CN114525293A - Novel CRISPR-Cas9 inhibitor protein and method for applying CRISPR-Cas9 inhibitor protein in chemical controllable gene editing through modification - Google Patents

Abstract

The invention discloses a novel CRISPR-Cas9 arrestin and a method for applying the alteration of the arrestin to chemically controllable gene editing. The inventor discovers nine novel II-A type Acr (AcrIIA24-32) protein families from mobile elements of Streptococcus, and the Acr proteins can effectively inhibit II-A type Cas9 ortholog proteins in bacteria and eukaryotic cells. In addition, acriia25.1 and acriia32.1 can inhibit both DNA binding and DNA cleavage activities of SpyCas9, showing a novel and unique inhibition mechanism. Based on this, the inventors developed a novel chemically inducible iAcr system, and established an efficient method of chemically controllable gene editing. The invention has important application value.

Description

Novel CRISPR-Cas9 inhibitor protein and method for applying CRISPR-Cas9 inhibitor protein in chemical controllable gene editing through modification

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a novel CRISPR-Cas9 arrestin and a method for applying the alteration of the novel CRISPR-Cas9 arrestin to chemically controllable gene editing.

Background

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) thereof constitute a prokaryotic adaptive immune defense system against invasive genetic elements including phages and plasmids. The CRISPR-Cas mediated defense process can be divided into three phases: (1) an adaptation stage: when the bacteriophage invades, a part of sequence information of the bacteriophage is inserted into a CRISPR locus of bacteria as a new spacer to form immunological memory; (2) and (3) an expression stage: systematically expressing the Cas gene and processing to form mature CRISPRRNA(crRNA); (3) and (3) interference stage: the Cas protein recognizes and destroys the target nucleic acid under the guidance of crRNA, forming an immune defense. At present, based on phylogenetic trees and action mechanisms, CRISPR-Cas systems are divided into 2 types (Class 1 and Class 2) and 6 types. The Class 1 system (including I, III and type IV) uses multi-subunit Cas protein complexes to target foreign nucleic acids; while the Class 2 system (including types II, V and VI) relies on a single effector protein (e.g., Cas9) to achieve interference and is therefore widely used. By far, various subtypes of Class 2CRISPR-Cas system have been identified and developed as powerful gene editing tools, mainly including CRISPR-Cas9, Cas12a (Cpf1), Cas13a (C2C2), and the like. They play an increasingly important role in the fields of scientific research, agriculture, animal husbandry, clinical detection and treatment and the like. Among them, Cas9(SpyCas9) from streptococcus pyogenes (streptococcus pyogenes) is the most widely used Cas effector and has been developed as a series of powerful gene editing tools.

However, despite the great development of CRISPR gene editing technology in recent years, it still faces an unsolved off-target problem, which hinders the application of CRISPR gene editing technology. To address this issue, some studies have further reduced off-target effects by engineering Cas proteins or guide RNAs to increase the specificity of targeted recognition, or by controlling the expression of Cas protein or guide RNA abundance versus time. In recent years, a series of proteins called anti-CRISPR (Acr) have been found in prophages or phages, which can inhibit the activity of various CRISPR-Cas systems, and some Acr proteins (such as AcrIIA4) have been shown to reduce off-target effects in cells by inhibiting Cas9 activity. These characteristics make Acr useful as a potential "switch" tool for regulating CRISPR-based applications, and therefore Acr exploration and molecular mechanism research are of great significance in the field of gene editing.

The Acr protein is a strategy developed by phage to antagonize the CRISPR-Cas system of prokaryotes, i.e. to inactivate the CRISPR-Cas system of prokaryotes by encoding a protein (i.e. Acr protein). Acr proteins have been found to be widely distributed among bacteriophages, prophages, and invasive genetic elements, are generally small in molecular weight, about 50-200 amino acids, and exhibit a high degree of sequence diversity. To date, 88 different types of Acr protein families have been identified that are capable of broadly inhibiting type I, II, III, V and VI CRISPR-Cas systems. Since Acr proteins are very diverse and exhibit a high degree of sequence diversity, it is difficult to search for Acr proteins by conventional methods of sequence homology alignment. Scientists have sought and confirmed Acr proteins using a variety of bioinformatic and biochemical approaches, including bioinformatic approaches based on Guilt-by-association, Self-targeting, Machine-learning, and biochemical approaches by phase-first and High-throughput. Notably, 23 AcrIIA families have been discovered by a variety of methods. Currently, the molecular mechanisms of only a small fraction of AcrIIA proteins are elucidated in detail. Studies have shown that these AcrIIA proteins mainly inhibit the third stage of CRISPR-Cas9 immunity, the interference stage, and can be roughly divided into three strategies: (1) interfering with the loading of Cas and crRNA, such as AcrIIA 15; (2) preventing binding of the Cas effector complex to the targeting nucleic acid, such as AcrIIA 4; (3) preventing cleavage of the target nucleic acid by Cas, such as AcrIIA 14. Considering that Acr protein can inhibit the CRISPR-Cas9 system through different strategies and develop a corresponding gene editing regulation tool, the research of the Acr molecular mechanism becomes a research hotspot in the field of gene editing.

