SaCas9 protein variant with high activity and high fidelity and application thereof

Abstract

The invention belongs to the field of gene editing, and particularly relates to a high-activity and high-fidelity saCas9 protein variant and application thereof. The saCas9 protein variant comprises mutations on the following amino acid residues: R654, at least one selected from the group consisting of Y239, N413 and Q414, and optionally at least one selected from the group consisting of Y211, R245, R314 and N419. Compared with the existing Cas9 protein, the saCas9 protein variant disclosed by the invention has the advantage that the cutting activity is remarkably improved while high fidelity is ensured.

Description

[0001] Cross-reference information

[0002] This application claims priority to Chinese patent application No. 2023116519332 filed on December 4, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of gene editing, and specifically relates to a saCas9 protein variant with high activity and high fidelity and an application thereof. Background Art

[0004] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated Cas proteins serve as the natural immune system of microorganisms, which can resist the invasion of bacteriophages in bacteria and fungi. CRISPR-Cas proteins can recognize and capture exogenous nucleic acids, thereby specifically cutting exogenous nucleic acids or silencing the expression of exogenous genes. Since Zhang Feng et al. first applied CRISPR-Cas9 protein to the field of gene editing in 2013, CRISPR-Cas9 has been widely used in disease treatment, agriculture, animal husbandry and other fields. However, the existing CRISPR-Cas9 and its homologs still need to be improved in terms of cutting activity and specificity. Summary of the invention

[0005] The present invention first provides a saCas9 protein variant, which has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence of SEQ ID NO: 1, and relative to the amino acid position of SEQ ID NO: 1, the saCas9 protein variant comprises mutations on the following amino acid residues: R654, and at least one selected from Y239, N413, Q414, and optionally at least one selected from Y211, R245, R314, N419; optionally, the saCas9 protein variant also comprises one or more of a nuclear localization sequence, a cell penetrating peptide sequence, and an affinity tag.

[0006] The present invention further provides a fusion protein, comprising: the saCas9 protein variant, and an effector domain fused to the saCas9 protein variant; optionally, the Cas9 protein and the effector domain are fused via a linker.

[0007] The present invention further provides an isolated nucleic acid encoding the saCas9 protein variant or the fusion protein.

[0008] The present invention further provides a recombinant vector comprising the isolated nucleic acid.

[0009] The present invention further provides a host cell comprising the isolated nucleic acid.

[0010] The present invention further provides a complex, which comprises: the saCas9 protein variant or the fusion protein; and a gRNA bound to the saCas9 protein variant or the fusion protein.

[0011] The present invention further provides a composition comprising: mRNA encoding the saCas9 protein variant or the fusion protein; and gRNA bound to the saCas9 protein variant or the fusion protein; optionally, the mRNA and the gRNA are encapsulated by nanolipid particles.

[0012] The present invention further provides a kit comprising: the isolated nucleic acid described above; and a nucleic acid encoding a gRNA, wherein the gRNA can bind to the saCas9 protein variant or fusion protein encoded by the isolated nucleic acid.

[0013] The present invention further provides an in vitro method for changing cell genes, comprising: expressing the following a) and b) in a target cell; or contacting the target cell with the following a) and b);

[0014] a) the saCas9 protein variant or the fusion protein,

[0015] b) having a gRNA complementary to the target sequence.

[0016] The present invention further provides an in vitro method for changing a double-stranded DNA molecule, comprising: contacting the target double-stranded DNA molecule with the following a) and b);

[0017] a) the saCas9 protein variant or the fusion protein,

[0018] b) having a gRNA complementary to the target sequence.

[0019] The present invention further provides the use of the saCas9 protein variant, or the fusion protein, or the isolated nucleic acid, or the recombinant vector, or the host cell, or the complex, or the composition, or the kit in the preparation of products for changing cell genes or double-stranded DNA molecules.

[0020] Compared with the existing Cas9 protein, the saCas9 protein variants of the present invention significantly improve the cleavage activity while ensuring high fidelity. In addition, some variants of the present invention further improve the specificity of the Cas9 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 Schematic diagram of the three-dimensional structure of saCas9 (Figure A) and the action site of the nucleic acid complex (Figure B) in the examples of the present invention.

[0023] Figure 2 The on-target editing efficiency and off-target editing efficiency of different Cas9 proteins targeting exogenous EMX1-1 in the examples of the present invention.

[0024] Figure 3 The on-target editing efficiency and off-target editing efficiency of different Cas9 proteins containing Q414A / R654A mutations targeting exogenous EMX1-1 in the embodiment of the present invention. In this figure, "QR" refers to Q414A / R654A; as an example, "Y211HQR" refers to Y211H / Q414A / R654A.

[0025] Figure 4 The cutting efficiency of different Cas9 proteins targeting endogenous EMX1-1 in the examples of the present invention.

[0026] Figure 5 The cutting efficiency of different Cas9 proteins targeting VEGFA in the examples of the present invention.

[0027] Figure 6 These are the target site editing ratios and off-target editing ratios of different Cas9 proteins targeting EMX1-1 and VEGFA in the examples of the present invention.

[0028] Figure 7The editing frequencies of target sites of different Cas9 proteins targeting EMX1-1 (Figure A) and VEGFA (Figure B) in the examples of the present invention.

[0029] Figure 8 The editing efficiency of different Cas9 proteins on the target genes EMX1 and TTR in the embodiments of the present invention is shown in Figure 2. The selected guide RNAs are EMX1-6, TTR-1, TTR-12, TTR-19, and TTR-20, respectively.

[0030] Fig. 9 The editing efficiency and off-target efficiency of different mutants based on the saCas9-KKH mutant in the examples of the present invention.

[0031] Fig.10 The target site editing ratio and off-target editing ratio of the target gene TTR by different Cas9 proteins in the examples of the present invention are shown.

[0032] Fig.11 The target site editing frequencies of the target gene TTR by different Cas9 proteins in the examples of the present invention; wherein Figures A to C respectively show the target site editing frequencies targeting TTR-1, TTR-12 and TTR-20.

[0033] Fig.12 It is the editing frequency of the target site when the hafCas9 mutant mRNA in the embodiment of the present invention is combined with the guide RNA in different modified forms.

[0034] Fig.13 is the humanized TTR in the embodiment of the present invention V50M Mouse construction strategy.

