and the editing efficiency is detected after 3 days.

In this example, TTR-sg12 and TTR-sg20 were used as experimental groups, and VEGFA-sg8 was used as a positive control group. The modified nucleotide sequences are shown in Table 2 below. It was found that the three head-to-tail modified sgRNAs can produce effective editing, and the editing efficiency is 2.3-11.4 times that of natural sgRNA. The results are shown in Figure 2 (C). The above results show that the 102nt sgRNA can effectively mediate gene editing of saCas9 using head-to-tail modifications. Therefore, the 102nt head-to-tail modification pattern with corresponding position modifications shown in the sequence in Table 2 is referred to as Mod001 modification in this article, that is, the nucleotides at positions -21, -20, -19, 79, 80, and 81 have 2'-O-methyl modifications, and the nucleotides at positions -21, -20, -19, 78, 79, and 80 have PS modifications at the 3' end.

Table 2

Example 4: saCas9 modified guide RNA length

Previous studies have reported that the editing efficiency of saCas9 guide RNA is highest when the length is 21-23nt. Since the head and tail modification of sgRNA may affect the binding of guide RNA to saCas9 and thus affect the cutting efficiency, sg12 is taken as an example in this embodiment to explore the effect of guide RNAs of different lengths on the cutting efficiency in the head and tail modified sgRNAs. The modified nucleotide sequences are shown in Figure 3 (A) and Table 3 below. Among them, the head and tail modification pattern of the 20nt guide RNA modified at the corresponding position shown in SEQ ID NO: 14 in Table 3 is referred to as Mod002, and the head and tail modification pattern of the 22nt guide RNA modified at the corresponding position shown in SEQ ID NO: 15 in Table 3 is referred to as Mod003. The results show that the cutting efficiency of the 21nt guide RNA in the modified sgRNA is significantly higher than that of the 22nt and 20nt guide RNAs, as shown in Figure 3 (B).

Table 3

Example 5: Heavy modification pattern of sgRNA of saCas9

In this example, sg12-21nt is used as an example to further study the effect of heavy modification on the editing efficiency of sgRNA. The modified nucleotide sequence is shown in Figure 4 and Table 4 below. In particular, Mod13 removes 4 base Us on the basis of Mod11, and moves the 3' end modification forward to the bases at positions 75-77 in the stem loop 2 region to explore whether the 4 base Us added at the 3' end are necessary; in addition, Intellia therapeutics' Spcas9 TTR sgRNA G000480 is used as a positive control. In this article, the modification patterns with corresponding position modifications shown in each sequence in Table 4 are referred to as Mod004-Mod013, respectively.

Table 4

According to the results shown in Figure 5, compared with the natural unmodified sgRNA, the editing efficiency mediated by Mod006, Mod008, Mod009, Mod010, Mod011, Mod012 and Mod013 modified sgRNAs increased by 8-26 times. The editing efficiency mediated by Mod013-modified sgRNA increased by about 1.5 times. In other words, heavily modified sgRNA significantly improved the editing efficiency.

Example 6: Editing efficiency of other saCas9 sgRNAs after modification

TTR-sg1-21nt sgRNA was modified by Mod001, Mod011 and Mod013. The modified nucleotide sequences are shown in Table 5 below. They were co-transfected into Huh7 cells with saCas9 mRNA at different doses, and the editing efficiency was detected after 3 days. As shown in Figure 6, compared with the natural TTR-sg1-21nt sgRNA, the editing efficiency mediated by sgRNA modified by Mod001, Mod011 and Mod013 increased by 6.7-15 times. Compared with the sgRNA modified by the head and tail, the editing efficiency mediated by sgRNA modified by Mod011 and Mod013 increased by 1.3-1.5 times. In other words, the modification provided by the present invention can be applied to different saCas9 sgRNAs.

Table 5

In addition, the amplification primers used in the examples are summarized as follows:

Table 6

Primers for amplifying cas9:

T7-AG-F:CGTACAGAAGCTAATACGACTCACTATAAGGA(SEQ ID NO: 50)

BZ_Tail_2：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTCCTACTCAGGCTTTATTCAAAGACCA(SEQ ID NO: 51)

The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims (32)