Some Acr proteins of these CRISPR-Cas systems have been identified so far, and scientists believe that still extensive Acr proteins should be present and not found. This not only hinders us from understanding the evolutionary competition mode between phage and host, but also hinders the development of novel Acr-based gene editing methods. Especially considering the off-target effect of Cas9, it would be particularly attractive to develop Acr protein as an effective gene editing tool to regulate the activity of Cas9 to solve the safety problem of Cas9 application. However, in the field of gene editing, the available Acr proteins and strategies to utilize Acr to modulate Cas9 activity remain limited. Therefore, the method for discovering the novel CRISPR-Cas9 arrestin and modifying the Acr protein to be applied to controllable gene editing has important significance for the field of gene editing.

Disclosure of Invention

The invention aims to discover a novel CRISPR-Cas9 inhibitor protein and establish a controllable gene editing method according to the novel CRISPR-Cas9 inhibitor protein.

The invention firstly protects the application of AcrIIA protein in inhibiting Cas9 gene editing, regulation and/or imaging in bacteria, cells or eukaryotes;

the AcrIIA protein can be AcrIIA24 protein, AcrIIA25 protein, AcrIIA25.1 protein, AcrIIA26 protein, AcrIIA27 protein, AcrIIA28 protein, AcrIIA29 protein, AcrIIA30 protein, AcrIIA31 protein, AcrIIA31.1 protein, AcrIIA32 protein or AcrIIA32.1;

the AcrIIA24 protein may be a1) or a2) or a3) or a4) as follows:

a1) the amino acid sequence is protein shown as SEQ ID NO. 1;

a2) 1, the N end or/and the C end of the protein shown in SEQ ID NO. 1 is connected with a label to obtain fusion protein;

a3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by a1) or a2) and has the function of inhibiting the activity of the Cas9 protein;

a4) 1, and has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO, and has the function of inhibiting the activity of the Cas9 protein;

the AcrIIA25 protein may be b1) or b2) or b3) or b4) as follows:

b1) the amino acid sequence is protein shown as SEQ ID NO. 2;

b2) 2, the N end or/and the C end of the protein shown in SEQ ID NO. 2 is connected with a label to obtain fusion protein;

b3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by b1) or b2) and has the function of inhibiting the activity of the Cas9 protein;

b4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 2 and has the function of inhibiting the activity of Cas9 protein;

the acriia25.1 protein may be c1) or c2) or c3) or c4) as follows:

c1) the amino acid sequence is protein shown as SEQ ID NO. 3;

c2) a fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 3;

c3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by c1) or c2) and has the function of inhibiting the activity of the Cas9 protein;

c4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 3 and has the function of inhibiting the activity of Cas9 protein;

the AcrIIA26 protein may be d1) or d2) or d3) or d4) as follows:

d1) the amino acid sequence is protein shown as SEQ ID NO. 4;

d2) a fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 4;

d3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by d1) or d2) and has the function of inhibiting the activity of the Cas9 protein;

d4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 4 and has the function of inhibiting the activity of Cas9 protein;

the AcrIIA27 protein may be e1) or e2) or e3) or e4) as follows:

e1) the amino acid sequence is the protein shown in SEQ ID NO. 5;

e2) a fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 5;

e3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by e1) or e2) and has the function of inhibiting the activity of the Cas9 protein;

e4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 5 and has the function of inhibiting the activity of Cas9 protein;

the AcrIIA28 protein may be f1) or f2) or f3) or f4) as follows:

f1) the amino acid sequence is the protein shown in SEQ ID NO. 6;

f2) fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 6;

f3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by f1) or f2) and has the function of inhibiting the activity of the Cas9 protein;

f4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 6 and has the function of inhibiting the activity of Cas9 protein;

the AcrIIA29 protein may be g1) or g2) or g3) or g4) as follows:

g1) the amino acid sequence is the protein shown in SEQ ID NO. 7;

g2) fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 7;

g3) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in g1) or g2) and has the function of inhibiting the activity of Cas9 protein;

g4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 7 and has the function of inhibiting the activity of Cas9 protein;

the AcrIIA30 protein may be h1) or h2) or h3) or h4) as follows:

h1) the amino acid sequence is the protein shown in SEQ ID NO. 8;

h2) fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 8;

h3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by h1) or h2) and has the function of inhibiting the activity of the Cas9 protein;

h4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 8 and has the function of inhibiting the activity of Cas9 protein;

the AcrIIA31 protein may be i1) or i2) or i3) or i4) as follows:

i1) the amino acid sequence is protein shown as SEQ ID NO. 9;

i2) fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 9;

i3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in i1) or i2) and has the function of inhibiting the activity of the Cas9 protein;

i4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 9 and has the function of inhibiting the activity of Cas9 protein;

the acriiaa 31.1 protein may be j1) or j2) or j3) or j4) as follows:

j1) the amino acid sequence is protein shown as SEQ ID NO. 10;