[0035] Fig.14 The mRNA of the hafCas9 mutant and the TTR-12 guide RNA with different modifications in the embodiment of the present invention are delivered to the humanized TTR in the form of nanoliposomes (LNPs). V50M Figure 3 The cleavage efficiency of TTR gene in liver (Figure A) and TTR protein content in serum (Figure B) of mice after injection of PBS. V50M Mice, the rest were injected with different nanoliposomes of TTR V50M Mouse. The contents of LNP01-LNP03 are hafCas9 mRNA and different modified TTR-12 guide RNAs, and the content of LNP04 is spCas9 mRNA and TTR guide RNA reported by Intellia therapeutics. DETAILED DESCRIPTION

[0036] 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. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0037] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0039] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0040] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0041] The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values ​​that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.

[0042] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0043] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0044] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0045] In the present invention, "optionally", "optional", "optionally", "optionally", "optional", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel schemes of "yes" or "no". If multiple "optional" or "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.

[0046] In the present invention, the term "saCas9" refers to Staphylococcus aureus Cas9 (Staphylococcus aureusCas9), which generally contains a RUVC domain (RuvC-like domain) and a REC domain (processing enzyme-related domain). Among them, the RUVC domain is generally located at the C-terminus of the Cas protein, and its main function is to cut the DNA chain near the target DNA site of the CRISPR-Cas system. The REC domain is generally located at the N-terminus of the Cas protein, which has the function of binding to RNA molecules. By binding to guide RNA (sgRNA), a Cas protein-guide RNA complex is formed. Then, this complex is complementary to a specific sequence in the target DNA, and the chain breakage or repair of the target DNA is caused by the cutting action of the RUVC domain, thereby achieving deletion, insertion or modification of the gene.

[0047] In the present invention, the term "spCas9" refers to the Cas9 protein derived from Streptococcus pyogenes. It is the earliest studied and applied Cas9 protein and is also the most widely used Cas9 protein.

[0048] In the present invention, the term "sgRNA" is a single-stranded guide RNA molecule, including a guide RNA sequence complementary to the target DNA and a sgRNA backbone.

[0049] In the present invention, the term "TTR" refers to transthyretin.

[0050] In the present invention, the term "ATTR" refers to a disease caused by transthyretin amyloid deposition.

[0051] The "mutation" mentioned in the present invention refers to changing the amino acid at the site to an amino acid different from the natural amino acid. For example, the 654th position is changed to any amino acid except R, and the 414th position is changed to any amino acid except Q.

[0052] In the present invention, "the amino acid position relative to..." means that the amino acid position makes an equal contribution to the function of the compared proteins (enzymes). For example, for a reference amino acid sequence (i.e., the amino acid sequence of SEQ ID NO: 1), when the amino acid sequence of the comparison object is arranged in a manner that allows for optimal comparison while taking into account the partial homology of the primary structure (amino acid sequence) (in this case, gaps can be introduced as needed to optimize the alignment), the amino acid at the position corresponding to the specific amino acid in the reference amino acid sequence can be determined as the "corresponding amino acid". Instead of comparing between primary structures, the comparison between stereostructures can be used, or not only the comparison between primary structures but also the comparison between stereostructures can be used to determine the "corresponding amino acid". By using stereostructure information, a highly reliable comparison result can be obtained. In this case, a method can be used to compare while comparing the atomic coordinates of the stereostructures of multiple enzymes. The stereostructure information of the mutant target enzyme can be obtained from a protein database commonly used in the art.

[0053] The following nomenclature is used for amino acid residue substitution in the present invention: original amino acid, position, substituted amino acid. As an example, the arginine (R) at position R654 is substituted with alanine (A) and is represented as R654A. Multiple amino acid residue substitutions are separated by a comma (""), such as Y239, N413, Q414.

[0054] In the present invention, when " / " is used to connect at least two amino acid residue substitutions, it means that the amino acid residues before and after the " / " connection occur simultaneously. As an example, when Q414A and R654A mutations occur simultaneously, it is expressed as Q414A / R654A; when Y211H, Q414A and R654A mutations occur simultaneously, it is expressed as Y211H / Q414A / R654A. Multiple different mutation schemes are separated by ",", such as Y239A / R654A, N413A / R654A, Q414A / R654A.

[0055] The "at least one" mentioned in the present invention includes 1 or more than 2 solutions. In some embodiments, those skilled in the art can select one amino acid site to mutate in "at least one selected from...", or can select multiple (such as 2, 3, or 4) amino acids to mutate in combination, both of which can enable the saCas9 protein variant to obtain better performance.

[0056] The "sequence identity" mentioned in the present invention refers to the degree of similarity and identity between two or more sequences. Sequence identity is usually evaluated by comparing the bases (DNA or RNA sequences) or amino acids (protein sequences) in the two sequences. In the DNA or RNA sequence identity comparison, commonly used methods include global comparison and local comparison. Global comparison is to compare the entire sequence, which is usually used to compare longer sequences. Local comparison is to find a part of the region in the sequence for comparison, which is commonly used to compare the conserved regions or specific functional regions in the sequence. The result of the comparison can be used to evaluate the consistency of the sequence by analyzing the variation information such as base matching, insertion, and deletion. In the protein sequence identity comparison, a commonly used method is to use a sequence alignment algorithm (such as BLAST, Clustal, etc.) to find the matching pattern and common conserved region between sequences. These algorithms can be compared according to information such as the similarity of amino acids, alternative groups, and generate scores and conservative analysis of the comparison. In the present invention, the similarity percentage is used as the result indicator of the sequence identity comparison, which represents the percentage of the same or similar bases or amino acids in the comparison of the two sequences.

[0057] saCas9 protein variants

[0058] The present invention first provides a saCas9 protein variant, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1, and relative to the amino acid position of SEQ ID NO: 1, the saCas9 protein variant comprises mutations on the following amino acid residues: R654, and at least one selected from Y239, N413, and Q414.

[0059] The present invention finds that by mutating the R654 site in the RUVC domain of the Cas9 protein and at least one of the Y239, N413, and Q414 in the REC domain, the cutting activity of the Cas9 protein can be significantly improved while maintaining high fidelity.

[0060] In some embodiments, the saCas9 protein variant comprises mutations at the following amino acid residues: R654, and at least one selected from Y239, N413, Q414, and at least one selected from Y211, R245, R314, N419.

[0061] In some embodiments, the saCas9 protein variant has at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence of SEQ ID NO:1.

[0062] In some embodiments, the saCas9 protein variant further comprises one or more of a nuclear localization sequence, a cell penetrating peptide sequence, and an affinity tag.

[0063] In some embodiments, the mutation refers to changing the amino acid at the site to alanine (A), aspartic acid (D) or histidine (H).

[0064] In some embodiments, the saCas9 protein variant comprises the following mutations: R654A, at least one selected from Y239A, N413A, and Q414A.

