WO2017215619A1 - Fusion protein producing point mutation in cell, and preparation and use thereof - Google Patents

Abstract

Provided are a fusion protein producing a point mutation in a cell, and preparation and use thereof. The fusion protein includes or is formed by a cytosine deaminase and Cas enzyme in which the nuclease activity is deficient but the helicase activity retains. Also provided are the coding sequence of the fusion protein, a polynucleotide sequence containing the coding sequence, a nucleic acid construct containing the polynucleotide sequence, the corresponding host cell, a method for producing the point mutation in the cell, and a kit, etc. The method can achieve site-directed mutagenesis, and at the same time, achieve high mutation efficiency and multiple mutation combinations in a particular gene region.

Description

Fusion protein for generating point mutation in cells, preparation thereof and use thereof Technical field

The present invention relates to fusion proteins which produce point mutations in cells, their preparation and use.

Background technique

There is a close relationship between genotype and phenotype. In nature, spontaneous mutations cause genotypic changes that produce multiple phenotypes. In the laboratory, mutations are still made to diversify genes, produce a variety of phenotypes, and thus screen out functional mutants, study the relationship between genes and functions, and obtain more functional proteins. In nature, the frequency of spontaneous mutations is extremely low. In common organisms, the spontaneous mutation rate of human genome is 5.0×10 ^-10 , the spontaneous mutation rate of mouse genome is 1.8×10 ^-10 , the spontaneous mutation rate of E. coli genome is 5.4×10 ^-10 , and the spontaneous mutation rate of HIV is 3×10 ^-5 , the spontaneous mutation frequency of the organism increases with the decrease of the biological genome [Holmes E C. The comparative genomics of viral emergence [J]. Proceedings of the National Academy of Sciences, 2010, 107 (4) :1742-1746]. But this low level of gene mutation frequency does not produce a sufficient number of phenotypes to study the relationship between genes, phenotypes and function.

In order to increase the frequency of gene mutations, the existing methods in the laboratory mainly include in vivo mutation methods and in vitro mutation methods. In vivo point mutation method: 1. Physical method: ultraviolet radiation, mutation frequency is 1 × 10 ^-10 [Packer M S, Liu D R. Methods for the directed evolution of proteins [J]. Nature Reviews Genetics, 2015]. 2. Chemical method: ENU is an alkylating agent that transfers ethyl groups to the oxygen and nitrogen atoms of DNA, causing mismatches, base substitutions or deletions, with a mutation frequency of 1-1.5×10 ^-5 [FILBY.ZEBRAFISH :METHODS AND PROTOCOLS.METHODS IN MOLECULAR BIOLOGY‐By GJLieschke, AC Oates and K. Kawakami. [J]. Journal of Fish Biology, 2010, 76(7): 1874-1876]. Although ENU is easy to obtain, it is sensitive to light, heat, and pH, limiting its application. Both methods can change the mutation frequency by dose, but the point mutations caused are random, the mutation frequency is low, the mutation map is not uniform, and it is harmful to the organism [Guénet J L.Chemical mutagenesis of the mouse genome:an overview[ J]. Genetica, 2004, 122(1): 9-24]. 3. Biological methods: transposons, the basic unit of autonomous replication and displacement on chromosomal DNA, can cause insertional mutations, can lead to gene knock-out by gene insertion, gene activation, and can select different insertions by selecting different vectors. Site, but its mutation rate is lower than ENU. In each cell cycle, only 3×10 ^-5 insertion events can occur, and host is required to simultaneously express transposase to complete transposition [Kitada K, Ishishita S, Tosaka K, et al. Transposon-tagged mutagenesis in the rat. [J]. Nature Methods, 2007, 4(2): 131-133].

In the immune system, B cells in the germinal center can produce multi-component antibodies by high-frequency mutation of somatic cells to resist the invasion of pathogens [Odegard VH, Schatz D G. Targeting of somatic hypermutation. [J]. Nature Reviews Immunology, 2006, 6(8): 573-583]. High-frequency mutations in somatic cells refer to non-template point mutations in the immunoglobulin heavy light chain variable region, which are associated with B cell affinity maturation [Odegard V H et al., supra]. The enzyme that mediates this process is activation-induced cytosine deaminase (AID). AID is a cytosine deaminase belonging to the APOBEC family, an RNA editing enzyme family: N-terminal nuclear localization signal, C-terminal nuclear export signal, and its catalytic domain is shared by APOBEC family [Zhenming X, Hong Z , Pone EJ, et al. Immunoglobulin class-switch DNA recombination: induction, targeting and beyond. [J]. Nature Reviews Immunology, 2012, 12(7): 517-31]. It is generally believed that the N-terminal structure is necessary for SHM. The expression of AID is restricted to the B cells of the germinal center, and its function of point mutation is conditional. It must act on single-stranded DNA and has sequence preference. The hotspot domain is RGYW [Kiyotsugu Y, Il-Mi O, Tomonori E, et al. AID Enzyme-Induced Hypermutation in an Actively Transcribed Gene in Fibroblasts [J]. Science, 2002, 296 (5575): 2033-2036]. R stands for A/G, Y stands for C/T, and W stands for A/T. It can be seen that the function of AID is related to the primary structure of DNA. First, the deamination of cytosine on single-stranded DNA is changed to U to form a UG mismatch. If UG is not repaired, a CT GA conversion mutation will be formed during DNA replication. In addition, U can be excised by UNG (uracil DNA glycosidase) to form a pyrimidine-free site, and four bases are randomly incorporated [Odegard V H et al., supra]. The point mutations produced by the above process are significant for somatic high frequency mutations and can produce diverse antibodies. However, the frequency of point mutations caused in vivo is 1×10 ^-4 -1×10 ^-3 , and the sites are random [Masatoshi A, Nesreen H, Andre S, et al.Accumulation of the FACT complex, as well as Histone H3.3, serves as a target marker for somatic hypermutation. [J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110 (19): 7784-7789], still unable to meet the experimental screening mutation Required for the body.

Summary of the invention

The first aspect herein provides a fusion protein comprising a Cas enzyme having a cytosine deaminase and a nuclease activity loss, retaining an understanding of the chymase activity.

In one or more embodiments, the fusion protein is formed by a Casase that lacks cytosine deaminase and nuclease activity, retains knowledge of the chymase activity.

In one or more embodiments, the Cas enzyme is selected from the group consisting of: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof or modified forms thereof.

In one or more embodiments, the nuclease activity of the Cas enzyme is partially deleted such that the Cas enzyme only causes DNA single strand breaks; or the nuclease activity of the Cas enzyme is all deleted, causing DNA double stranding fracture.

In one or more embodiments, the Cas enzyme is a Cas9 enzyme selected from the group consisting of: Cas9 (SpCas9) from S. pyogenes, Cas9 (SaCas9) from S. aureus, and Cas9 from S. thermophilus ( St1Cas9).

In one or more embodiments, the Cas enzyme is a Cas9 enzyme, the two endonuclease catalytic domains of the enzyme are mutated in RuvC1 and/or HNH, resulting in loss of nuclease activity of the enzyme .

In one or more embodiments, both the RuvC1 and HNH of the Cas9 enzyme are mutated, resulting in a loss of the nuclease activity of the enzyme, retaining an understanding of the chymase activity.

In one or more embodiments, the 10th amino acid asparagine of the Cas9 enzyme is mutated to alanine or other amino acid, and the amino acid histidine at position 841 is mutated to alanine or other amino acid.

In one or more embodiments, the amino acid sequence of the Cas9 enzyme is set forth in SEQ ID NO: 2, pp. 42-1452, or as shown in SEQ ID NO: 72, amino acid residues 42-1419.

In one or more embodiments, the cytosine deaminase is a full length cytosine deaminase or a fragment thereof, wherein the fragment comprises at least an NLS domain, a catalytic domain, and an APOBEC-like cytosine deaminase Domain.

In one or more embodiments, the cytosine deaminase undergoes a substitution mutation at amino acid residues 10, 82, and 156.

In one or more embodiments, the substitution mutations are K10E, T82I, and E156G.

In one or more embodiments, the fragment comprises at least amino acid residues 9-182 of the AID, eg, at least amino acid residues 1-182 of the AID.

In one or more embodiments, the amino acid sequence of the cytosine deaminase is as shown in amino acids 1457-1654 of SEQ ID NO: 2, or as amino acid residues 1447-1629 of SEQ ID NO: 68 Show.

In one or more embodiments, the fragment comprises at least amino acid residues 1465-1638 of SEQ ID NO: 2, eg, at least amino acid residues 1457-1638 of SEQ ID NO: 2.

In one or more embodiments, the fragment consists of amino acid residues 1-182, consists of amino acid residues 1-164, or consists of amino acid residues 1-109.

In one or more embodiments, the fusion protein further comprises one or more of the following sequences: A head, a nuclear localization sequence, and an amino acid residue or amino acid sequence introduced for the purpose of constructing a fusion protein, promoting expression of a recombinant protein, obtaining a recombinant protein that is automatically secreted outside the host cell, or facilitating purification of the recombinant protein.

In one or more embodiments, the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 2, 4, 66, 68, 70 or 72, or as shown in amino acids 26-1654 of SEQ ID NO: 2. Or as shown in SEQ ID NO: 4, positions 26-1638, or as shown in SEQ ID NO: 68, amino acids 26-1629, or as SEQ ID NO: 70, amino acids 26-1629, or as SEQ ID NO: 72 is shown in amino acids 26-1638.

A second aspect of the invention provides a polynucleotide sequence selected from the group consisting of:

(1) a polynucleotide sequence encoding the fusion protein of the first aspect of the invention;

(2) (1) The complementary sequence of the sequence.