j2) 10 in SEQ ID NO, and the N end or/and the C end of the protein is connected with a label to obtain a fusion protein;

j3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in j1) or j2) and has the function of inhibiting the activity of the Cas9 protein;

j4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 10 and has the function of inhibiting the activity of Cas9 protein;

the AcrIIA32 protein can be k1) or k2) or k3) or k4) as follows:

k1) the amino acid sequence is protein shown as SEQ ID NO. 11;

k2) fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 11;

k3) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by k1) or k2) and having a function of inhibiting the activity of the Cas9 protein;

k4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 11 and has the function of inhibiting the activity of Cas9 protein;

the acriiaa 32.1 protein may be l1) or l2) or l3) or l4) as follows:

l1) the amino acid sequence is the protein shown in SEQ ID NO. 12;

l2) fusion protein obtained by attaching labels to the N-terminal or/and C-terminal of the protein shown in SEQ ID NO. 12;

l3) is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in l1) or l2) and has the function of inhibiting the activity of the Cas9 protein;

l4) has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 12 and has the function of inhibiting the activity of the Cas9 protein.

Wherein SEQ ID NO 1 consists of 104 amino acid residues. SEQ ID NO 2 consists of 96 amino acid residues. SEQ ID NO 3 consists of 101 amino acid residues. SEQ ID NO 4 consists of 183 amino acid residues. SEQ ID NO 5 consists of 79 amino acid residues. SEQ ID NO 6 consists of 88 amino acid residues. SEQ ID NO 7 consists of 241 amino acid residues. SEQ ID NO 8 consists of 73 amino acid residues. SEQ ID NO 9 consists of 69 amino acid residues. SEQ ID NO 10 consists of 125 amino acid residues. SEQ ID NO 11 consists of 141 amino acid residues. SEQ ID NO 12 consists of 95 amino acid residues.

In order to facilitate purification of the proteins of a1), b1), c1), d1), e1), f1), g1), h1), i1), j1), k1) or l1), the amino-or carboxy-termini of the proteins shown in SEQ ID NO:1-SEQ ID NO:12, respectively, are attached tags as shown in Table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
FLAG
		8	DYKDDDDK
Strep-tagII				8	WSHPQFEK
	c-myc	10	EQKLISEEDL

The protein of a3), b3), c3), d3), e3), f3), g3), h3), i3), j3), k3) or l3), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is the substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The protein in a3), b3), c3), d3), e3), f3), g3), h3), i3), j3), k3) or l3) can be artificially synthesized, or the coding gene can be synthesized first and then biologically expressed.

The gene encoding the protein of a3), b3), c3), d3), e3), f3), g3), h3), i3), j3), k3) or l3) described above can be obtained by deleting one or several amino acid residues of the codon in the DNA sequence encoding the protein, and/or by performing missense mutation of one or several base pairs, and/or by connecting the coding sequence of the tag shown in table 1 to the 5 'end and/or 3' end thereof.

The term "homology" as used herein refers to sequence similarity to a native amino acid sequence. "homology" includes amino acid sequences having 60% or more, or 65% or more, or 70% or more, or 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more homology with the amino acid sequences shown in SEQ ID NO 1 to SEQ ID NO 12 of the present invention. Homology can be assessed using computer software (sequence alignment software such as BLAST). Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.

The application of any one of the AcrIIA proteins in inhibiting the activity of the Cas9 protein also belongs to the protection scope of the invention.

In the above applications, the inhibition of the activity of the Cas9 protein can be shown as inhibition of the binding of the Cas9 protein to DNA and/or inhibition of the cleavage activity of the Cas9 protein to substrate DNA.

The invention also protects a system of the chemical induction type iAcr; the system adopts the AcrIIA25.1 and/or the AcrIIA32.1 as the protein of the chemically inducible anti-CRISPR.

In the above system, the use of said AcrIIA25.1 as a protein of chemically inducible anti-CRISPR can be specifically carried out by using iA25.1(Intein-AcrIIA25.1(S76)) in Table 4.

In the above system, the AcrIIA32.1 is used as the protein of the chemically inducible anti-CRISPR, and specifically, iA32.1(Intein-AcrIIA32.1(T40)) in Table 4 can be used.

Any of the above systems rely on 4-hydroxy tamoxifen for regulation.

The application of any of the above systems in the implementation of chemically controlled gene editing also falls within the scope of the present invention.

The invention also provides a chemically controllable gene editing method; the method employs any of the above-described chemically inducible iAcr systems for gene editing.

The method may rely on 4-hydroxy tamoxifen for modulation.

The application of any one of the AcrIIA proteins in the preparation of a chemically-induced iAcr system also belongs to the protection scope of the invention.

The application of any one of the AcrIIA proteins in chemically controllable gene editing also belongs to the protection scope of the invention.

In any of the above applications, the AcrIIA protein may specifically be the AcrIIA25.1, the AcrIIA32.1, iA25.1(Intein-AcrIIA25.1(S76)) or iA32.1(Intein-AcrIIA32.1(T40)) in Table 4.