[0065] In some embodiments, the saCas9 protein variant comprises the following mutations: R654A, at least one selected from Y239A, N413A, Q414A, and at least one selected from Y211H, R245A, R314A, N419D.

[0066] In some embodiments, the saCas9 protein variant comprises mutations at the following amino acid residues: R654 and Q414.

[0067] In some embodiments, the saCas9 protein variant comprises mutations at the following amino acid residues: R654 and Q414, and at least one selected from Y211, R314, and N419.

[0068] In some preferred embodiments, the saCas9 protein variant comprises the following mutations: R654A and Q414A.

[0069] In some preferred embodiments, the saCas9 protein variant comprises the following mutations: R654A and Q414A, and at least one selected from Y211H, R314A, and N419D.

[0070] In some embodiments, the saCas9 protein variant comprises any of the following mutations:

[0071] Y239A / R654A, N413A / R654A, Q414A / R654A, Y211H / Q414A / R654A, R314A / Q414A / R654A, Q414A / N419D / R654A, Y211H / R314A / Q414A / R654A, or Y211H / Q414A / N419D / R654A. All of them have better cleavage activity.

[0072] In some more preferred embodiments, the saCas9 protein variant comprises any of the following mutations: Q414A / R654A, Y211H / Q414A / R654A, R314A / Q414A / R654A, Q414A / N419D / R654A, Y211H / R314A / Q414A / R654A, or Y211H / Q414A / N419D / R654A. It has better cleavage activity and higher fidelity.

[0073] In some further preferred embodiments, the saCas9 protein variant comprises any of the following mutations: Q414A / R654A, Y211H / Q414A / R654A, R314A / Q414A / R654A, or Y211H / Q414A / N419D / R654A. It has stable and excellent cleavage activity when applied to a variety of different target proteins.

[0074] In some most preferred embodiments, the saCas9 protein variant comprises the following mutations:

[0075] Q414A / R654A. It can better improve the fidelity, cleavage activity and specificity of Cas9 protein. In the present invention, the saCas9 protein variant is also called hafCas9.

[0076] In some embodiments, the saCas9 protein variant further comprises one or more mutations in the PI domain and / or the WED domain. When the mutations in the RUVC domain and the REC domain provided by the present invention are combined with the mutations with performance optimization effects occurring in the PI domain (PAM-interacting domain, a domain that interacts with PAM) and / or the WED domain (Wedges and Edgesdomain, also known as REC lobe, is a specific binding site of Cas9 with a target DNA sequence during recognition of the target sequence), the activity and specificity of the mutant can be further improved.

[0077] In some preferred embodiments, the saCas9 protein variant further comprises at least one mutation selected from E782K, K929R, N968K, N986R, D987N, D987S, L988R, L988T, L989R, R991L, R991K, R991I, I992V, V994L, and R1015H.

[0078] In some more preferred embodiments, the saCas9 protein variant further comprises any of the following mutations: E782K / N968K / R1015H (KKH variant), N986R, N986R / R991L, R991K, R991K / D987N, D987N / L988R / R991K / V994L,

[0079] D987S / L988T / L989R / R991I / I992V, E782K / K929R / R1015H (KRH variant), or E782K / K929R / N968K / R1015H (KRKH variant). The mutation combinations mentioned in the present invention can improve the cleavage activity and specificity of PAM mutants, especially variants containing the above mutations.

[0080] In some embodiments, the saCas9 protein variant further comprises the following mutations, E782K / N968K / R1015H (KKH variant).

[0081] In some embodiments, the saCas9 protein variant containing E782K / N968K / R1015H (KKH variant) comprises the sequence shown in SEQ ID NO: 2; or has at least 50%, at least 60%, at least 70%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the sequence shown in SEQ ID NO: 2. The mutation site mentioned in the present invention can be further introduced on the basis of the sequence.

[0082] In some embodiments, the saCas9 protein variant comprises the sequence shown in SEQ ID NO:3; or has a sequence identity of at least 50%, at least 60%, at least 70%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% to the sequence shown in SEQ ID NO:3.

[0083] In some embodiments, the saCas9 protein variant comprises other conservative mutations in addition to the mutations mentioned in the present invention, so that the saCas9 protein variant has up to 5%, 10%, 15% or 20% difference in amino acid residues from any of the sequences shown in SEQ ID NOs: 1 to 3. The "conservative substitution" mentioned herein is also referred to as substitution by "homologous" amino acid residues, which refers to substitutions in which amino acid residues are replaced by amino acid residues with similar side chains, for example, amino acids with basic side chains (such as lysine, arginine and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), non-charged polar side chain amino acids (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chain amino acids (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain amino acids (such as threonine, valine, isoleucine) and aromatic side chain amino acids (such as tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions generally have little effect on the activity of the resulting protein.

[0084] In a preferred embodiment, the saCas9 protein variant comprising other conservative mutations still retains the activity of the saCas9 protein variant, for example, retains its nuclease activity, and / or retains its ability to guide RNA to interact with target DNA.

[0085] In some embodiments, SV40 (Simian Virus 40) NLS and Nucleoplasmin NLS are fused to the N-terminus and C-terminus of the saCas9 protein variant, respectively; or, SV40 NLS is fused to both the N-terminus and C-terminus of the saCas9 protein variant. In some specific embodiments, the amino acid sequence of the SV40 NLS is shown in SEQ ID NO: 4. In some specific embodiments, the amino acid sequence of the Nucleoplasmin NLS is shown in SEQ ID NO: 5.

[0086] In some embodiments, the saCas9 protein variant comprises a cell-penetrating peptide that facilitates delivery to the intracellular space, such as a HIV-derived TAT peptide, a membrane-penetrating peptide, a transit peptide, or an hCT-derived cell-penetrating peptide.

[0087] In some embodiments, the saCas9 protein variant comprises a portion with high affinity for a ligand, such as GST, FLAG, or a hexahistidine sequence. Such affinity tags can assist in purifying the recombinant variant protein.

[0088] Fusion Protein

[0089] The present invention further provides a fusion protein, comprising: the saCas9 protein variant described in any of the aforementioned schemes, and an effector domain fused to the saCas9 protein variant.

[0090] In some embodiments, the Cas9 protein and the effector domain are fused via a linker. The linker that can be used for these fusion proteins (or between fusion proteins in a connected structure) can include any sequence that does not interfere with the function of the fusion protein. In a preferred embodiment, the linker is short, such as 2 to 20 amino acids, and contains amino acids with a high degree of freedom, such as glycine, alanine, and serine.