A third aspect of the invention provides a nucleic acid construct comprising the polynucleotide sequence of the second aspect herein.

In one or more embodiments, the nucleic acid construct is an expression vector for expression of a fusion protein described herein in a host cell.

A fourth aspect of the invention provides a host cell comprising a fusion protein, a coding sequence thereof or a nucleic acid construct as described herein.

A fifth aspect herein provides a method of producing a point mutation in a cell, the method comprising the step of expressing a fusion protein and sgRNA described herein in said cell.

In one or more embodiments, the methods comprise the step of transferring a fusion protein described herein, or an expression vector thereof, and sgRNA or an expression vector thereof into the cell, followed by screening to obtain the desired mutated nucleic acid sequence.

In one or more embodiments, the sgRNA comprises a target binding region and a Cas protein recognition region, the target binding region being capable of specifically binding to a nucleic acid sequence to be mutated, the Cas protein recognition region being capable of being The Cas enzyme recognizes and binds.

In one or more embodiments, the target binding region of the sgRNA specifically binds to a template strand of a nucleic acid sequence to be mutated, and the contralateral region of the sgRNA binding region on the template strand is immediately adjacent to the anterior region sequence recognized by the Cas protein Adjacent motifs, or bases separated by 10 or less.

In one or more embodiments, the gene to be mutated encodes a functional protein.

In one or more embodiments, the functional protein includes proteins involved in the development, progression, and metastasis of diseases, proteins involved in cell differentiation, proliferation, and apoptosis, proteins involved in metabolism, development-related proteins, and Drug targets and so on.

In one or more embodiments, the functional protein is selected from the group consisting of antibodies, enzymes, lipoproteins, hormone proteins, transport and storage proteins, motor proteins, receptor proteins, and membrane proteins.

A sixth aspect of the invention provides a kit comprising a fusion protein, polynucleotide sequence or nucleic acid construct as described herein.

A seventh aspect of the invention provides the use of a fusion protein, polynucleotide sequence or nucleic acid construct as described herein for producing a point mutation in a cell, or in the preparation of a composition or kit for producing a point mutation in a cell Applications.

DRAWINGS

Figure 1: A and C are PCR-amplified AID (lane 1) and AIDX fragment (lane 1); B is pEntr11-dCas9-AID plasmid agarose gel, in which one lane is pEntr11 empty plasmid, 2 The plasmid is pEntr11-dCas9 plasmid, the 3-7 lanes are pEntr11-dCas9-AID plasmid; D is the PCR result of pEntr11-dCas9-AIDX plasmid bacterial solution, and the amplified fragment is AIDX. Lanes 1-5 in D represent 5 different positive clones, respectively, and No. 6 is an empty plasmid as a negative control.

Figure 2: A, 1 and 2 lanes are respectively PCR-amplified dCas9-AID and dCas9-AIDX fragments; B, enzymatically cleavage of MO91 empty-loaded plasmid, one of which is BglII single-cut, and the other is MO91 empty Plasmid, 3 lanes are BglII and XhoI double digestion; C, MO91-dCas9-AIDX plasmid bacterial solution PCR results, the amplified fragment is AIDX; D, MO91-dCas9-AID plasmid bacterial solution PCR results, amplified The fragment is an AID.

Figure 3: A, 1 is the 3*flag+NLS fragment amplified by PCR, and 2 and 3 lanes are BglII single-cutting MO91-dCas9-AID plasmid and MO91-dCas9-AIDX plasmid, respectively, and 4 lanes are MO91- dCas9-AID plasmid control; B, 1-4 lanes are MO91-dCas9 (3*flag, NLS)-AID plasmid, lane 5 is MO91-dCas9-AID plasmid, and lane 6-9 is MO91-dCas9 (3*flag, NLS)-AIDX plasmid.

Figure 4: Sequence of the EGFP reporter, the stop codon is shown in bold. The designed sgRNA is indicated by an arrow.

Figure 5: Schematic representation of the pattern of the reporter plasmid.

Figure 6: Flow cytometry reporting cell line. The three curves from left to right indicate Thy1.1 expression levels of unstained controls, reporter negative cells, and reporter positive cells, respectively.

Figure 7: Comparison of dCas9-AID, dCas9-AIDX, AID and AIDX point mutation efficiencies in reporter cells.

Figure 8: Optimization of dCas9-AID point mutation efficiency in reporter cells. A, dCas9-AID induces GFP expression; B, a schematic of different AID variants and the efficiency of their induction of point mutations; C, dCas9-AIDX induces point mutations requiring cytosine deaminase activity of AID.

Figure 9: Point mutation frequency distribution of dCas9-AIDX and AID on EGFP and cMyc genes.

Figure 10: dCas9-AIDX randomly mutates C and G bases to three other bases. A, statistics of base mutation types; B, dCas9-AIDX induces point mutation mechanism.

Figure 11: UGI increases the frequency of base substitutions in the dCas9-AIDX system, revealing that dCas9-AIDX is genetically The trajectory of the action, and the direction of the base mutation is more singular.

Figure 12: dCas9-AIDX not only acts on exogenous genes, but also on endogenous genes.

Figure 13: Structural functional domain of the AID.

Figure 14: Experimental procedure (a) and results (b-d) of the application of dCas9-AIDX to the Gleevec resistance screening of the K562BCR-ABL gene.

Figure 15: TAM (targeting cytosine deaminase AID-mediated gene mutation technique) mutating amino acids of the anti-HEL-IgG1 variable region.

Figure 16: TAM induces base mutations in the anti-HEL-IgG1 variable region (top panel) and can repeatedly induce base mutations in the IgGl CDRs (bottom panel).

Figure 17: The affinity of the mutated antibody for HEL is increased by more than 10 fold.

Figure 18: Results of expression of nCas9-AIDX in bacteria. The boxed box is a band of nCas9-AIDX fusion protein.

Figure 19: Functional test results for different fusion proteins. For each set of data, the three pillars from left to right represent the results of MO91-AIDX-XTEN-dCas9, MO91-dCas9-XTEN-AIDX, and MO91-dCas9-AIDX.

Figure 20: Functional test results for different fusion proteins. For each set of data, the three pillars from left to right represent the results of MO91-dCas9-AIDX, MO91-dCas9-XTEN-AIDX (K10E T82I E156G) and MO91-dCas9-XTEN-AIDX.

Figure 21: Functional verification results of the nCas9-AIDX fusion protein.

detailed description

This document relates to a fusion protein of Cas protein with nuclease activity and cytosine deaminase AID or a mutant thereof. Under the guidance of sgRNA, the fusion protein is recruited to a specific DNA sequence, and AID or its mutant deamination of cytosine to produce uracil, which is then randomly mutated into other bases during DNA repair. High mutation efficiency is obtained while achieving site-directed mutagenesis.

With regard to the content of the Cas/sgRNA, in addition to the description herein, see also CN 201380049665.5 and CN 201380072752.2, the entire contents of each of which are hereby incorporated by reference.

Cas protein

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a genetic editing system in which bacteria resist viral invasion or evade mammalian immune responses. The system has been modified and optimized and is now widely used in in vitro biochemical reactions, cell and individual gene editing.

Typically, a complex of a Cas protein with endonuclease activity and its specifically recognized sgRNA is complementary paired with a template strand in the target DNA by a pairing region of the sgRNA, and the double stranded DNA is cleaved by Cas at a specific position. It should be understood that "Cas protein" and "Cas enzyme" are used interchangeably herein.

The above-described property of Cas/sgRNA is utilized herein to utilize sgRNA specific binding to a target to position Cas to a desired position at which the cytosine is deaminated by AID or a mutant thereof in the fusion protein. Partial or complete deletions of nuclease activity suitable for use in the present invention, particularly partial or complete deletion of endonuclease activity, but retaining knowledge of the chymase activity may be derived from various Cas proteins and variants thereof well known in the art. Including but not limited to Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, a homolog thereof or a modified form thereof.

In some embodiments, a Cas9 enzyme lacking nuclease activity and a single-stranded sgRNA specifically recognized by the same are used. The Cas9 enzyme may be a Cas9 enzyme from a different species including, but not limited to, Cas9 (SpCas9) from S. pyogenes, Cas9 (SaCas9) from S. aureus, and Cas9 (St1 Cas9) from Streptococcus thermophilus, and the like. Various variants of the Cas9 enzyme can be used as long as the Cas9 enzyme specifically recognizes its sgRNA and lacks nuclease activity.

The Cas protein with nuclease activity deletion can be prepared by methods well known in the art, including but not limited to deletion of the entire catalytic domain of the endonuclease in the Cas protein or mutation of one or several amino acids in the domain. Thereby producing a Cas protein lacking in nuclease activity. The mutation may be one or several (for example, two or more, three or more, four or more, five or more, more than ten, to the entire catalytic domain) deletion or substitution of amino acid residues, or one or several new amino acids. Insertion of residues (for example, one or more, two or more, three or more, four or more, five or more, ten or more, or 1 to 10, and 1 to 15). Deletion of the above domains or mutation of amino acid residues can be carried out by methods conventional in the art, and whether the Cas protein after the mutation also has nuclease activity. For example, for Cas9, its two endonuclease catalytic domains, RuvC1 and HNH, can be mutated, for example, the 10th amino acid of the enzyme (in the RuvC1 domain) is mutated to alanine or other An amino acid that mutates the histidine of amino acid 841 (located in the HNH domain) to alanine or other amino acids. These two mutations cause Cas9 to lose endonuclease activity. Preferably, the Cas enzyme is completely nuclease free. In one or more embodiments, the amino acid sequence of the nuclease-free Cas9 enzyme used herein is set forth in SEQ ID NO: 2, pp. 42-1452. In other embodiments, the Cas enzyme portion used herein lacks nuclease activity, ie, the Cas enzyme can cause DNA single strand breaks. A representative example of such a Cas enzyme can be shown as amino acid residues 42-14-19 of SEQ ID NO:72.