The inventor of the application finds nine novel II-A type Acr proteins (AcrIIA24-32) and three Aca (Acr-assailated) (Aca11-13) protein families which are widely distributed in Streptococcus mobile elements by using a guilt-by-assailation method and widely distributed AcrIIA6 as an initial marker. AcrIIA24-32 was found to be a specific inhibitor of the streptococcal CRISPR-Cas9 system, and can effectively inhibit type II-A Cas9 proteins in bacteria and human cells, including SpyCas9, Streptococcus thermophilus Cas9(CR1/St1Cas9 and CR3/St3Cas 9). Among these Acrs, AcrIIA26, AcrIIA27, AcrIIA30, and AcrIIA31 strongly block Cas9 binding to DNA, while AcrIIA24 blocks Cas 9's DNA cleavage activity. Notably, acriiaa 25.1 and acriiaa 32.1 inhibited DNA binding and DNA cleavage activity of SpyCas9, showing a novel and unique inhibition mechanism. The inventors of the present application have also developed a number of chemically inducible iAcr (indelible anti-CRISPR) systems based on AcrIIA25.1 and AcrIIA32.1, which are fusions of Acr protein and 4-hydroxy tamoxifen (4-HT, 4-hydroxytamoxifen) reactive intein (intein). The iAcr system can effectively control CRISPR-Cas9 mediated gene editing activity after 'translation' in human cells, and shows strong application prospect. The application expands the diversity of II-A type Acr proteins, Acr inhibition mechanisms, and chemical controllable gene editing methods based on Acr. The invention has important application value.

Drawings

FIG. 1 is an identification of nine novel AcrIIA protein families (AcrIIA 24-32). (A) Schematic representation of candidate acr, aca and adjacent genes in streptococcal phage and prophage genomes. The Acr gene is shown numerically in the dark reading frame. The arrows indicate the protein sequence homology relationship (shown as a percentage) between the acr loci. The Aca genes are shown in white reading frame. Other adjacent genes are shown in grey and some known gene structural information (e.g., AP2 DNA binding domain) is annotated according to the NCBI website. Asterisks indicate genes detected in e.coli plasmid interference experiments. (B) Schematic diagram of interference experiment of plasmid and plasmid of Escherichia coli for analyzing Acr activity in Escherichia coli. Cas9, Acr, and pT plasmids carry compatible replicons and resistance genes. AmpR, ampicillin resistance; KanR, kanamycin resistance; ChlR, chloramphenicol resistance. (C) The bar graph shows the efficiency of Acr to inhibit Cas9 orthologs (SpyCas9, St1Cas9, and St3Cas 9). # indicates lower than detection standard. n is 3. Values are shown as mean ± SEM.

FIG. 2 shows the distribution of AcrIIA24-32 homologous proteins. A minimal evolutionary phylogenetic tree of AcrIIA24-32 homologous proteins (A-I), the protein sequences were determined by BLASTp search. The Acr protein analyzed in this study was labeled behind the species.

FIG. 3 shows that AcrIIA24-32 is an inhibitory protein specific to the Streptococcus CRISPR-Cas9 system. A is a plaque determination experiment schematic diagram. It was investigated whether AcrIIA24-32 has broad spectrum inhibitory activity against a variety of II-A, II-B, II-C type Cas9 homologous proteins. Coli harbors plasmids expressing Cas9, sgRNA, and Acr, followed by infection with T4 phage. The T4 phage gene 23 design was targeted by multiple Cas9 homologous proteins (abbreviated by strain name). B plaque assay experiments using 10-fold serial dilutions of T4 phage (black circles) to assess the inhibitory effect of different Acr proteins on different types of Cas9 orthologous proteins, including type II-a (SpyCas9, St1Cas9, St3Cas9 and SaCas9), type II-B (FnCas9) and type II-C (NmeCas9) CRISPR-Cas9 systems.

FIG. 4 shows that AcrIIA24-32 can inhibit type II-A Cas9 homologous protein in vitro. A is a DNA cutting experiment schematic diagram. Cas9RNP complex targets DNA substrates in the presence or absence of Acr. B-D is targeting of linearized plasmid DNA using SpyCas9(B), St3Cas9(C), and St1Cas9(D) RNP complexes in the presence or absence of Acr. The open arrows indicate uncut linearized plasmid DNA. Solid arrows indicate the cleavage products. Gel images represent three independent replicates.

Figure 5 is that AcrIIA24-32 can inhibit Cas 9-mediated gene editing in human cells. A is a schematic T7E1 experiment for testing the inhibitory activity of Acr on Cas9 protein in HEK293T cells. Plasmids encoding Cas9, sgRNA, and Acr were co-transfected into human cells and subsequently analyzed by T7E1 experiments. (B-G) is a representative gel image of T7E1 experiments to show inhibitory activity of Acrs on SpyCas9(B), St3Cas9(D) and St1Cas9 (F). Target sites for the human genes AAVS1 (targeted by SpyCas9) and DYRK1A (targeted by St1Cas9 and St3Cas9) are shown at the top of each gel image, with PAM highlighted by underlining. The subtype and number of Acr are shown above the glue figure; a, AcrIIA; c, AcrIIC. Open arrows indicate T7E1 undigested bands (not edited). Solid arrows indicate T7E1 digested bands (edited). The editing efficiency is shown as "indel (%)" at the bottom of each lane. SpyCas9(C), St3Cas9(E), and St1Cas9(G) mediated gene editing efficiency was quantified in the presence of different Acrs. n-3, values show mean ± SEM.