[0091] In some embodiments, the effector domain includes one or more of APOBEC1 (Apolipoprotein B mRNA Editing Catalytic Subunit 1), APOBEC3 (Apolipoprotein B mRNA Editing Catalytic Subunit 3), TadA8e, KRAB (Krüppel-Associated Box), VP64, reverse transcriptase, and transposon (also known as jumping element). The effector domains mentioned above can be derived from different species.

[0092] In some embodiments, the fusion protein is a gene editing tool derived from a saCas9 variant. As an example, the gene editing tool can be a base editor (such as ABE, CBE, AYBE, ACBE, CGBE, GBE, etc.) or a Prime editor. The saCas9 variant of the present invention can improve the editing efficiency and specificity of the above-mentioned gene editing tools.

[0093] Isolated nucleic acids

[0094] The present invention further provides an isolated nucleic acid encoding the saCas9 protein variant described in any of the preceding schemes, or the fusion protein described in any of the preceding schemes.

[0095] In some embodiments, the isolated nucleic acid comprises any one of SEQ ID NOs: 10-12, 16-18; or has a sequence identity of at least 50%, at least 60%, at least 70%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% to any one of SEQ ID NOs: 10-12, 16-18.

[0096] In some specific embodiments, the DNA sequence encoding the saCas9 protein variant is shown in any one of SEQ ID NOs: 10 to 12.

[0097] In some specific embodiments, the mRNA sequence encoding the saCas9 protein variant is shown in any one of SEQ ID NOs: 10 to 12.

[0098] Recombinant vector

[0099] The present invention further provides a recombinant vector comprising the isolated nucleic acid described in any of the aforementioned schemes.

[0100] The "recombinant vector" of the present invention refers to a nucleic acid molecule capable of transporting a nucleic acid inserted therein to a target. Those skilled in the art can select an appropriate vector according to the purpose of use (cloning, protein expression) and the type of host cell. The vector of the present invention is preferably an expression vector. "Expression vector" refers to a vector capable of introducing a nucleic acid inserted therein into a target cell (host cell) and capable of expressing it in the cell. An expression vector generally comprises a promoter sequence necessary for the expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like. In addition, an expression vector comprising a selection marker can also be used. When using the expression vector, the selection marker can be used to confirm the presence (and extent) of the introduction of the expression vector.

[0101] Host cells

[0102] The present invention further provides a host cell comprising the isolated nucleic acid described in any of the aforementioned schemes.

[0103] In order to use the saCas9 protein variants of the present invention, it may be desirable to express them from nucleic acids encoding them. This can be done in a variety of ways. For example, nucleic acids encoding Cas9 variants can be cloned into intermediate vectors for transformation into prokaryotic or eukaryotic cells for replication and / or expression. Intermediate vectors are typically prokaryotic vectors, such as plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of nucleic acids encoding Cas9 variants or for production of Cas9 variants. Nucleic acids encoding Cas9 variants can also be cloned into expression vectors for administration to plant cells, animal cells, preferably mammalian cells or human cells, fungal cells, bacterial cells, or protozoan cells.

[0104] Compound

[0105] The present invention further provides a complex, which comprises: the saCas9 protein variant described in any of the aforementioned schemes or the fusion protein described in any of the aforementioned schemes; and a gRNA bound to the saCas9 protein variant or the fusion protein.

[0106] In some embodiments, the gRNA is about 15 to 250 (preferably 15 to 200, more preferably 15 to 150) nucleotides long and comprises a sequence of at least 10 consecutive nucleotides complementary to the target sequence.

[0107] 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 bp in length. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 consecutive nucleotides complementary to the target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in a mammalian genome. In some embodiments, the target sequence is a sequence in a human genome.

[0108] In some embodiments, the gRNA is a gRNA modified with 2'-MOE (2'-O-methoxyethyl ester modification) and a phosphorothioate bond.

[0109] In some specific embodiments, the gRNA is the modified saCas9 sgRNA mentioned in CN202310828316.9.

[0110] In some preferred embodiments, the gRNA is any gRNA mentioned in Table 6.

[0111] Composition

[0112] The present invention further provides a composition comprising: mRNA encoding the saCas9 protein variant described in any of the above schemes or the fusion protein described in any of the above schemes; and gRNA bound to the saCas9 protein variant or the fusion protein. The composition can efficiently cut the target gene in mammalian cells and humanized mouse models, and reduce off-target efficiency.

[0113] In some embodiments, the mRNA and the gRNA are encapsulated by nanolipid particles.

[0114] In some embodiments, the mRNA comprises any one of SEQ ID NOs: 16 to 18; or has a sequence identity of at least 50%, at least 60%, at least 70%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% to any one of SEQ ID NOs: 16 to 18.

[0115] In some embodiments, the mRNA comprises a 5'UTR, a coding sequence (CDS) of the saCas9 protein variant or the fusion protein, a 3'UTR and a PolyA tail.

[0116] In some embodiments, the mRNA is capped with 5' Cap1.

[0117] In some embodiments, uracil in the mRNA is replaced by N1-methyl-pseudouracil.

[0118] In some embodiments, the gRNA is a 2'-MOE-modified and phosphorothioate-modified gRNA.

[0119] In some specific embodiments, the gRNA is the modified saCas9 sgRNA mentioned in CN202310828316.9.

[0120] In some preferred embodiments, the gRNA is any gRNA mentioned in Table 6.

[0121] In some embodiments, the composition can efficiently knock out the TTR gene for the treatment of ATTR.

[0122] Reagent test kit

[0123] The present invention further provides a kit comprising: the isolated nucleic acid described in any of the aforementioned schemes; and a nucleic acid encoding a gRNA, wherein the gRNA can bind to the saCas9 protein variant or fusion protein encoded by the isolated nucleic acid.

[0124] In some embodiments, the kit comprises a nucleic acid construct and an expression construct; wherein the nucleic acid construct comprises the isolated nucleic acid described in any of the foregoing schemes and a heterologous promoter for driving the nucleic acid to express the sequence; the expression construct comprises a gRNA, and the expression construct further comprises a cloning site, the positioning of which allows a nucleic acid sequence identical or complementary to the target sequence to be cloned into the gRNA backbone.

[0125] In some embodiments, the isolated nucleic acid and the nucleic acid encoding the gRNA are loaded on the same expression vector (such as an adenovirus vector).

[0126] In some embodiments, the gRNA is a 2'-MOE-modified and phosphorothioate-modified gRNA.

[0127] In some specific embodiments, the gRNA is the modified saCas9 sgRNA mentioned in CN202310828316.9.