The function of the Cas/sgRNA complex requires a protospacer adjacent motif (PAM) in the non-template strand (3' to 5') of the DNA. Different Cas enzymes, their corresponding PAMs are not identical. For example, the PAM for SpCas9 is typically NGG; the PAM for the SaCas9 enzyme is typically NNGRR; the PAM for the St1Cas9 enzyme is typically NNAGAA; wherein N is A, C, T or G and R is G or A.

In certain preferred embodiments, the PAM for the SaCas9 enzyme is NNGRRT. In certain preferred embodiments, the PAM for SpCas9 is TGG.

sgRNA

sgRNA usually consists of two parts: a target binding region and a Cas protein recognition region. The target binding region and the Cas protein recognition region are usually joined in the 5' to 3' direction.

The target binding region is typically 15 to 25 bases in length, more typically 18 to 22 bases, such as 20 bases. The target binding region specifically binds to the template strand of DNA, thereby recruiting the fusion protein to a predetermined site. Typically, the contralateral region of the sgRNA binding region on the DNA template strand is in close proximity to the PAM, or is separated by a few bases (eg, within 10, or within 8 or within 5). Therefore, when designing sgRNA, the PAM of the enzyme is usually determined according to the Cas enzyme used, and then a site which can be used as a PAM is found on the non-template strand of DNA, and then the non-template strand (3' to 5') PAM is used. Fragments 15 to 25 bases long, more usually 18 to 22 bases long downstream of the PAM site or within 10 (eg, within 8 or less, etc.) of the PAM site A sequence that is the target binding region of sgRNA.

The Cas protein recognition region of sgRNA is determined based on the Cas protein used, which is well known to those skilled in the art.

Therefore, the sequence of the target binding region of the sgRNA herein is that the DNA strand containing the PAM site recognized by the selected Cas enzyme is immediately downstream of the PAM site or within 10 of the PAM site (for example, 8 or less, 5 Fragments of 15 to 25 bases in length, usually 18 to 22 bases in length; the Cas protein recognition region is specifically recognized by the selected Cas enzyme.

The sgRNA can be prepared by methods conventional in the art, for example, by conventional chemical synthesis methods. The sgRNA can also be transformed into cells via an expression vector to express the sgRNA in the cell. Expression vectors for sgRNA can be constructed using methods well known in the art.

Activation-induced cytosine deaminase (AID)

AID is a cytosine deaminase belonging to the APOBEC family, an RNA editing enzyme family: a nuclear localization signal at the N-terminus and a nuclear export signal at the C-terminus. The catalytic domain is shared by the APOBEC family. It is generally believed that the N-terminal structure is required for somatic hypermutation (SHM). AID function is deamination of cytosine, cytosine The pyridine becomes uracil, and subsequent DNA repair can turn uracil into other bases. It will be appreciated that cytosine deaminase, or fragments or mutants thereof that retain the biological activity of cytosine deamination, cytosine to uracil, are well known in the art.

The structural functional domain of the AID is shown in Figure 14. Among them, amino acids 9-26 are nuclear localization (NLS) domains, especially amino acids 13-26 are involved in DNA binding, amino acids 56-94 are catalytic domains, amino acids 109-182 are APOBEC-like domains, and amino acids 193-198 are The nuclear export (NES) domain, amino acids 39-42 interact with catenin-like protein 1 (CTNNBL1), and amino acids 113-123 are hotspot recognition loops.

A full length sequence of AID (as indicated by amino acids 1457-1654 of SEQ ID NO: 2) can also be used herein, and fragments of AID can also be used. Preferably, the fragment comprises at least an NLS domain, a catalytic domain and an APOBEC-like domain. Thus, in certain embodiments, the fragment comprises at least amino acid residues 9-182 of the AID (ie, amino acid residues 1465-1638 of SEQ ID NO: 2). In other embodiments, the fragment comprises at least amino acid residues 1-182 of the AID (ie, amino acid residues 1457-1638 of SEQ ID NO: 2). For example, in certain embodiments, an AID fragment as used herein consists of amino acid residues 1-182, consists of amino acid residues 1-164, or consists of amino acid residues 1-190. Thus, in certain embodiments, the AID fragment used herein consists of amino acid residues 1457-1638 of SEQ ID NO: 2, amino acid residues 1457-1642 of SEQ ID NO: 2, or SEQ ID NO: 2 Amino acid residue composition of 1457-1646.

Variants of AID that retain their cytosine deaminase activity can also be used herein. For example, such variants may correspond to a wild-type sequence of AIDs having from 1 to 10, such as 1-8, 1-5 or 1-3 amino acid variations, including deletions, substitutions and mutations of amino acids. Preferably, these amino acid variations do not occur within the above-described NLS domain, catalytic domain and APOBEC-like domain, or even within these domains do not affect the original biological function of these domains. For example, it is preferred that these variations do not occur at positions 24, 27, 38, 56, 58, 87, 90, 112, 140, etc. of the AID amino acid sequence. In certain embodiments, these variations also do not occur within amino acids 39-42, amino acids 113-123. Thus, for example, variation can occur among amino acids 1-8, amino acids 28-37, amino acids 43-55, and/or amino acids 183-198. In certain embodiments, the variation occurs at positions 10, 82, and 156. For example, substitution mutations occur at positions 10, 82, and 156, and such substitution mutations can be K10E, T82I, and E156G. In these embodiments, the amino acid sequence of an exemplary AID mutant comprises the amino acid sequence set forth at positions 1447-1629 of SEQ ID NO: 68, or the amino acid set forth at positions 1447-1629 of SEQ ID NO:68. Residue composition.

Fusion protein

Provided herein are fusion proteins comprising Cas enzyme and AID. In the fusion protein herein, the Cas enzyme is usually at the N-terminus of the amino acid sequence of the fusion protein, and the AID is at the C-terminus. In certain embodiments, provided herein are fusion proteins formed primarily of Cas enzyme and AID. It should be understood that a fusion protein or similar expression "formed primarily by" herein does not mean that the fusion protein includes only Cas enzyme and AID, and the definition is understood to mean that the fusion protein may include only Cas enzyme and AID, or It may also contain other components that do not affect the targeting of the Cas enzyme in the fusion protein and the function of the AID mutant target sequence, including but not limited to various linker sequences, nuclear localization sequences, and gene cloning operations, as described below, And/or an amino acid sequence introduced in the fusion protein for the purpose of constructing a fusion protein, promoting expression of a recombinant protein, obtaining a recombinant protein that is automatically secreted outside the host cell, or facilitating detection and/or purification of the recombinant protein.

The Cas enzyme can be fused to the AID via a linker. The linker may be a peptide of 3 to 25 residues, for example, a peptide of 3 to 15, 5 to 15, 10 to 20 residues. Suitable examples of peptide linkers are well known in the art. Typically, the linker contains one or more motifs that are repeated before and after, and the motif typically contains Gly and/or Ser. For example, the motif can be SGGS, GSSGS, GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motif is contiguous in the linker sequence with no amino acid residues inserted between the repeats. The linker sequence may comprise 1, 2, 3, 4 or 5 repeat motifs. In certain embodiments, the linker sequence is a polyglycine linker sequence. The amount of glycine in the linker sequence is not particularly limited, but is usually 2 to 20, for example, 2 to 15, 2 to 10, and 2 to 8. In addition to glycine and serine, the linker may also contain other known amino acid residues such as alanine (A), leucine (L), threonine (T), glutamic acid (E), styrene Amino acid (F), arginine (R), glutamine (Q), and the like. In certain embodiments, the linker sequence is XTEN, the amino acid sequence of which is set forth in amino acid residues 183-198 of SEQ ID NO:66.

As an example, a linker can be composed of the following amino acid sequences: G(SGGGG) ₂ SGGGLGSTEF (SEQ ID NO: 21), RSTSGLGGGS (GGGGS) ₂ G (SEQ ID NO: 22), QLTSGLGGGS (GGGGS) ₂ G (SEQ ID NO: 23) ), GGGS (SEQ ID NO: 24), GGGGS (SEQ ID NO: 25), SSSSG (SEQ ID NO: 26), GSGSA (SEQ ID NO: 27), GGSGGGGGGSGGGGSGGGGS (SEQ ID NO: 28), SSSSGSSSSGSSSSG (SEQ) ID NO: 29), GGSGAGSGSAGSGSA (SEQ ID NO: 30), GGSGGGGSGGGGSGG (SEQ ID NO: 31), SEQ ID NO: 72, amino acid residues 1420-1456, and the like.

It will be appreciated that in gene cloning procedures, it is often desirable to design a suitable cleavage site which necessarily introduces one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest. In order to construct a fusion protein, promote expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus of the recombinant protein or within the protein. Other suitable regions, for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, End extension, etc. Thus, the amino terminus or carboxy terminus of the fusion protein herein may also contain one or more polypeptide fragments as a protein tag. Any suitable label can be used in this article. For example, the tag may be FLAG (DYKDDDDK, SEQ ID NO: 32), HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B , gE and Ty1. These tags can be used to purify proteins.