Figure 6 shows Acrs inactivates Cas9 in human cells using multiple strategies. A is a schematic diagram of a plasmid designed for human telomere localization fluorescence imaging to study the inhibition strategy of Acrs on the II-A type Cas9 orthologous protein. The plasmid encoding Cas9 fluorescent protein, their respective telomere-targeting sgRNA plasmids, and the plasmid of Acr protein (labeled with blue fluorescent protein TagBFP) were co-transfected into the U20S cell line. S _ (d) Cas9- (mCherry)₃Three plasmids, including Spy _ dCas9- (mCherry), were used in this experiment₃、St1_dCas9-(mCherry)₃And St3_ dCas9- (mCherry)₃. B is a sequence targeting human telomeres for Cas9 homologous proteins (Nme, Spy, St1 and St3) in U2OS cells. C is Nme _ dCas9- (sfGFP)₃、Spy_dCas9-(mCherry)₃Representative images of U2OS cells co-transfected with Acr plasmid. The fluorescent channels are shown at the top of the figure, and the different Acr proteins are shown to the right of each row. The scale bar represents 10 μm. D is for Spy _ dCas9- (mCherry) in the presence of a different Acr protein₃The cells forming telomeres were quantified. The method is carried out by having Nme _ dCas9- (sfGFP)₃And Spy _ dCas9- (mCherry)₃Percentage of co-localized telomeric imaging cells. n-the number of cells scored under each condition. E-H for transfection St3_ dCas9- (mCherry)₃(E) Or St1_ dCas9- (mCherry)₃(G) And Nme _ dCas9- (sfGFP)₃And representative images of U2OS cells after different Acr plasmids. The scale bar represents 10 μm. Quantitation of St3_ dCas9- (mCherry) under each condition using the same method as in D₃(F) And St1_ dCas9- (mCherry)₃(H) The proportion of cells forming telomeres.

Figure 7 shows that Acrs has no effect on Cas 9-sgrnanp complex formation. A and B are EMSA experiments to test the effect of AcrIIA25.1, AcrIIA26, AcrIIA27, AcrIIA28 and AcrIIA32.1 on SpyCas9-sgRNARNP complex formation. The assay was analyzed on a non-denaturing polyacrylamide gel with sgRNA stained by SYBR gold. The order of addition of the different reaction components (Acrs, SpyCas9, and sgRNA) is shown above the dashed box. C is when AcrIIA24 was added before or after the addition of sgRNA, EMSA assays were performed to analyze the effect of AcrIIA24 protein on the binding of St3Cas9 to sgRNA. D is the detection of binding of St1Cas9 to sgRNA using EMSA experiments in the presence or absence of AcrIIA30 or AcrIIA31.

Figure 8 shows that Acrs exhibits multiple mechanisms of action to inhibit Cas9 in vitro. A-F EMSA experiments were performed to analyze the effect of different Acr proteins on the binding of Cas9RNP to DNA, including AcrIIA25.1(A), AcrIIA26(B), AcrIIA27(C), AcrIIA32.1(D), AcrIIA24(E), AcrIIA30, AcrIIA31(F), Cas9RNP (256nM) and Acr gradients (0.125, 0.25, 0.5, 0.1, 0.2, 0.4, 0.8 and 1.6. mu.M), before or after addition of target DNA. Experiment ofPerformed on a non-denaturing gel and the targeting DNA was labeled with Cy 5. Three independent replicates of the experiment were performed and representative picture presentations were selected. G is obtained by adding additional Mg in EMSA²⁺To restore the cleavage activity of Cas9 on DNA to perform DNA cleavage experiments. RT, room temperature. H and I are DNA cleavage experiments performed to analyze the effect of Acrs on Cas9 cleavage DNA activity under different conditions indicated by G. SpyCas9RNP (500nM), St1Cas 9RNP (500nM), Acrs (10. mu.M) and substrate DNA (50nM) (non-target strand labeled with Cy 3). The experiment was repeated 3 times and a representative gel pattern is shown. J summarizes the different inhibitory mechanisms of the anti-CRISPR proteins identified in this study. AcrIIA26, AcrIIA27, AcrIIA30, and AcrIIA31 block binding of Cas9 to DNA, while AcrIIA24 inhibits the DNA cleavage activity of Cas 9. Notably, acriiaa 25.1 and acriiaa 32.1 can inhibit both DNA binding and DNA cleavage activity of Cas 9.