[0128] In some preferred embodiments, the gRNA is any gRNA mentioned in Table 6.

[0129] In vitro methods

[0130] The present invention further provides an in vitro method for changing cell genes, comprising: expressing the following a) and b) in a target cell; or contacting the target cell with the following a) and b);

[0131] a) the saCas9 protein variant described in any of the foregoing schemes or the fusion protein described in any of the foregoing schemes,

[0132] b) having a gRNA complementary to the target sequence.

[0133] In some embodiments, the cell is a stem cell.

[0134] In some embodiments, the stem cells are mesenchymal stem cells or induced pluripotent stem cells.

[0135] The present invention further provides an in vitro method for changing a double-stranded DNA molecule, comprising: contacting the target double-stranded DNA molecule with the following a) and b);

[0136] a) the saCas9 protein variant described in any of the foregoing schemes or the fusion protein described in any of the foregoing schemes,

[0137] b) having a gRNA complementary to the target sequence.

[0138] In some embodiments, the gRNA is a 2'-MOE-modified and phosphorothioate-modified gRNA.

[0139] In some specific embodiments, the gRNA is the modified saCas9 sgRNA mentioned in CN202310828316.9.

[0140] In some preferred embodiments, the gRNA is any gRNA mentioned in Table 6.

[0141] application

[0142] The present invention further provides the use of the saCas9 protein variant described in any of the preceding schemes, or the fusion protein described in any of the preceding schemes, or the isolated nucleic acid described in any of the preceding schemes, or the recombinant vector described in any of the preceding schemes, or the host cell described in any of the preceding schemes, or the complex described in any of the preceding schemes, or the composition described in any of the preceding schemes, or the kit described in any of the preceding schemes in the preparation of products for changing cell genes or double-stranded DNA molecules.

[0143] In some embodiments, the product can efficiently knock out the TTR gene and is therefore used to treat ATTR.

[0144] Example

[0145] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0146] The guide RNA sequences, target sites and amplification primer information of off-target sites involved in the following examples are shown in Tables 1 to 3 below.

[0147] Table 1 Guide RNA sequences targeting endogenous genes

[0148]

[0149]

[0150] Table 2 Amplification primers for target gene loci

[0151]

[0152] Table 3 Primers for amplification of target and off-target sites of EMX1-1, VEGFA-8, TTR-1, TTR-12 and TTR-20

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] For experimental methods in the following examples where specific conditions are not specified, reference is made to the instructions given in the present invention first, and may also be made to experimental manuals or conventional conditions in the art, or to other experimental methods known in the art, or to conditions recommended by manufacturers.

[0159] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0160] Example 1 Engineered saCas9 has higher activity (EMX1 and VEGFA target sequences)

[0161] According to the three-dimensional structure of saCas9 (PDB: 5CZZ), the binding sites of the nucleic acid binding region, the guide RNA and the target DNA were analyzed, and the amino acids to be mutated were selected, such as Figure 1 As shown. Mutation primers were designed near the mutated amino acid, and saCas9 point mutants were constructed by overlap PCR. Guide RNAs targeting endogenous genes such as VEGFA and EMX1 were selected from F. An Ran, 2015, Nature. The forward and reverse strand oligonucleotides were synthesized by Jin Weizhi, annealed and connected to the linearized vector px601 (Addgene NO.61591) after BsaI digestion. The specific sequence of the guide RNA is shown in Table 1. The guide RNA of spCas9 was designed at the same position and connected to the BsmbI digestion site of the lentiCRISPR v2 plasmid (Addgene NO.52961).

[0162] The wild-type saCas9 plasmid or saCas9 mutant plasmid and the plasmid carrying the inactivated GFP expression sequence were co-transfected into HEK293 cells using Lipofectamine3000. When saCas9 cuts the inserted targeting sequence in the inactivated GFP sequence, it activates the GFP sequence reconstruction and GFP expression is restored. 72 hours after transfection, the proportion of GFP-positive cells was analyzed using flow cytometry. The results are shown in Figure 2 and Figure 3 .

[0163] Figure 2The results showed that when the insertion sequence was a target sequence, multiple saCas9 mutants including Q414A, Y239A, R314A, Y239A / R654A, R314A / R654A, and Q414A / R654A had cleavage activities close to or higher than the wild-type saCas9 protein. When the insertion sequence was an off-target sequence, multiple saCas9 mutants including Q414A, Y239A, R314A, N413A, R654A, Y239A / R654A, R314A / R654A, N413A / R654A, and Q414A / R654A had cleavage activities lower than the wild-type saCas9, among which the cleavage activity of Q414A / R654A was lower than that of the known saCas9 high-fidelity variant saCas9-HF.

[0164] Figure 3 The results showed that when one or two additional point mutations were added to the Q414A / R654A mutant, the targeted editing efficiency of multiple mutants was higher than that of wild-type saCas9 and the off-target editing was lower than that of wild-type saCas9, including Q414A / R654A, Y211H / Q414A / R654A, R314A / Q414A / R654A, and N419D / Q414A / R654A.

[0165] The wild-type saCas9 plasmid and saCas9 mutant plasmid were transfected into HEK293 cells using Lipofectamine3000. Genomic DNA was collected 3 days after transfection, and PCR primers were designed near the target site (primer sequences are shown in Table 2) to amplify the edited products. The PCR products were purified and recovered, and Sanger sequencing was used, and the cutting efficiency was analyzed using TIDE. The results are shown in Figure 4 and Figure 5 .

[0166] Figure 4The results showed that when targeting the endogenous gene EMX1, the cutting activity of multiple saCas9 mutants including Q414A / R654A, N413A / R654A, Y211H / Q414A / R654A, R314A / Q414A / R654A, and N419D / Q414A / R654A was higher than that of the wild-type saCas9 protein. The activities of multiple saCas9 mutants including Q414A / R654A, Y239A / R654A, N413A / R654A, Y211H / Q414A / R654A, R314A / Q414A / R654A, and Y211H / N419D / Q414A / R654A are higher than the reported high-fidelity mutant HF (R245A / N413A / N419A / R654A, PNAS, 2019). The activities of multiple saCas9 mutants include: Q414A / R654A, Y211H / Q414A / R654A, R314A / Q414A / R654A, and Y211H / R314A / Q414A / R654A, which are higher than the reported high-fidelity mutant Q414A (PLOS BIOLOGY, 2020). Among them, the cleavage activities of Q414A / R654A, R314A / Q414A / R654A, and Y211H / R314A / Q414A / R654A mutants are higher than spCas9. The editing efficiency is shown in Table 4.