The fusion proteins herein may also contain a nuclear localization sequence (NLS). Nuclear localization sequences of various sources and various amino acids well known in the art can be used. Such nuclear localization sequences include, but are not limited to, the NLS of the SV40 viral large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 33); the NLS from the nuclear protein, for example, having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 34) Nuclear protein dichotomous NLS; NLS from c-myc having the amino acid sequence PAAKRVKLD (SEQ ID NO: 35) or RQRRNELRSRSP (SEQ ID NO: 36); NLS from hRNPA1M9 having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 37); sequence from the IBB domain of the input protein-α RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 38); sequence of fibroid T protein VSRKRPRP (SEQ ID NO: 39) and PPKKARED (SEQ ID NO: 40); mouse c Sequence of -ablIV SALIKKKKKMAP (SEQ ID NO: 41); sequence of influenza virus NS1 DRLRR (SEQ ID NO: 42) and PKQKKRK (SEQ ID NO: 43); sequence of hepatitis virus delta antigen RKLKKKIKKL (SEQ ID NO: 44) ; sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO: 45); sequence of human poly(ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 46); and sequence of steroid hormone receptor (human) glucocorticoid RKCLQAGMNLEARKTKK (SEQ I D NO: 47); etc. In certain embodiments, the sequence shown by amino acid residues 26-33 of SEQ ID NO: 2 is used herein as the NLS. The NLS may be located at the N-terminus and C-terminus of the fusion protein; it may also be located in the fusion protein sequence, such as the N-terminus and/or C-terminus of the Cas9 enzyme in the fusion protein, or the N-terminus and/or C of the AID located in the fusion protein. end.

The accumulation of the fusion protein of the invention in the nucleus can be detected by any suitable technique. For example, a detection marker can be fused to the Cas enzyme such that the location of the fusion protein within the cell can be visualized when combined with means for detecting the location of the nucleus (eg, a dye specific for the nucleus, such as DAPI). In certain embodiments, 3*flag is used herein as a marker, and the peptide sequence can be as shown in amino acid residues 1-23 of SEQ ID NO:2. It will be understood that, in general, if a marker sequence is present, the marker sequence is typically at the N-terminus of the fusion protein. The tag sequence can be directly linked to the NLS or can be joined by a suitable linker sequence. The NLS sequence can be ligated directly to the Cas enzyme or AID, or it can be ligated to the Cas enzyme or AID by a suitable linker sequence.

Thus, in certain embodiments, the fusion proteins herein consist of a Cas enzyme and an AID. In other embodiments, the fusion protein herein is formed by the Cas enzyme linked to the AID via a linker. In certain embodiments, The fusion protein NLS, Cas enzyme, AID, and optional linker sequences between the Cas enzyme and the AID are comprised herein. In certain embodiments, the Cas enzyme in the fusion protein is a Cas9 enzyme as described hereinbefore. In certain particular embodiments, the amino acid sequence of the AID in the fusion protein is set forth in amino acid residues 1457-1654 of SEQ ID NO:2. In other specific embodiments, the amino acid sequence of the AID in the fusion protein is set forth in amino acid residues 1457-1646 of SEQ ID NO:4. In other specific embodiments, the amino acid sequence of the AID in the fusion protein is set forth in amino acid residues 1447-1629 of SEQ ID NO:68.

In certain embodiments, the amino acid sequence of the fusion protein herein is as set forth in SEQ ID NO: 2, 4, 66, 68, 70 or 72, or as shown in amino acids 26-1654 of SEQ ID NO: 2, or As shown in SEQ ID NO: 4, positions 26-1638, or as shown in SEQ ID NO: 68, amino acids 26-1629, or as SEQ ID NO: 70, amino acids 26-1629, or as SEQ ID NO: 72 is shown in amino acids 26-1638.

Polynucleotide sequence, host and protein expression

Included herein are polynucleotide sequences encoding the fusion proteins herein. The polynucleotides herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

The nucleotide sequences described herein can generally be obtained by PCR amplification. In particular, primers can be designed according to the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and using a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template, The relevant sequences were amplified. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order. For example, in certain embodiments, the polynucleotide sequence encoding a fusion protein described herein is set forth in SEQ ID NO: 1, 3, 65, 67, 79 or 71, or as SEQ ID NO: 1 73-4965 Shown as a base, or as shown in bases 73-4917 of SEQ ID NO: 3, or as bases 76-4890 of SEQ ID NO: 67, or as SEQ ID NO: 70, 76- The 4890 bases are shown, or as shown in SEQ ID NO: 72, positions 76-4917.

Also included herein are nucleic acid constructs comprising the polynucleotides. The nucleic acid construct contains the coding sequences for the fusion proteins described herein, as well as one or more regulatory sequences operably linked to the sequences. The coding sequences for the fusion proteins of the invention can be manipulated in a variety of ways to ensure expression of the proteins. The nucleic acid construct can be manipulated depending on the identity or requirements of the expression vector prior to insertion of the nucleic acid construct into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The control sequence can be a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutated, truncated and hybrid promoters, and may be derived from an extracellular or heterologous source encoding the host cell. Or Gene acquisition of intracellular polypeptides.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a suitable leader sequence, an untranslated region of the mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.

In certain embodiments, the nucleic acid construct is a vector. For example, a polynucleotide sequence herein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element. The expression vector may also include a ribosome binding site for translation initiation and a transcription terminator. The polynucleotide sequences described herein are operably linked to a suitable promoter in an expression vector to direct mRNA synthesis via the promoter. Representative examples of such promoters are: lac or trp promoter of E. coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti- Promoters for the expression of LTRs of transcriptional viruses and other known controllable genes in prokaryotic or eukaryotic cells or their viruses. The marker gene can be used to provide phenotypic traits for selection of transformed host cells including, but not limited to, dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or for the large intestine Bacillus tetracycline or ampicillin resistance. When a polynucleotide described herein is expressed in a higher eukaryotic cell, transcription will be enhanced if an enhancer sequence is inserted into the vector. An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.

One of ordinary skill in the art will recognize how to select appropriate vectors, promoters, enhancers, and host cells. Expression vectors containing the polynucleotide sequences described herein and appropriate transcription/translation control signals can be constructed using methods well known to those of skill in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

The vectors described herein can be transformed into a suitable host cell to enable expression of the fusion proteins described herein. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; a filamentous fungal cell, or a higher eukaryotic cell, such as a mammalian cell. The host cell can also be a plant cell. Representative examples of host cells are: Escherichia coli; Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, filamentous fungi; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, Or animal cells of Bowes melanoma cells, etc. In addition to the cells used to express the fusion protein, others include A polynucleotide sequence or vector and a cell of sgRNA or an expression vector thereof, such as a cell for the preparation of a point mutant protein, are also within the scope of the host cells described herein.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl ₂ method, and the procedures used are well known in the art. Another method is to use MgCl ₂ . Conversion can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.

After transformation of the host cell, the obtained transformant can be cultured in a conventional manner to allow it to express the fusion protein described herein. The medium used in the culture may be selected from various conventional media depending on the host cell used. The recombinant fusion proteins herein can be isolated and purified using various separation methods known in the art. These methods are well known to those skilled in the art and include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic bacteria, super treatment, ultracentrifugation, molecular sieve chromatography ( Gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Thus, host cells comprising a fusion protein described herein, a coding sequence or expression vector thereof, and optionally sgRNA or an expression vector thereof, are also included herein. Such host cells can constitutively express the fusion proteins described herein, and can also express the fusion proteins described herein under certain conditions of induction. Methods for constitutively expressing a host cell or expressing a fusion protein of the invention under inducing conditions are well known in the art. For example, in certain embodiments, an expression vector of the invention is constructed using an inducible promoter to effect inducible expression of the fusion protein.

Composition, kit

The fusion protein herein, its coding sequence or expression vector, and/and sgRNA, its coding sequence or expression vector can be provided in the form of a composition. For example, the composition may contain an expression vector for the fusion protein and sgRNA or sgRNA herein, or an expression vector containing the fusion protein herein and an expression vector for sgRNA or sgRNA. In the composition, the fusion protein or its expression vector, or sgRNA or its expression vector may be provided as a mixture, or may be packaged separately. The composition may be in the form of a solution or it may be in a lyophilized form.

The composition can be provided in a kit. Accordingly, provided herein are kits containing the compositions described herein. Alternatively, a kit is provided herein, comprising the expression vector of the fusion protein and sgRNA or sgRNA herein, or an expression vector comprising the expression vector of the fusion protein herein and sgRNA or sgRNA. In the kit, the fusion protein or its expression vector, or sgRNA or its expression vector can be packaged separately or in the form of a mixture. Also included in the kit are reagents for transferring the fusion protein or its expression vector and/or sgRNA or expression vector thereof into a cell, and instructions for directing the skilled person to perform the transfer. Alternatively, the kit can also include instructions for the skilled artisan to practice the various methods and uses described herein using the components contained in the kit. Kit Other reagents, such as reagents for PCR, etc., are also included.

Method and use

A third aspect herein provides a method of producing a point mutation in a cell, the method comprising the step of expressing a fusion protein and sgRNA described herein in said cell. In certain embodiments, a fusion protein of the invention, or an expression vector thereof, and sgRNA or an expression vector thereof, are introduced into the cell. Where the cell constitutively expresses a fusion protein described herein, only the corresponding sgRNA or its expression vector can be transferred into the cell. In the case of cell-inducible expression of the fusion protein described herein, the cells may also be incubated with the inducer after administration of the sgRNA, or the cells may be subjected to corresponding inducing measures (eg, illumination). The fusion protein or its expression vector and/or sgRNA or expression vector thereof can be transferred into a cell using conventional transfection methods. For example, in certain embodiments, upon transfection, a plasmid DNA-liposome complex is first prepared, and then the plasmid DNA-liposome complex and the corresponding sgRNA are co-transfected into cells. After obtaining a cell in which a point mutation is produced, the cell can be cultured under conditions suitable for the growth of the cell and expression of a desired protein, and the resulting mutant can be isolated and analyzed by various conventional methods such as a high-throughput method.