Fig. 9 is a diagram of various mechanisms by which Acrs inhibits St3Cas 9. DNA cleavage assays were performed to analyze the effect of AcrIIA24, AcrIIA25, and acriiaa 32.1 on the DNA cleavage activity of St3Cas9 under different conditions, as shown in fig. 8, G. St3Cas 9RNP (500nM), Acrs (10. mu.M) and substrate DNA (50nM) (target strand labeled Cy 5). The experiment was repeated 3 times and a representative gel pattern is shown.

FIG. 10 shows the establishment of a chemically inducible iAcr (inductively anti-CRISPR) system to achieve chemically controllable gene editing. A is a schematic diagram of an iAcr system. The insertion of ligand-dependent inteins into Acr proteins inactivates Acr. Binding of 4-HT can trigger self-splicing of intein proteins and restore Acr activity to inhibit Cas 9. B is a schematic of the BFP-to-GFP reporter system using guided editing to examine the inhibitory activity of intein-Acr hybrids on Cas9 in human cells. HEK293T cells with chromosomally integrated BFP (HEK293T-BFP cells) were transfected with plasmids encoding the guide editor, the BFP-targeting pegRNA and the intein-Acr hybrid in the presence or absence of 4-HT (1 μ M). The percentage of GFP positive cells was calculated by flow cytometry 72 hours after transfection. PE can convert BFP to GFP by replacing CC with GT resulting in a single H66Y amino acid substitution. The target and PAM sequences are shown shaded and underlined, respectively. C is the comparison of the efficiency of BFP conversion to GFP under different conditions. intein-Acr variants are identified by residues that are replaced by intein. Wild-type Acrs included C1(AcrIIC1), a4(AcrIIA4), a5(AcrIIA5), a25.1(AcrIIA25.1) and a32.1(AcrIIA32.1) as controls. n-3, values show mean ± SEM. . D is a representative gel image of the T7E1 assay to show inhibitory activity of Acr and iAcr proteins on SpyCas9 in the presence or absence of 4-HT. HEK293T cells were transfected with Cas9(1 μ g), sgRNA (0.5 μ g) and Acr (0.5 or 0.25 μ g) plasmids. Targeting sequences are shown at the top of each gel image, with PAM highlighted by underlining. The editing efficiency is shown as "indel (%)" at the bottom of each lane. E and F are representative gel images of T7E1 assays to study the inhibitory activity of Acr and iAcr proteins on SpyCas9(E) or St3Cas9(F) in the presence or absence of 4-HT. HEK293T cells were transfected with Cas9(1 μ g), sgRNA (0.5 μ g) and Acr (0.25 μ g) plasmids. Targeting sequences for human EMX1 (targeted by SpyCas9) and DYRK1A (targeted by St3Cas9) are shown on top of each gel, with PAM highlighted by underlining. The editing efficiency is shown as "indel (%)" at the bottom of each lane.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The names, NCBI accession numbers, and amino acid sequences of the Acr and Aca proteins in the examples below are shown in table 2.

TABLE 2

Examples of the following,

Materials and methods

1. Microorganisms

Coli strains (TOP10 or Mach1-T1, Biomed) were used for plasmid amplification and plasmid interference experiments. Coli strains (T7 Express, Biomed) were used for protein expression and plaque assay analysis. Coli are usually (unless otherwise stated) cultured in Lysogenic Broth (LB) medium at 37 ℃ containing the appropriate antibiotics (if required): ampicillin (50. mu.g/ml), kanamycin (50. mu.g/ml) or chloramphenicol (25. mu.g/ml).

2. Cell lines

HEK293T and HEK293T-BFP cells in DMEM medium (Gibco) containing 10% (vol/vol) fetal bovine serum (FBS, Gibco) at 37 deg.C with 5% CO₂Culturing in an incubator.

U2OS cells were placed in 10% FBS-containing McCoy's 5A (modified) medium (Gibco) at 37 ℃ in 5% CO₂Culturing in an incubator.

3. Bioinformatics analysis

The BLASTp program was used to search the non-redundant protein database for AcrIIA6(accession number: AVO22749.1) homologous proteins to manually check whether acr and aca genes from neighboring candidate genes are likely. HHpred of the MPI bioinformatics kit was used to identify DNA binding domains from neighboring genes, where aca candidates were screened. BLASTp search was performed using the aca gene to screen for acr candidate genes and further validated by biochemical analysis.

For homologous protein distribution and phylogenetic analysis of Acr proteins, homologous protein sequences of Acrs were obtained by BLASTp program using non-redundant protein databases. Sequences with high homology were determined (E-value <0.001, query coverage > 70%), and distance trees were generated using the fast minimum evolutionary tree method, 0.85 maximum sequence differences, and the Grishin (protein) distance model.