[0167] Table 4 Editing efficiency of different Cas9 proteins targeting EMX1-1

[0168] all variants Mean SD p value (vs WT) p value (vs HF) WT 9.2 1.4 HF 2.867 1.601 0.00670825 Q414A 9.733 1.168 0.639004083 0.003878 Q414A / R654A 16.47 1.25 0.002574394 0.000316 R314A / R654A 6.1 2.427 0.127799309 0.126369 Y239A / R654A 5.967 0.3215 0.017559892 0.030271 N413A / R654A 10.3 3.637 0.650540738 0.031682 Y211H / Q414A / R654A 13.7 4.513 0.17440856 0.017274 R245A / Q414A / R654A 2.667 1.343 0.004304071 0.876376 R314A / Q414A / R654A 17.9 1.153 0.001146559 0.00019 N419D / Q414A / R654A 16.17 2.103 0.008801453 0.000955 Y211H / R314A / Q414A / R654A 7.4 1.664 0.225005558 0.027277 Y211H / N419D / Q414A / R654A 8 1.473 0.364247187 0.015015 Y211H / R245A / Q414A / R654A 3.6 0.4 0.002637836 0.484422 R314A / Q414A / N419D / R654A 3.333 1.002 0.004121587 0.690694 R314A / R245A / Q414A / R654A 7.167 3.258 0.376823113 0.109471 R245A / N419D / Q414A / R654A 3.967 0.8622 0.005282864 0.353886 spCas9 13.8 1.572 0.01934475 0.001079

[0169] Figure 5 The results showed that when targeting the endogenous gene VEGFA, the cleavage activity of multiple saCas9 mutants Q414A / R654A was higher than that of the wild-type saCas9 protein. The activities of multiple saCas9 mutants included: Q414A / R654A, Y211H / Q414A / R654A, R314A / Q414A / R654A, N419D / Q414A / R654A were higher than the reported high-fidelity mutant HF, and Q414A / R654A was higher than the reported high-fidelity mutant Q414A. Among them, the cleavage activity of Q414A / R654A, Y211H / Q414A / R654A, R314A / Q414A / R654A, and N419D / Q414A / R654A mutants was higher than that of spCas9. Since the Q414A / R654A mutant showed superior characteristics to the wild-type Cas9 protein when targeting EMX1 and VEGFA, it was named hafCas9. The editing efficiency of different mutants targeting VEGFA is shown in Table 5.

[0170] Table 5 Editing efficiency of different Cas9 proteins targeting VEGFA-8

[0171] all variants Mean SD p value (vs WT) p value (vs HF) WT 28.53 2.601 HF 16.93 2.031 0.003678 Q414A 23.47 0.8963 0.033207 0.006991 Q414A / R654A 36.43 3.443 0.033816 0.001075 R314A / R654A 8.3 1.114 0.000244 0.002962 Y239A / R654A 15.03 2.631 0.003206 0.378153 N413A / R654A 16.8 9.107 0.098438 0.981439 Y211H / Q414A / R654A 21.07 1.644 0.01366 0.051903 R245A / Q414A / R654A 11.53 2.301 0.00106 0.038105 R314A / Q414A / R654A 24.67 1.75 0.099483 0.007509 N419D / Q414A / R654A 23.67 5.05 0.211986 0.098794 Y211H / R314A / Q414A / R654A 10.7 1.967 0.000693 0.018799 Y211H / N419D / Q414A / R654A 11.47 2.346 0.001079 0.037964 Y211H / R245A / Q414A / R654A 12 1.153 0.000548 0.021598 R314A / Q414A / N419D / R654A 11.4 2.193 0.000951 0.032696 R314A / R245A / Q414A / R654A 10.77 1.955 0.000697 0.019286 R245A / N419D / Q414A / R654A 8.633 0.6658 0.000212 0.002543 spCas9 15.63 1.222 0.001475 0.395867

[0172] Example 2 Engineered saCas9 mutants have higher specificity

[0173] Using Cas-OFFinder 2.4( http: / / www.rgenome.net / cas-offinder) predicted EMX1-1 and potential off-target sites of VEGFA-8 guide RNA. The off-target sites were ranked according to the number of bases mismatched with the target sequence and the number of bulges generated with the guide RNA, and the top ten off-target sites were selected. Primer 3.0 was used to design amplification primers near the potential off-target sites, and barcodes and adapters for next-generation sequencing were added. The specific primer sequences are shown in Table 3. The amplified products were used to construct a DNA library and submitted to the Illumia NovaSeq 6000 sequencing platform. The sequencing data were analyzed by fastq-multx( https: / / github.com / brwnj / fastq-multx ) are split into different groups according to the barcodes and finally processed by CRISPResso2 ( https: / / github.com / pinellolab / CRISPResso2 ) Analyze the editing frequency of each site. According to the detected editing frequencies of target sites and off-target sites, calculate the target site editing ratio, i.e., on-target ratio, and off-target editing ratio, i.e., off-target ratio.

[0174] The target site editing ratio and off-target editing ratio of wild-type saCas9, HF, Q414A, and hafCas9 mutant targeting EMX1 and VEGFA are shown in Figure 6 The specific editing frequency is shown in Figure 7 . These genomic high-throughput sequencing results show that compared with wild-type saCas9, the hafCas9 mutant significantly increased the target site editing ratio, while reducing the off-target editing ratio and improving specificity. Specifically, the hafCas9 mutant significantly increased the editing frequency of the target genes EMX1 and VEGFA, while reducing non-specific editing at multiple off-target sites. However, traditional high-fidelity mutants, especially HF, did not increase the target site editing / off-target editing ratio. The HF mutant reduced the editing frequency at certain off-target sites, but reduced the editing frequency at the target site more. The Q414A mutant did not significantly improve its specificity at the EMX1-1 site, but significantly improved its specificity at the VEGFA site. However, the hafCas9 mutant has better specificity at these two sites than these two mutants.