Thus, the methods described herein for generating point mutations in cells can also be used to generate mutant libraries, and then the mutants in the library can be isolated and screened using conventional techniques to obtain mutants having the desired biological function. Accordingly, the invention also provides a method of constructing a mutant library, the method comprising the step of expressing a fusion protein and sgRNA described herein in said cell.

One or more sgRNAs can be designed for the same site to be mutated. When designing multiple sgRNAs, the target binding regions of the various sgRNAs designed are different, but have the same Cas protein recognition region. The one or more sgRNAs can then be transferred into the cell along with the corresponding fusion protein.

The cell can be any cell of interest, including prokaryotic cells and eukaryotic cells, such as plant cells, animal cells, microbial cells, and the like. Particularly preferred are animal cells, such as mammalian cells, rodent cells, including humans, horses, cows, sheep, rats, rabbits, and the like. Microbial cells include cells from a variety of microbial species well known in the art, especially those having microbial species of medical research value, production value (e.g., production of fuels such as ethanol, protein production, lipids such as DHA production). The cells may also be cells of various organ origin, such as cells from human liver, kidney, skin, and the like. The cells may also be various mature cell lines currently marketed, such as 293 cells, COS cells. In certain embodiments, the cell is a cell from a healthy individual; in other embodiments, the cell is a cell from a diseased tissue of a diseased individual, such as a cell from an inflammatory tissue, a tumor cell, an induced pluripotent stem cell, and the like. . The cells may also be cells that have been genetically engineered to have a particular function (eg, to produce a protein of interest) or to produce a phenotype of interest. In other words, the gene or nucleic acid sequence to be mutated may be naturally present in the cell for the cell (endogenous) The gene or nucleic acid sequence may also be a foreign-transferred (exogenous) gene or nucleic acid sequence. The extraneously transferred gene or nucleic acid sequence can be integrated into the genomic sequence of the cell or independently of the genome and stably expressed.

For different cells, expression vectors expressing the fusion proteins and sgRNAs herein can be designed using known techniques to render these expression vectors suitable for expression in such cells. For example, a promoter that facilitates expression in the cell and other related regulatory sequences can be provided in an expression vector. These can be selected and implemented by the technician according to the actual situation.

Nucleic acid sequences which are expected to produce point mutations can be any nucleic acid sequence of interest, such as a gene sequence, particularly various diseases, or related to the production of various proteins of interest, or various biological functions of interest. A related gene or nucleic acid sequence. Such gene or nucleic acid sequences of interest include, but are not limited to, nucleic acid sequences encoding various functional proteins. Herein, a functional protein refers to a protein capable of performing physiological functions of an organism, including a catalytic protein, a transport protein, an immune protein, and a regulatory protein. In certain embodiments, the functional proteins include, but are not limited to, proteins involved in the development, progression, and metastasis of diseases, proteins involved in cell differentiation, proliferation, and apoptosis, proteins involved in metabolism, development-related proteins. , as well as various drug targets and so on. For example, the functional protein may be an antibody, an enzyme, a lipoprotein, a hormone protein, a transport and storage protein, a motor protein, a receptor protein, a membrane protein, or the like. Thus, mutant libraries, polynucleotides, nucleic acid constructs, cells, methods, and the like, as described herein, can be used to construct mutant libraries and further screen for proteins with new or greater functions, such as antibodies, enzymes, or other functional proteins. Wait.

Mutations can be made on the nucleic acid sequence of interest using the methods described herein, or at specific sites in the nucleic acid sequence of interest. For the former, the PAM site on the template strand can be searched according to the Cas enzyme used, and the PAM site is immediately downstream of the PAM site or within 10 cells (for example, within 8, within 5 or 3). A fragment of 15 to 25 bases in length, usually 18 to 22 bases in length, is designed as the target recognition region of the sgRNA to design the sgRNA recognized by the Cas enzyme. For the latter, a site that can serve as a PAM can be found near the specific site, and the Cas enzyme capable of recognizing the PAM can be selected according to the PAM, and the fusion protein of the present invention containing the Cas enzyme and correspondingly designed and prepared according to the description herein sgRNA.

The method herein may be an in vitro method or an in vivo method. When performed in vivo, the fusion protein or expression vector thereof and sgRNA or expression vector thereof can be transferred into a subject, such as a corresponding tissue cell, by a means well known in the art, and the sensation can be screened by observing the phenotypic change of the animal. A functional variant of interest. It should be understood that in vivo experiments, the subject may be a variety of non-human animals, particularly various non-human model organisms conventionally employed in the art. In vivo experiments should also meet ethical requirements.

The invention will be elucidated below in the context of specific embodiments. It is to be understood that the examples are merely illustrative and not limiting of the scope of the invention. The experimental methods in the following examples that do not specify the specific conditions are usually in accordance with the conventional strips. The conditions are as described in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, or as recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

Example 1: Construction of pEntr11-dCas9-AID plasmid and pEntr11-dCas9-AIDX plasmid

1. The cDNA reverse-transcribed from the A20 cell line (purchased in the cell bank of the Chinese Academy of Sciences' Type Culture Collection Committee) as a template, using the primers shown in SEQ ID NOS: 5 and 6, and SEQ ID NOS: 5 and Primers amplify the full-length AID sequence and the AIDX fragment (truncate from amino acid residue 183) (see Figure 1, A and C);

2. Construction of the pEntr11-dCas9-TET1CD plasmid:

(1) a dCas9 gene fragment was amplified from dCas9 plasmid (Addgene) by PCR;

(2) The dCas9 gene fragment and the pEntr11 plasmid (Invitrogen) were digested with restriction endonucleases BamHI and NcoI to recover the above fragment;

(3) ligating the digested dCas9 fragment and the pEntr11 vector, and then transforming the ligation product into TOP10 competent cells;

(4) Select positive clones, extract the plasmid and send it to sequencing verification, thus completing the construction of pEntr11-dCas9 plasmid;

(5) Amplifying the TET1CD target gene fragment by PCR;

(6) The pEntr11-dCas9 plasmid was digested with restriction endonucleases BamHI and XhoI, and the fragment was recovered;

(7) The TBi1CD was cloned into the pEntr11-dCas9 plasmid by the Gibson Assembly method, and the construction of the pEntr11-dCas9-TET1CD plasmid was completed.

3. The pEntr11-dCas9-TET1CD plasmid, AID and AIDX fragments were digested with restriction endonucleases BamHI and XhoI, and then the pEntr11-dCas9 vector and AID and AIDX fragments were recovered;

4. Linking the digested AID and AIDX fragments to the pEntr11-dCas9 vector, respectively, and then transforming the ligation product into TOP10 competent cells;

5. Positive clones were selected, plasmids were extracted and sequenced, and the construction of pEntr11-dCas9-AID and pEntr11-dCas9-AIDX plasmids was completed (Fig. 1, B and D).

Example 2: Construction of MO91-dCas9-AID plasmid and MO91-dCas9-AIDX plasmid

1. The dCas9-AID fragment and the dCas9-AIDX fragment were amplified from the pEntr11-dCas9-AID plasmid and the pEntr11-dCas9-AIDX plasmid using the primers shown in SEQ ID NOS: 8 and 9 (Fig. 2, A);

2. The MO91 plasmid (Addgene Plasmid #19755) and the AID and AIDX fragments were digested with restriction endonucleases BglII and XhoI, and then the vector, AID fragment and AIDX fragment were recovered (Fig. 2, B);

3. The AID fragment and the AIDX fragment after digestion are ligated to the MO91 vector, and then the ligated product is transformed into Stbl3 competent cells;

4. Positive clones were selected, plasmids were extracted and sequenced, and the construction of MO91-dCas9-AID and MO91-dCas9-AIDX plasmids was completed (Fig. 2, C and D).

Example 3: Construction of MO91-dCas9 (3*flag, NLS)-AID plasmid and MO91-dCas9 (3*flag, NLS)-AIDX plasmid

Using pCW-Cas9 plasmid (Wuhan Yuling Biotechnology Co., Ltd.) as a template, primers were designed to amplify 3*flag+NLS fragments, and 3*flag+NLS fragments were cloned into MO91-dCas9-AID plasmid by Gibson Assembly method. And the dCas9 N-terminus of the MO91-dCas9-AIDX plasmid, the MO91-dCas9 (3*flag, NLS)-AID plasmid and the MO91-dCas9 (3*flag, NLS)-AIDX plasmid (Fig. 3) were constructed.

Example 4: Establish an effective reporting system indicating the efficiency of AID point mutations

The level of point mutations at the genomic level needs to be detected by a simple and intuitive method. The present invention mainly uses flow analysis techniques to indirectly detect the level of point mutations at the protein level. The human insertion insertion stop codon (TAG) in the EGFP gene, EGFP could not be expressed normally. When the fusion protein of this example acts on the stop codon in the EGFP gene, the stop codon is mutated and the EGFP gene mutation is normally expressed. Therefore, the higher the level of EGFP expression, the higher the efficiency of point mutations.