4. Plasmid interference experiments in E.coli

The DNA sequence encoding the Acr protein was synthesized by bmede and ligated into the pBAD24 vector. Using CaCl₂Heat shock procedure plasmids were transformed into E.coli. Briefly, E.coli TOP10 or Mach1-T1 strains harboring Acr plasmids were cultured overnight in LB medium containing 0.2% arabinose and then used as competent cells. Transformation was then performed with 25ngpT and 25ng Cas9 plasmids (with either matched or mismatched spacers). After 2 hours of recovery in LB medium containing 0.2% arabinose, the cells were seeded on LB agar plates containing 50. mu.g/ml ampicillin, 50. mu.g/ml kanamycin, 25. mu.g/ml chloramphenicol, 1mM IPTG and 0.2% arabinose, and incubated at 37 ℃ for 24-32 hours. Clones were photographed using a gel scanner (Tanon3500) and counted by ImageJ software. The inhibitory activity of each Acr was calculated by the transformation ratio between the pT-targeted and non-pT Cas9 plasmids.

5. Plaque assay

Coli (T7 Express, Biomed) cells were co-transformed with a plasmid expressing Cas9-sgRNA targeting phage T4 and a compatible plasmid encoding the Acr protein. Both the Cas9 plasmid and the empty pBAD24 plasmid (without Acr) for the non-targeted phage T4 served as controls. Coli containing Acr and Cas9 plasmids were cultured in LB medium containing 50. mu.g/ml ampicillin and 25. mu.g/ml chloramphenicol and grown overnight at 37 ℃. The next morning, the overnight cultures were inoculated in fresh LB medium containing antibiotics and grown for two hours at 37 ℃. Subsequently, 1mM IPTG was added to induce expression of Cas9 protein. After two hours, 0.2% arabinose was added to induce Acr protein expression. After a further two hours, 200. mu.l of the culture were mixed with 4ml of melted LB-agar (0.7%, supplemented with 10mM MgSO₄) Mixing, pouring into a container containing 10mM MgSO₄And 0.2% arabinose (1.5% containing 1mM IPTG, 50. mu.g/ml ampicillin and 25. mu.g/ml chloramphenicol) on plates. Next, a 10-fold dilution of phage T4 was spotted onto the surface of the dish. The plates were incubated overnight at 37 ℃ and photographed using a gel scanner (Tanon 3500).

6. Protein expression and purification

DNA sequences encoding St1Cas9, St3Cas9, or Acr proteins were integrated into pET28a vectors for expression of proteins in e.coli (T7 Express, Biomed). Coli cells were induced to express proteins routinely in LB medium containing 1mM IPTG and 50. mu.g/ml kanamycin, and cultured at 18 ℃ for 16 hours. Cells were harvested and resuspended in lysis buffer (50mM Tris-HCl, pH 8.0, 10mM imidazole, 0.5mM TCEP-NaOH and 500mM NaCl) containing 1mM PMSF and lysozyme. After sonication and centrifugation, the cell supernatant was bound to Ni-NTA agarose (QIAGEN) and the bound proteins were eluted with 500mM imidazole. An Amicon Ultra centrifugal filter (Millipore) was used to concentrate the protein and buffer exchanged to storage buffer (20mM HEPES-NaOH, pH 7.5, 5% (v/v) glycerol, 300mM NaCl and 1mM DTT). For Acr protein, after overnight incubation with Tobacco Etch Virus (TEV) protease at 4 ℃, a second round of Ni-NTA purification was performed to isolate unlabeled Acr protein.

7. In vitro DNA cleavage experiments

The names and nucleotide sequences of the substrate DNAs are shown in Table 3.

TABLE 3

Note: denotes fluorescein.

All sgrnas in the assay were prepared using the in vitro T7 transcription kit (Invitrogen) according to the manufacturer's manual, and transcription templates were generated using linearized sgRNA plasmids. SpyCas9 protein was purchased from Invitrogen. For FIG. 4, plasmid pC002 was constructed and further linearized by the restriction endonuclease NotI (NEB). Cleavage reactions were performed using NEBuffer3.1 with Cas9 protein (500nM), sgRNA (500nM), target DNA substrate (30 ng/. mu.l) and excess Acr protein (10. mu.M) in a total volume of 10. mu.l. Cas9 protein was complexed with sgrnas for 10 min at 37 ℃. Acr protein was then added and incubated for an additional 20 minutes at room temperature. The target DNA was added and incubated at 37 ℃ for 10 minutes. The reaction was stopped by adding 1. mu.l of Proteinase K (PK). The products were analyzed on a 1% agarose/1 XTAE (Tris-acetate-EDTA) gel.

For FIGS. 8 and 9, fluorescently labeled substrate DNA was used for the cleavage assay and was prepared by annealing synthetic oligonucleotides for the target strand and non-target strand labeled with Cy5 or Cy 3. The course of the cleavage assay is shown in FIG. 9, G. Briefly, Cas9 protein (500nM), and sgRNA (500nM) were mixed for 10 min at 37 ℃ to form Cas9RNP complexes using 1 × binding buffer (150mM KCl, 5mM EDTA, 5mM MgCl)₂1mM DTT, 5% (v/v) glycerol, 50 μ g/ml heparin, 0.01 % Tween 20 and 100 μ g/ml BSA, 20mM Tris-HCl at pH 7.6) to eliminate the cleavage activity of Cas 9. Acr protein (10. mu.M) and substrate DNA (50nM) were then added in different order and incubated for 20 min at room temperature. Subsequently, MgCl was added₂(10mM) to restore DNA cleavage activity of Cas9, and then incubated at room temperature for an additional 20 min. The reaction was stopped by addition of Gel Loading Buffer II (Invitrogen) and incubated for 6 min at 85 ℃. The product was analyzed on a 12% denaturing PAGE gel and visualized by Typhoon7000 (GE).