[0175] Example 3. Engineered saCas9 mutants have higher cleavage activity against multiple endogenous genes

[0176] In order to explore the editing activity of engineered saCas9 mutants at other endogenous gene sites, this example selected another guide RNA EMX1-6 (Nature, 2016) of the EMX1 gene and 4 guide RNAs of TTR (selected from CN202310828316.9). First, these 5 guide RNAs were connected to different saCas9 mutant plasmids and transfected into HEK293 cells using PEI. Genomic DNA was extracted 3 days after transfection, and the editing product was amplified using target site amplification primers. After the amplified product was sequenced by Sanger, the target site editing frequency was analyzed using TIDE. The guide RNA sequences and target site amplification primers are shown in Table 1 and Table 2, respectively. The analysis results are shown in Figure 8 , the results showed that compared with wild-type saCas9, the hafCas9 mutant significantly improved the editing activity at 80% (4 / 5) of the sites. Compared with the HF mutant, the hafCas9 mutant significantly improved the editing activity at all sites. Compared with the Q414A mutant, the hafCas9 mutant significantly improved the editing activity at 80% (4 / 5) of the sites. These results show that the editing activity of the hafCas9 mutant is better than that of the wild-type saCas9 and the reported high-fidelity mutants.

[0177] Example 4. Engineering saCas9 mutants significantly improves the cleavage activity and specificity of saCas9-KKH mutants

[0178] In order to explore the applicability of engineered saCas9 mutants in other saCas9 mutants, this example transplanted the engineered saCas9 mutant to a PAM extension mutant of saCas9, saCas9-KKH, and selected three targeting sites, designed targeted guide RNAs, and connected them to different saCas9 mutant plasmids. The plasmid was transfected into HEK293 cells using PEI for 72 hours, and genomic DNA was extracted. Specific amplification primers were designed based on the targeting sites and off-target sites predicted by Cas-OFFinder 2.4. The amplified bands were purified and recovered for high-throughput sequencing, and the sequencing targeted editing efficiency and off-target editing efficiency were analyzed using CRISPResso2 (http: / / crispresso.pinellolab.org / submission). The analysis results are shown in Fig. 9 The results showed that compared with the wild-type saCas9-KKH, the hafCas9-KKH mutant significantly improved the on-target editing efficiency and significantly reduced the off-target editing efficiency, and the off-target editing efficiency was lower than that of the known saCas9-KKH-Q414A and saCas9-KKH-HF high-fidelity mutants.

[0179] Example 5. Engineered saCas9 mutants significantly improve the cleavage activity and specificity of the TTR gene

[0180] Next-generation sequencing was used to analyze the target site editing frequency and off-target editing frequency of the three sites targeting TTR by wild-type saCas9, HF, and hafCas9 mutants, and the target site editing ratio and off-target editing ratio were calculated. The specific primer sequences are shown in Table 3. The target site editing ratio results are shown in Fig.10 The results show that compared with wild-type saCas9, the hafCas9 mutant significantly increased the editing rate of the target site and reduced the off-target editing rate. Specifically, the hafCas9 mutant significantly increased the frequency of editing at the target site and reduced nonspecific editing at multiple off-target sites. The results are shown in Fig.11 . The traditional high-fidelity mutant HF did not increase the ratio of target site editing / off-target editing. The HF mutant reduced the editing frequency at some off-target sites, but reduced the editing frequency at the target site more. The specificity of the hafCas9 mutant at these three sites was better than that of the HF mutant.

[0181] Example 6. Engineering saCas9 mRNA significantly improves the cleavage activity of the TTR gene

[0182] After the wild-type saCas9 and Q414A / R654A mutant sequences were connected to a vector containing a T7 promoter, the T7-saCas9-polyA fragment was amplified by PCR, and saCas9 was transcribed into mRNA using RNA polymerase II using this amplified product as a template.

[0183] The relevant DNA sequences are shown in SEQ ID NOs: 6-12, wherein SEQ ID NOs: 6, 8, and 9 are three DNA sequences of T7-saCas9-polyA, SEQ ID NO: 7 is the sequence of the T7 promoter, and SEQ ID NOs: 10 to 12 are three DNA sequences of T7-hafCas9-polyA, respectively.

[0184] In this embodiment, saCas9 mRNA comprises a 5'-end Cap1 cap and a 3'-end polyA, and uracil is replaced by N1-methyl pseudo-uracil. The relevant mRNA sequences are shown in SEQ ID NOs: 13-18. Among them, SEQ ID NOs: 13 to 15 are the mRNA sequences of saCas9 corresponding to SEQ ID NOs: 6, 8, and 9, respectively, and SEQ ID NOs: 16 to 18 are the mRNA sequences of hafCas9 corresponding to SEQ ID NOs: 10 to 12, respectively.

[0185] RNAimax was used to co-transfect in vitro transcribed saCas9 mRNA (SEQ ID NO: 13), hafCas9 mRNA (SEQ ID NO: 16) and different modified forms of TTR-sg12 guide RNA (sequence in Table 6) in CN202310828316.9 into human Huh7 cell line. Genomic DNA was extracted 3 days after transfection, and the editing frequency of the target site was analyzed by Sanger sequencing and TIDE. The sequencing results show that the editing frequency of the target site can be significantly increased when the hafCas9 mutant mRNA is combined with different modified forms of guide RNA. The results are shown in Fig.12 This indicates that when the hafCas9 mutant is delivered transiently, its editing activity is significantly better than that of the wild-type saCas9.

[0186] Table 6 gRNA sequences

[0187]

[0188]

[0189] Note: In Table 6, m represents 2'-MOE modification and * represents phosphorothioate bond modification.

[0190] Example 7. Engineered saCas9 mRNA can efficiently cleave the liver TTR gene in vivo

[0191] The in vitro transcribed hafCas9 mRNA (SEQ ID NO: 16) was respectively packaged with TTR-sg12-Mod001, TTR-sg12-Mod011, TTR-sg12-Mod013 (sequences in Table 6) in CN202310828316.9 into cationic nanolipid particles (LNPs), and the packaged mixtures were respectively called LNP01, LNP02, and LNP03. At the same time, spCas9 mRNA (SEQ ID NO: 19) was packaged into cationic nanolipid particles with the TTR guide RNA reported by Intellia therapeutics, and the packaged mixture was called LNP04. In this embodiment, the cationic nanolipid particles are prepared by Acuitas cationic lipids (ALC-0366), DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5.

[0192] Four nanolipid particles were injected into 5-month-old humanized TTR mice via tail vein. V50M Mice that can highly express human TTR mutant protein in liver cells. The construction strategy of the mice is shown in Fig.13. 100-200 μl of orbital blood was drawn 4 hours, 7 days and 14 days after LNP injection, and the TTR level in serum was detected using a human TTR ELISA kit (Aviva Systems Biology, Cat#OKIA00081). 14 days after injection, the mice were killed, and DNA from the mouse liver was extracted and Sanger sequencing was used to detect the editing efficiency of the TTR gene. The sequencing results show that the three Q414A / R654A mRNA-encapsulated LNPs can efficiently cut the TTR gene in the liver, and the editing efficiency is comparable to the spCas9 effect reported by Intellia therapeutics. The results are shown in Fig.14 A in the figure. The ELISA results show that LNP01, LNP02, and LNP03 can all reduce the TTR protein content in serum, indicating that they can effectively reduce the expression of TTR protein, with protein knockout efficiencies greater than 70%, greater than 90%, and close to 100%, respectively. Among them, LNP03 has the highest protein knockout efficiency and is comparable to that of LNP04. The results are shown in Fig.14 B. These results indicate that the LNP composition containing hafCas9 can efficiently cleave TTR in vivo, reduce harmful TTR protein expression, and can be used to treat ATTR.