In this example, the EGFP gene containing the stop codon (sequence shown in Figure 4) was inserted into the MO405-thy1.1 plasmid (Addgene), and MSCV initiated gene expression. Infecting 293T with this plasmid, specifically including:

1. Laying 293T, the cell density reaches 90% when intoxicating;

2. After 24 hours, the poisoning method is the same as the transfection method;

3. Change the liquid 24 hours after the poisoning;

4, 24 hours after the poisoning, the first time to take the poison, add polyglycolamine 1ug / ml, 800g, 90min, 6-8h after the change;

5, 48h after the poisoning, the second time to take the poison, add polyglycolamine 1ug / ml, 800g, 90min, 6-8h after the change;

6. After the cells have grown to a sufficient number, flow staining (PE-thy1.1), sorting th1.1 positive cells as reporter cells. The result is shown in Figure 6. A schematic diagram of the reported cells is shown in Figure 5.

Example 5: Preparation of sgRNA

1. Find a 20 bp target sequence. If the starting base of the 20 bp target sequence is not G, a G is added to its 5' end to enable efficient transcription by the RNA polymerase III U6 promoter. It should be noted that the target sequence cannot contain a recognition site for XhoI or NheI.

2. The sgRNA was cloned into pLX (Addgene 50662) to obtain pLX sgRNA. The following four primers are required, wherein R1 and F2 are sgRNA specific:

F1: AAACTCGAGTGTACAAAAAAGCAGGCTTTAAAG (SEQ ID NO: 10)

_{R1: rc (GN 19) GGTGTTTCGTCCTTTCC} (SEQ ID NO: 11)

F2: GN ₁₉ GTTTTAGAGCTAGAAATAGCAA (SEQ ID NO: 12)

R2: AAAGCTAGCTAATGCCAACTTTGTACAAGAAAGCTG (SEQ ID NO: 13)

Among them, GN ₁₉ = new target sequence, rc (GN ₁₉ ) = reverse complement of the new target sequence.

3. Amplify pLX sgRNA using F1+R1 and F2+R2, respectively;

4. The product obtained by two times of gel purification is combined and used for F1+R2 for the third PCR;

5. The product obtained by PCR using the NheI and XhoI digestion step 4;

6. Ligation and transformation to prepare an expression vector for sgRNA.

The base sequences of the target binding regions of the four sgRNAs are as follows:

Example 6: CRISPR-Cas9 improves AID point mutation efficiency

2000试剂稀释在

培养基中，分别将MO91-dCas9(3*flag,NLS)-AID质粒或MO91-dCas9(3*flag,NLS)-AIDX质粒稀释在

培养基中，然后将稀释的质粒分别加到稀释的

2000试剂中(1：1)孵育30分钟。之后将该质粒DNA-脂质体复合物和实施例5制备的针对EGFP终止密码子的4个sgRNA共同转染实施例4所构建的报告细胞。作为对照，仅用所述质粒DNA-脂质体复合物转染实施例4所构建的报告细胞。加嘌呤霉素2ug/ml和杀稻瘟菌素20ug/ml进行培育，筛选3d，分别在转染后第4天和第7天流式分析EGFP表达水平。Transfection was carried out by culturing the reporter cells constructed in Example 4 to a confluency of 70-90%. When transfected, first prepare a plasmid DNA-liposome complex, including four times the amount

2000 reagent diluted in

In the medium, dilute the MO91-dCas9 (3*flag, NLS)-AID plasmid or the MO91-dCas9 (3*flag, NLS)-AIDX plasmid, respectively.

In the medium, the diluted plasmid is then separately added to the diluted

Incubate for 30 minutes in 2000 reagents (1:1). The plasmid DNA-liposome complex and the 4 sgRNAs against the EGFP stop codon prepared in Example 5 were then co-transfected with the reporter cells constructed in Example 4. As a control, the reporter cells constructed in Example 4 were transfected only with the plasmid DNA-liposome complex. Incubation was carried out by adding 2 ug/ml of puromycin and 20 ug/ml of blasticidin, and screening for 3d, and analyzing the expression level of EGFP on the 4th and 7th day after transfection, respectively.

As a result, as shown in Fig. 7, the %EGFP+ of AID and AIDX were 0.14% and 0.30%, respectively. The %EGFP+ of dCas9-AID+sgRNA and dCas9-AIDX+sgRNA were 2.14% and 4.36%, respectively.

The results showed that the fusion of AID or AIDX with dCas9, under the guidance of sgRNA, would make the AID point mutation function in the specific part of the AID under the targeting effect of sgRNA, and increase its concentration and increase its mutation. effectiveness.

Example 7: CRISPR-Cas9 improves AID point mutation efficiency and optimization

The expression vector of sgRNA and dCas9-AID was co-transduced in the reporter cells constructed in Example 4 in the same manner as in Example 6. The sgRNA was divided into two groups, one of which was a control sgRNA against AAVS1, and the target binding regions thereof were as follows: GATTCCCAGGGCCGGTTAATG (SEQ ID NO: 18); GTCCCCTCCACCCCACAGTG (SEQ ID NO: 19); and GGGGCCACTAGGGACAGGAT (SEQ ID NO: 20). The other group is the sgRNA group against EGFP (SEQ ID NOS: 14-17). At the same time, the control group was set to single-turn AID in the reporter cells. An expression vector for the control sgRNA was constructed as described in Example 5.

On the 8th day after transfection, FACS was measured, and the EGFP%+ of the AID group was only 0.13%, while the EGFP%+ of the dCas9-AID+sgRNA group was 2.1% (Fig. 8, A), and the EGFP%+ had a 16-fold increase. To further optimize the efficiency of the dCas9-AID system, dCas9 was fused to different AID mutants: AID-FL (full length), AID-CD (catalytic domain only), P182X (from amino acid residue 183) Short), R186X (truncated from amino acid residue at position 187), R190X (truncated from amino acid residue at position 191). Each dCas9-AID expression vector and sgRNA were co-transformed in the reporter cells, with dCas9-R186X being the most efficient (Figure 8, B and C). The experiments of Examples 8-13 were therefore carried out using dCas9-R186X, and in these examples, dCas9-R186X was simply referred to as dCas9-AIDX.

In order to prove that the DCas9-AID system is indeed fused with dCas9, the entire system has a base substitution function, and a functional mutant of Cas9, dCas9, dCas9-AIDX is separately co-transferred in the reporter cells [R186X(E58Q) ], dCas9-AIDX and sgRNA, only the dcas9-AIDX and sgRNA groups have EGFP%+, while the other groups are all 0 (Fig. 8, C). It also proves that it is indeed the fusion of AID and dCas9 that the entire system has a base replacement function.

Example 8: CRISPR-Cas9 limits AID point mutation to sgRNA targeting sites

To investigate whether CRISPR-Cas9 can limit the AID point mutation function to the sgRNA targeting site, use the genomic DNA of the reporter system constructed in Example 4 as a template, PCR the EGFP containing the stop codon, construct a library, and use cMyc as For the control gene, Miseq sequencing was performed. The result is shown in Figure 9. According to the sequencing results of the reporter cells, although the Miseq has high sequencing throughput and filters out low-quality readings, it still has a test. The order base mutation frequency was 0.25% for EGFP and 0.15% for cMyc. However, even with basal level interference, the frequency of EGFP gene mutations in the dCas9-AIDX+sgRNA group was significantly higher than that in the AIDX group, which also proved that CRISPR-Cas9 increased the efficiency of AID point mutation. Moreover, these high frequency mutation sites are mainly concentrated in the targeting site of sgRNA, and almost no point mutation occurs in the cMyc gene. After demonstrating that dCas9 is fused to AID, sgRNA targets dCas9-AID to the targeting site of sgRNA, so that AID will only act on the target site of sgRNA, producing point mutations without causing very much other gene loci. Great change; and can greatly increase the frequency of point mutations.

Example 9: dCas9-AIDX randomly mutates C and G bases to three other bases

AIDX itself will mutate C to T and G to A. After the fusion of dCas9 and AIDX, the mutation direction of C and G became more uniform compared with the AIDX group.

At the same time, the role of the AID itself is dependent on the WRCY of the hotspot motif (W stands for A/T, R stands for A/C, Y stands for C/T), and the most preferred motif is AGCT. After the fusion of dCas9 and AIDX, the preference of this motif will obviously disappear. Therefore, the inventors have proposed a hypothesis that under normal circumstances, AID will deamination of cytosine to form uracil, which is repaired by DNA replication, and this ug mismatch is retained, and mutations of C to T and G to A occur, and The U base can be excised by base excision repair, and then four bases are inserted. Therefore, the fusion of dCas9 and AID is likely to inhibit the DNA replication pathway, promote base excision repair, and make the mutation direction more uniform (Fig. 10, b).

In addition, statistical analysis of the Miseq data showed that the type of point mutations caused by the AIDX and dCas9-AIDX+sgRNA groups on EGFP was basically consistent with the report. The C and G base mutations accounted for the majority, and A and T accounted for a small proportion. And the main mutation of G to T, C mutation to A. However, in the dCas9-AIDX group, the ratio of G mutation to T and C increased, and the ratio of C mutation to G or A increased. Therefore, dCas9-AIDX can produce a more uniform type of mutation (Fig. 10, a).

Example 10: UGI increases the base substitution frequency of the dCas9-AIDX system, reveals the trajectory of dCas9-AIDX on the gene, and makes the base mutation direction more singular.

UGI is an inhibitor of UNG, a phage protein that protects its genome from host UNG when it invades E. coli (Fig. 11, a). Three plasmids were co-transduced in the reporter cells, expressing dCas9-AIDX, a single sgRNA (target binding region GCCTCGAACTTCACCTCGGCG, SEQ ID NO: 16) and UGI (protein sequence: UniProtKB-P14739) to enhance a single sgRNA throughout the system. Mutation efficiency. The results showed a 10-fold increase in the highest point mutation efficiency (Fig. 11, b).