A modified saCas9 sgRNA, characterized in that the saCas9 sgRNA comprises a guide RNA of 20 to 22 nucleotides in length and a sgRNA backbone of 77 nucleotides in length, and the nucleotide sequence of the saCas9 sgRNA comprises at least one chemical modification. The saCas9 sgRNA according to claim 1, wherein the at least one chemical modification is selected from at least one of 2’-O-methyl modification, 2’-F modification, 2’-MOE modification and thiophosphate bond modification. The saCas9 sgRNA according to claim 1, wherein the 2’-O-methyl modification, 2’-F modification or 2’-MOE modification is present on at least 1, at least 2, at least 3 or at least 4 nucleotides out of the 7 nucleotides at the 5’ end and/or 3’ end of the saCas9 sgRNA, respectively. The saCas9 sgRNA according to claim 1, wherein the 2’-O-methyl modification, 2’-F modification or 2’-MOE modification is present on at least 3 or at least 4 nucleotides at the 5’ end and/or 3’ end of the saCas9 sgRNA, respectively. The saCas9 sgRNA according to claim 1, wherein the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification is present on at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of the nucleotides at positions -14 to -9, -3, -1, 1, 3 to 9, 11 to 27, 29 to 33, 36 to 39, 41, 42, 44 to 47, 49 to 77 of the saCas9 sgRNA. The saCas9 sgRNA according to claim 5, wherein the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification is present on at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides among the nucleotides at positions 11-22 and 62-69 of the saCas9 sgRNA. The saCas9 sgRNA according to claim 6, wherein the 2’-O-methyl modification, 2’-F modification or 2’-MOE modification is present on all nucleotides at positions 11-22 of the saCas9 sgRNA. The saCas9 sgRNA according to claim 6, wherein the 2’-O-methyl modification, 2’-F modification or 2’-MOE modification is present on all nucleotides at positions 62-69 of the saCas9 sgRNA. The saCas9 sgRNA according to claim 6, wherein the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification is present on all nucleotides at positions 11-22 and 62-69 of the saCas9 sgRNA. The saCas9 sgRNA according to claim 1, wherein the modification of the thiophosphate bond exists between at least 2, at least 3, at least 4 or at least 5 nucleotides out of the 7 nucleotides at the 5' end and/or 3' end of the saCas9 sgRNA, respectively. The saCas9 sgRNA according to claim 10, wherein the modification of the thiophosphate bond exists between at least 4 or at least 5 nucleotides at the 5' end and/or 3' end of the saCas9 sgRNA. The saCas9 sgRNA according to claim 1, wherein the modified saCas9 sgRNA is modified by Mod001-Mod013. The saCas9 sgRNA according to claim 1, wherein the sgRNA backbone comprises a repeat sequence/inverted repeat sequence region, a stem-loop 1 region, a connecting region, and a stem-loop 2 region in sequence from 5' to 3' direction. The saCas9 sgRNA according to claim 1, wherein the saCas9 sgRNA further comprises a nucleotide sequence UUUU at the 3’ end. A saCas9 sgRNA targeting TTR, characterized in that the saCas9 sgRNA comprises a guide RNA at the 5' end, and the sequence of the guide RNA is at least 20, at least 21, or all 22 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-9. The saCas9 sgRNA according to claim 15, wherein the sequence of the guide RNA is at least 20, at least 21, or all 22 consecutive nucleotides at the 3’ end of the nucleotide sequence shown in any one of SEQ ID NOs: 1-9. The saCas9 sgRNA according to claim 15, wherein the saCas9 sgRNA further comprises a sgRNA backbone of 77 nucleotides in length. The saCas9 sgRNA according to claim 15, wherein the nucleotide sequence of the saCas9 sgRNA contains at least one chemical modification. The saCas9 sgRNA according to claim 18, wherein the at least one chemical modification is selected from at least one of 2’-O-methyl modification, 2’-F modification, 2’-MOE modification and thiophosphate bond modification. The saCas9 sgRNA according to claim 19, wherein the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification is present at the 5' end and/or the 3' end of the saCas9 sgRNA, respectively. at least 1, at least 2, at least 3 or at least 4 of the nucleotides. The saCas9 sgRNA according to claim 19, wherein the 2’-O-methyl modification, 2’-F modification or 2’-MOE modification is present on at least 3 or at least 4 nucleotides at the 5’ end and/or 3’ end of the saCas9 sgRNA, respectively. The saCas9 sgRNA according to claim 19, wherein the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification is present on at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of the nucleotides at positions -14 to -9, -3, -1, 1, 3 to 9, 11 to 27, 29 to 33, 36 to 39, 41, 42, 44 to 47, and 49 to 77 of the saCas9 sgRNA. The saCas9 sgRNA according to claim 22, wherein the 2'-O-methyl modification, 2'-F modification or 2'-MOE modification is present on at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides among the nucleotides at positions 11-22 and 62-69 of the saCas9 sgRNA. The saCas9 sgRNA according to claim 23, wherein the 2’-O-methyl modification, 2’-F modification or 2’-MOE modification is present on all nucleotides at positions 11-22 of the saCas9 sgRNA. The saCas9 sgRNA according to claim 23, wherein the 2’-O-methyl modification, 2’-F modification or 2’-MOE modification is present on all nucleotides at positions 62-69 of the saCas9 sgRNA. The saCas9 sgRNA according to claim 23, wherein the 2’-O-methyl modification, 2’-F modification or 2’-MOE modification is present on all nucleotides at positions 11-22 and 62-69 of the saCas9 sgRNA. The saCas9 sgRNA according to claim 19, wherein the modification of the thiophosphate bond exists between at least 2, at least 3, at least 4 or at least 5 nucleotides out of the 7 nucleotides at the 5' end and/or 3' end of the saCas9 sgRNA, respectively. The saCas9 sgRNA according to claim 27, wherein the modification of the thiophosphate bond exists between at least 4 or at least 5 nucleotides at the 5' end and/or 3' end of the saCas9 sgRNA. The saCas9 sgRNA according to claim 14, wherein the nucleotide sequence of the saCas9 sgRNA is shown in any one of SEQ ID NOs: 12-25 and 27-29. A composition comprising the saCas9 sgRNA according to any one of claims 1-29, and saCas9 nuclease or mRNA encoding saCas9 nuclease. A kit comprising the saCas9 sgRNA according to any one of claims 1-29 or the composition according to claim 30, and optionally instructions for use. Use of the saCas9 sgRNA according to any one of claims 1 to 29, the composition according to claim 30, or the kit according to claim 31 in modifying target DNA.