[0193] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A saCas9 protein variant having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence of SEQ ID NO: 1, and relative to the amino acid position of SEQ ID NO: 1, the saCas9 protein variant comprises mutations at the following amino acid residues: R654, and at least one selected from Y239, N413, Q414, and optionally at least one of Y211, R245, R314, N419; optionally, the saCas9 protein variant further comprises one or more of a nuclear localization sequence, a cell penetrating peptide sequence, and an affinity tag.

2. The saCas9 protein variant according to claim 1, wherein The saCas9 protein variant comprises the following mutations: R654A, and at least one selected from Y239A, N413A, Q414A, and optionally at least one selected from Y211H, R245A, R314A, N419D.

3. The saCas9 protein variant according to claim 1, wherein The saCas9 protein variant comprises mutations at the following amino acid residues: R654 and Q414, and optionally at least one of Y211, R314, and N419; preferably, the saCas9 protein variant comprises the following mutations: R654A and Q414A, and optionally at least one of Y211H, R314A, and N419D.

4. The saCas9 protein variant according to claim 1, wherein The saCas9 protein variant comprises any of the following mutations: Y239A / R654A, N413A / R654A, Q414A / R654A, Y211H / Q414A / R654A, R314A / Q414A / R654A, Q414A / N419D / R654A, Y211H / R314A / Q414A / R654A, or, Y211H / Q414A / N419D / R654A; preferably, the saCas9 protein variant comprises the following mutation: Q414A / R654A.

5. The saCas9 protein variant according to any one of claims 1 to 4, wherein the saCas9 protein variant further comprises one or more mutations in the PI domain and / or the WED domain; preferably, the saCas9 protein variant further comprises at least one selected from E782K, K929R, N968K, N986R, D987N, D987S, L988R, L988T, L989R, R991L, R991K, R991I, I992V, V994L, R1015H one mutation; preferably, the saCas9 protein variant also comprises any of the following mutations: E782K / N968K / R1015H, N986R, N986R / R991L, R991K, R991K / D987N, D987N / L988R / R991K / V994L, D987S / L988T / L989R / R991I / I992V, E782K / K929R / R1015H, or E782K / K929R / N968K / R1015H.

6. The saCas9 protein variant according to claim 1, wherein the saCas9 protein variant comprises the sequence shown in SEQ ID NO: 3; or has a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% to the sequence shown in SEQ ID NO:

3.

7. The saCas9 protein variant according to any one of claims 1 to 6, wherein SV40 NLS and Nucleoplasmin NLS are fused to the N-terminus and C-terminus of the saCas9 protein variant, respectively; or, SV40 NLS is fused to both the N-terminus and C-terminus of the saCas9 protein variant; preferably, the amino acid sequence of the SV40 NLS is as shown in SEQ ID NO: 4, and preferably, the amino acid sequence of the Nucleoplasmin NLS is as shown in SEQ ID NO:

5.

8. A fusion protein comprising: the saCas9 protein variant according to any one of claims 1 to 7, and an effector domain fused to the saCas9 protein variant; optionally, the Cas9 protein and the effector domain are fused via a linker.

9. The fusion protein according to claim 8, wherein The effector domain includes one or more of APOBEC1, APOBEC3, TadA8e, KRAB, VP64, reverse transcriptase, and transposon.

10. An isolated nucleic acid encoding the saCas9 protein variant according to any one of claims 1 to 7, or the fusion protein according to claim 8 or 9.

11. The isolated nucleic acid of claim 10, comprising any one of SEQ ID NOs: 10 to 12, 16 to 18; or having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% to any one of SEQ ID NOs: 10 to 12, 16 to 18.

12. A recombinant vector comprising the isolated nucleic acid of claim 10 or 11.

13. A host cell comprising the isolated nucleic acid of claim 10 or 11.

14. A composite comprising: The saCas9 protein variant according to any one of claims 1 to 7 or the fusion protein according to claim 8 or 9; and A gRNA that binds to the saCas9 protein variant or the fusion protein; optionally, the gRNA is a gRNA modified by 2'-MOE and a phosphorothioate bond.

15. A composition comprising: An mRNA encoding the saCas9 protein variant according to any one of claims 1 to 7 or the fusion protein according to claim 8 or 9; and A gRNA that binds to the saCas9 protein variant or the fusion protein; Optionally, the mRNA and the gRNA are encapsulated by nanolipid particles; Optionally, the gRNA is a 2'-MOE-modified and phosphorothioate-modified gRNA.

16. The composition according to claim 15, wherein The mRNA comprises any one of SEQ ID NOs: 16 to 18; or has a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% to any one of SEQ ID NOs: 16 to 18.

17. A kit comprising: The isolated nucleic acid of claim 10 or 11; and A nucleic acid encoding a gRNA, wherein the gRNA can bind to the saCas9 protein variant or fusion protein encoded by the isolated nucleic acid; optionally, the gRNA is a gRNA modified by 2'-MOE and a thiophosphate bond.

18. An in vitro method for changing a cell gene, comprising: Expressing the following a) and b) in target cells; Or, contacting the target cell with the following a) and b); a) the saCas9 protein variant according to any one of claims 1 to 7 or the fusion protein according to claim 8 or 9, b) having a gRNA complementary to the target sequence.

19. An in vitro method for altering a double-stranded DNA molecule, comprising: contacting the target double-stranded DNA molecule with the following a) and b); a) the saCas9 protein variant according to any one of claims 1 to 7 or the fusion protein according to claim 8 or 9, b) having a gRNA complementary to the target sequence.

20. Use of the saCas9 protein variant according to any one of claims 1 to 7, or the fusion protein according to claim 8 or 9, or the isolated nucleic acid according to claim 10 or 11, or the recombinant vector according to claim 12, or the host cell according to claim 13, or the complex according to claim 14, or the composition according to claim 15 or 16, or the kit according to claim 17 in the preparation of a product for changing a cell gene or a double-stranded DNA molecule.

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