In addition, after the addition of UGI, the mutation direction of the whole system is more single, C to T, G to A. Same The trajectory of the action of dCas9-AIDX and the frequency of mutations caused by the whole system before and after the PAM sequence were counted. Figure 11 (c) is a statistic based on data for 4 sgRNAs designed for the EGFP site. N is the first base in NGG in the PAM sequence. The upstream is -, the downstream is +, the statistical results of the two sets of data are consistent, both of which cause mutations in the upstream 20 bp of the PAM, that is, in the prototype interval region, and the highest point of mutation is in the -12/-13 position of the PAM. UGI can increase the overall mutation frequency of AID, but it will increase the proportion of base substitutions and reduce the conversion ratio (Fig. 11, d).

Example 11: dCas9-AIDX can act not only on exogenous genes, but also on endogenous genes. The above experiments were all carried out in the reporter cells. In this example, the endogenous gene AAVS1 was selected as the target site, and three sgRNAs (SEQ ID NO: 18-20) were designed, and dCas9-AID and AAVS1 were co-transduced in 293T. The vector of three sgRNAs (as described in Example 7).

The result is shown in FIG. The dCas9-AID system can also generate base substitutions to the endogenous gene AAVS1, and this mutation is also concentrated in the sgRNA target site.

Example 12: Application of dCas9-AIDX to Gleevec resistance screening of K562BCR-ABL gene

K562 is a leukemia cell line derived from human chronic myeloid leukemia. There is a chromosome in this cell called the ph chromosome. The chromosome is transposed by the long arms of chromosomes 9 and 22. The ABL gene on chromosome 9 contains a tyrosine kinase active center, which is in a low activity state under normal conditions, and has a high activity when translocated into the BCR locus. Will cause a series of signal transduction, leading to cancer, so BCR-ABL is a proto-oncogene, the commonly used drug is Gleevec (Gleevec, the active ingredient is imatinib mesylate), the main mechanism of action is gleevec ATP can be competitively bound to ABL, resulting in a low activity of the ABL gene. However, in patient samples, point mutations, such as T315I, were found in the tyrosine kinase activity domain, causing the domain to lose the ability to bind to gleevec, resulting in gleevec resistance. In addition, base substitutions at other sites can also cause Gleevec resistance. The dCas9-AIDX system can be used to screen for Gleevec resistance sites and specific mutation types as a basis for designing next generation inhibitors.

First, in order to obtain K562 cells stably expressing dCas9-AIDX, we used the plasmid of interest MSCV-dCas9-AID-P182X-IRES-Thy1.1 to co-transfect 293T cells with the viral packaging plasmid pcl-10A1. 1×10 ⁶ 293T cells were plated in a well of a six-well plate 12-24 hours in advance, and cultured overnight with 2 ml of DMEM without anti-10% FBS, and the next day when the cells were grown to 80% density, 3 ug of the target plasmid was transfected. 1ug virus packaging plasmid, and 10ul transfection reagent LIPO2000. After 24 hours of transfection, the cells were cultured with 2 ml of anti-seeding solution, and virus was collected at 48 hours and 72 hours, respectively. Good collection 1000rpm virus immediate cell debris was removed by centrifugation for 5 minutes, the supernatant was added 2ul 10mg / ml Polybrene of infection 1x10 ⁵ K562 cells, 37 ℃, 900g speed rejection board 90 minutes. The cells were centrifuged 4 hours after infection, and the pellet was cultured with an anti-seeding solution. After two days of continuous infection, K562 cells need to be cultured for another two days. Flow cytometry is used to label cells expressing Thy1.1 surface molecules as PE ⁺ (antibody 1:200 dilution) and using single cell sorting technique. Two 96-well plates of PE-Thy1.1 ⁺ K562 single cells were obtained. After two weeks of culture, RNA of the cell population produced by each single cell clone was collected and subjected to RT-qPCR experiments. The cell line with the highest expression of dCas9-AIDX was used for subsequent screening of Gleevec resistance sites and mutation types.

At the same time, in order to screen out the Gleevec resistance site, we designed the sgRNA for the genomic region of Exon6, the sixth exon of ABL gene. A total of 16 sgRNAs were designed (target sequence sequences are shown in SEQ ID NOs: 49-64, respectively), of which 6 are targeted to intron regions adjacent to exon Exon6, and 10 are directly targeted to the Exon6 region. And covered 83% of the exon sequences. Since the mutation of T315I has been recognized as one of the most important mutations causing Gleevec resistance, one and only one of the sgRNAs we designed can cover the T315I mutation site (944C) and can be used as a positive control. At the same time, we designed three sgRNAs as negative controls for the genomic sequence of the AAVS1 gene unrelated to Gleevec resistance (target sequence sequences are shown in SEQ ID NOs: 18-20). These sgRNA sequences were all chemically synthesized, digested with BamH1 and HindIII, and finally cloned into the pSUPER-sgRNA vector carrying the H1 promoter. We used a phenol chloroform-ethanol sedimentation method to sediment an equal amount of 16 Exon6 sgRNA plasmids or 3 AAVS1 sgRNA plasmids so that the final concentration of the mixed plasmid was above 1.5 ug/ul. Subsequently, the K562 cell line stably expressing dCas9-AIDX was electroporated with ABL-Exon6 and AAVS1 mixed sgRNA libraries, respectively, and the instrument was used by the American Life Technology company Neoelectric transducer. 12-24 hours before electroporation, K562 cells were cultured in IMDM medium without anti-10% FBS. On the day of electroporation, two 1.2× ^{10 6} K562 cells were transfected with 8ug respectively on the condition of 1000V voltage, single pulse and 50ms shock time. Equally mixed ABL-Exon6 or AAVS1 sgRNA. Since the pSUPER-sgRNA plasmid vector carries the puromycin resistance gene, cells expressing sgRNA were screened 24 hours after transfection by adding 2 ug/ml puromycin. After treatment with puromycin for 48 hours, K562 cells continued to expand. On day 6 post-transfection, 2x10 ⁵ of cellular DNA and RNA were collected for high-throughput sequencing and used as an Input control. The remaining cells were split into two portions and treated with 10 uM Gleevec drug or with an equal volume of DMSO, respectively. Once every three days Ficoll, remove dead cells, until the cell number far lower than when 2x10 ^4. Under Gleevec drug treatment, control cells transfected with AAVS1sgRNA died almost completely in about 7-10 days, while cells transfected into ABL-Exon6sgRNA continued to proliferate. The cells of the experimental group proliferated to the order of 10 ^{7 on} the 36th to 40th day after transfection (Fig. 14, b). DNA and RNA from Gleevec-treated and DMSO-treated cells were simultaneously collected for high-throughput sequencing analysis. Sequencing results showed that there was a T315I mutation in 30% of the cells, and this mutation was a known drug-resistant mutation found in patients. In addition, several unreported point mutations were also found (Fig. 14 , c and d).

Example 13: Application of dCas9-AIDX to increase the affinity and specificity of antibodies in vitro

Antibodies can specifically recognize antigens as drug proteins for the treatment of various diseases. The affinity of an antibody is directly proportional to the somatic mutations produced in the germinal center in vivo. In general, high affinity antibodies have multiple somatic high frequency mutations. Therefore, dCas9-AIDX can be used to mutate antibody genes to screen for antibodies with stronger affinity or other characteristics (such as better specificity, etc.).

The protocol is as follows. The antibody molecule is stably expressed on the surface of 293T cells, and then sgRNA is designed for the antibody gene, and 293T cells are simultaneously transfected with dCas9-AIDX, and then the cell surface is stained. The stronger the stained cells, the mutant antibody molecules have Stronger affinity.

The present embodiment employs from Invitrogen stably expressing Flp-In ^TM -293 lacZ-ZeocinTM a cell fusion locus. First, a low-affinity cDNA sequence of mouse IgG1 antibody (K _D = 2.78E-09M) against chicken egg lysozyme (HEL) was synthesized, and the coding sequence of the H2Kk protein transmembrane region sequence was ligated to add H2Kk at the end of the antibody. The transmembrane region sequence of the protein was cloned into a cDNA sequence such as pcDNA5/FRT/GOI vector (Life Science Technology, USA). The vector into Flp-InMM-293 cells, using the Flp-In ^TM system Flp-In ^TM -293 cells contained the coding sequence containing the IgG1 Flp recombinase integrated into the target site by Flp recombinase lacZ- ZeocinTM fusion locus. Cells that were not successfully integrated were able to express anti-Zeocin proteins; after successful integration, anti-Zeocin proteins could not be expressed due to the lack of the initiation codon ATG, but were able to express hygromycin-resistant proteins. Therefore, hygromycin antibiotics were used to screen for IgG1-synthesized 293 cells in which only one copy of the anti-HEL-IgG1 gene was expressed per cell.

Next, 16 suitable PAM sequences were selected for each of the 3 CDRs of the IgG1 heavy and light chain, respectively, to design the sgRNA (SEQ ID NO: 73-88) shown below, such that the CDR of each heavy or light chain has at least 2 sgRNA overlays:

IgH

IgL

The sgRNA sequence was then cloned into the pSUPER-puro plasmid vector (Addgene). The MO91-dCas9 (3*flag, NLS)-AIDX plasmid constructed in Example 3 and the sgRNA library (ie, 16 sgRNAs were mixed together in equal amounts) or the sgRNA of the control gene AAVS1 were co-transfected into the IgG1-expressing IgG1 obtained previously. In 293 cells, after penicillin and blasticidin antibiotics were screened, PE anti-mouse IgG and Alex647-HEL were stained on the 7th day after transfection, and then flow sorted to sort out IgG strength. Cells that are unchanged and bind to the HEL antigen. After culture and proliferation, the high-throughput sequencing analysis of the mutations on the DNA was first performed, and the results were basically consistent with the mutations of the ABL gene or the GFP gene (Fig. 15). dCas9-AIDX induced base mutations in the anti-HEL IgG1 variable region and repeatedly induced base mutations in the IgG1 CDRs (Fig. 16).

Thereafter, the mutant cells were detected by flow cytometry using PE anti-mouse IgG1 and 647-HEL surface staining, and it was found that a small group of cells had unchanged IgG1 expression and increased binding to HEL. This group of cells was then subjected to flow sorting, and after sorting and amplification, compared with the cells before the mutation, it was found that the affinity of the mutant antibody to HEL was enhanced more than 10 times (Fig. 17).

Then, an appropriate amount of cells were extracted for genomic DNA for sequencing, and the main reason for the increase in affinity was that the glycine at position 52 of the light chain was mutated to aspartic acid (the base was changed from GGT to GAT, Fig. 15).

Example 14: Preparation of other fusion proteins

1, the construction of the plasmid

(1) synthesizing XTEN linker sequences by gene synthesis;

(2) the MO91-dCas9-AIDX plasmid obtained in the construction of Example 2 was digested with restriction endonuclease, and the vector, AIDX fragment and dCas9 fragment were recovered;

(3) ligating the AIDX fragment, dCas9 fragment and XTEN linker sequence into the MO91 vector, and then transforming the ligation product into Stbl3 competent cells;

(4) The positive clones were selected, the plasmid was extracted and sequenced, and the construction of the MO91-dCas9-XTEN-AIDX plasmid was completed.

The plasmids MO91-AIDX-XTEN-dCas9, MO91-dCas9-XTEN-AIDX (K10E T82I E156G) and MO91-nCas9-AIDX can be constructed by referring to the above steps and the methods of Examples 1 and 2.

When a 3*flag and/or NLS fragment needs to be cloned, the 3*flag and/or NLS fragment can be cloned into the above plasmid by the method of Example 3 to obtain SEQ ID NO: 66, 68, 70 and 72, respectively. A plasmid for the fusion protein shown. The AIDX in these fusion proteins is an AID fragment or a mutant thereof truncated from amino acid residue 183.

2. Expression and purification of recombinant protein

(1) constructing plasmid pET-nCas9-AIDX-6His according to a conventional method, and then transforming Escherichia coli BL21STAR-competent cells with the plasmid;

(2) The resulting expression strain was grown overnight in LB medium containing 100 μg/ml kanamycin at 37 °C. The cells were diluted 1:100 into 2xYT medium and grown at 37 °C to an OD600 = -0.6. The culture was cooled to 4 ° C in 2 hours, IPTG 0.5 mM was added, and the protein expression was induced for ~16 h;

(3) The cells were collected by centrifugation at 4000 g for 15 minutes and resuspended in lysis buffer;

(4) The cells were lysed with a cell disrupting agent (Union) at 800 bar for 5 minutes, and the lysate supernatant was separated by centrifugation for 15 minutes after centrifugation;

(5) The lysate was incubated with Ni-NTA (1 ml of slurry/L bacteria) (DP101, TransGen) for 1 hour at 4 ° C to capture the His-tagged fusion protein; the resin was transferred to the column and washed with cold Buffer (wide extent of color change not observed with Coomassie G250);

(6) His-tagged fusion protein was eluted in elution buffer and concentrated to a total volume of 1 ml by ultrafiltration (Amicon-Millipore, 100 kDa molecular weight cut-off);

(7) The protein was diluted to 20 ml in buffer A and loaded onto a Hi-Trap SP column (29051324, GE Healthcare) and eluted with a gradient of 100 mM-1 M NaCl;

(8) The eluted fraction containing nCas9-AIDX was concentrated to about 1 ml and passed through a Superdex 20010/300 GL column (17517501, GE Healthcare);

(9) The eluted protein was concentrated to about 3 mg/ml, rapidly frozen in liquid nitrogen and stored at -80 °C.

An electropherogram of the induction of nCas9-AIDX expression in bacteria is shown in Figure 18.

3. Functional testing of different fusion proteins

The function of the different fusion proteins of this example was tested in the same manner as in Example 10. The result is shown in Figure 19-21.

Claims (10)

A fusion protein, characterized in that the fusion protein contains a cytosine deaminase and a nuclease activity loss, retains a secase that understands the activity of a chymase, or is deficient in cytosine deaminase and nuclease activity, retaining understanding Enzymatically active Cas enzyme formation. The fusion protein according to claim 1, wherein The citase activity of the Cas enzyme is completely deleted, has no DNA double-strand break ability, or is partially deleted, and has only DNA single-strand break ability; and/or The Cas enzyme is selected from the group consisting of: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2 , Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2 , Csf3, Csf4, a homolog thereof or a modified form thereof; preferably, the Cas enzyme is a Cas9 enzyme, preferably selected from the group consisting of: Cas9 from S. pyogenes, Cas9 from S. aureus, and from Streptococcus thermophilus Cas9; and / or The cytosine deaminase is a full-length cytosine deaminase, or a fragment or mutant thereof that retains an enzyme activity, wherein the fragment includes at least an NLS domain, a catalytic domain, and an APOBEC-like cytosine deaminase. Domain; and/or The fusion protein further comprises one or more of the following sequences: a linker, a nuclear localization sequence, and, in order to construct a fusion protein, promote expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate recombinant protein. An amino acid residue or amino acid sequence introduced by purification. The fusion protein according to claim 2, wherein The Cas enzyme is a Cas9 enzyme, and the two endonuclease catalytic domains of the enzyme are mutated in the catalytic domain RuvC1 and/or HNH, resulting in loss of the enzyme nuclease activity and retention of the chymase activity; preferably, the Cas9 enzyme Both RuvC1 and HNH are mutated, resulting in deletion of the nuclease activity of the enzyme, retaining the understanding of the chymase activity; more preferably, the 10th amino acid asparagine of the Cas9 enzyme is mutated to alanine or other amino acid, amino acid 841 The histidine is mutated to alanine or other amino acid; more preferably, the amino acid sequence of the Cas9 enzyme is as shown in SEQ ID NO: 2, 42-1452, or as SEQ ID NO: 72, 42-1419. Base; and/or The fragment of the cytosine deaminase comprises at least amino acid residues 9-182 of the cytosine deaminase, for example, comprising at least amino acids 1-182; preferably, the fragment is from amino acid 1-182 Base composition, Or consisting of amino acid residues 1-186 or consisting of amino acid residues 1-190; alternatively, the amino acid sequence of the cytosine deaminase is represented by amino acids 1457-1654 of SEQ ID NO: 2, The fragment comprises at least amino acid residues 1465-1638 of SEQ ID NO: 2, for example comprising at least amino acid residues 1457-1638 of SEQ ID NO: 2, preferably the fragment is represented by SEQ ID NO: 2 Amino acid residue 1457-1638, amino acid residue 1457-1642 of SEQ ID NO: 2, or amino acid residue 1457-1646 of SEQ ID NO: 2; the mutant has positions 10, 82 and 156 In place of the mutation, preferably, the substitution mutations are K10E, T82I and E156G, more preferably, the mutant contains the amino acid sequence as shown in 1447-1629 of SEQ ID NO: 68, or by SEQ ID NO: 68 The amino acid residue shown in positions 1447-1629. The fusion protein according to claim 1, wherein the fusion protein has the amino acid sequence shown as SEQ ID NO: 2, 4, 66, 68, 70 or 72, or SEQ ID NO: 2, 26- Amino acid at position 1654, or as shown in positions 26-1638 of SEQ ID NO: 4, or as shown in amino acids 26 to 1629 of SEQ ID NO: 68, or amino acids 26 to 1629 of SEQ ID NO: Shown or as shown in amino acids 26-1638 of SEQ ID NO:72. A polynucleotide sequence selected from the group consisting of: (1) A polynucleotide sequence encoding the fusion protein of any one of claims 1 to 4; (2) (1) The complementary sequence of the sequence. A nucleic acid construct comprising the polynucleotide sequence of claim 5; preferably, the nucleic acid construct is an expression vector for expression of a fusion protein described herein in a host cell. A host cell comprising or expressing the fusion protein of claims 1-4, or the polynucleotide sequence of claim 5 or the nucleic acid construct of claim 6. A method of producing a point mutation in a cell, the method comprising the step of expressing the fusion protein of any one of claims 1 to 4 and the sgRNA in the cell, wherein the sgRNA comprises a target binding a region and a Cas protein recognition region capable of specifically binding to a nucleic acid sequence to be mutated, the Cas protein recognition region being capable of being recognized and bound by a Cas enzyme in the fusion protein. The method according to claim 8, wherein said method comprises transferring said fusion protein or an expression vector thereof and sgRNA or an expression vector thereof into said cell, and then screening to obtain a desired mutant nucleic acid The steps of the sequence; and Optionally, the target binding region of the sgRNA specifically binds to a template strand of the nucleic acid sequence to be mutated, and the contralateral region of the sgRNA binding region on the template strand is immediately adjacent to the prosthetic sequence adjacent motif recognized by the Cas protein, or Bases separated by 10 or less; and Optionally, the nucleic acid sequence to be mutated encodes a functional protein, preferably, the functional protein is selected from the group consisting of: antibodies, enzymes, lipoproteins, hormone proteins, transport and storage proteins, motor proteins, receptor proteins, and membranes protein. A kit comprising the fusion protein of any one of claims 1 to 4, the polynucleotide sequence of claim 5, or the nucleic acid construct of claim 6.

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