8. Construction of Intein-Acr (Intein-Acr) plasmid

The DNA sequence encoding the Acr protein (AcrIIA4, AcrIIA5, AcrIIA25.1, or AcrIIA32.1) was cloned into pcdna3.1 vector for expression in human cells.

The intein 37R3-2 sequence was synthesized and inserted into the Acr protein at the position described to construct the intein-Acr plasmid.

intein-Acr plasmids express intein-Acr variants. The names and amino acid sequences of the intein-Acr variants are shown in Table 4.

TABLE 4

Note: single underlining is intein 37R3-2, double underlining is Acr.

9. T7 Endonuclease I (T7E1) detection

Target sequences in the AAVS1, EMX1, and DYRK1A loci and primer sequences for PCR amplification are shown in table 5. For fig. 5, HEK293T cells cultured in 24-well plates were transfected with 1 μ g Cas9 plasmid, 0.5 μ g sgRNA plasmid, and 0.5 μ g acr plasmid per well using Lipofectamine LTX reagent (Invitrogen) according to the manufacturer's protocol. For fig. 10, HEK293T cells were transfected with 1 μ g Cas9 plasmid, 0.5 μ g sgRNA plasmid, 0.5 or 0.25 μ g acr (iacr) plasmid in 24-well plates with or without 4-HT (1 μ M, Selleck S7827) treatment. 72 hours after transfection, genomic DNA of the cells was extracted using DNeasy Blood and Tissue kit (QIAGEN) and PCR amplified using Q5 High-Fidelity 2X Master Mix (NEB). The PCR product mixed with NEBuffer 2 was denatured and annealed and incubated at 37 ℃ before adding T7 endonuclease I (NEB). The samples were separated in a 3% agarose/1 × TAE gel. Bands were quantified using ImageJ software.

The genome editing efficiency of mammalian cells is calculated as follows:

TABLE 5 primers and target sequences used in the detection of T7E1

10. Human cell telomere fluorescence imaging

For imaging, U2OS cells were cultured on a15 mm glass bottom (Electron Microscopy Sciences) in 24-well plates. Cell transfection was performed using Lipofectamine LTX reagent (Invitrogen) according to the manufacturer's manual, using 60ng of each dCas9 plasmid, 300ng of each sgRNA plasmid and 300ngAcr plasmid. 24 hours after transfection, cells were fixed with 4% paraformaldehyde (Beyotime) and observed and imaged using a Nikon A1R + confocal microscope and a 60 Xoily objective.

Cell quantification cells from each condition were coded by a marker number by one experimenter. While another experimenter, unaware of these conditions, observed and scored the cells under the microscope. For quantification, only TagBFP and mCherry fluorescence were expressed simultaneously with Nme _ dCas9- (sfGFP)₃Evaluation of the Presence or absence of Co-localization of S _ (d) Cas9- (mCherry) in cells forming green telomeres₃Red telomeres.

11. Gel migration assay (electrophoretic mobility shift assays, EMSA)

Rnamsa was performed by incubating Cas9 protein (256nM) and sgRNA (256nM) in the order shown in the legend in the presence or absence of Acrs (5 μ M). The reaction was performed in 1 Xbinding buffer (150mM KCl, 5mM EDTA, 5mM MgCl)₂1mM DTT, 5% (v/v) glycerol, 50. mu.g/ml heparin, 0.01 % Tween 20, 100. mu.g/ml BSA and 20mM Tris-HCl pH 7.6). sgRNAs and Acr proteins were added in different order and incubated at 37 ℃ for 10 min, respectively. Samples were analyzed on a 6% Tris-rate-EDTA (TBE) polyacrylamide gel and visualized using Typhoon7000(GE) SYBRgold (Invitrogen) staining.

The inventors performed DNAEMSA, briefly, Cas9-sgRNA complex was incubated in 1 × binding buffer at 37 ℃ for 10 min, at concentrations as shown in the figure examples. Subsequently, different concentrations of Acr protein were added and incubated at room temperature for 20 minutes, then 20nM of fluorescently labeled substrate DNA (Cy 5-labeled target strand) was added to the mixture, followed by incubation at 37 ℃ for 10 minutes. In parallel experiments, fluorescently labeled substrate DNA was added and incubated at 37 ℃ for 10 minutes, then different concentrations of Acr protein were added and incubated at room temperature for 20 minutes. The samples were analyzed by biphasic polyacrylamide (6% in the upper half of the gel and 12% in the lower half of the gel)/0.5 × TBE gel electrophoresis. The gel was visualized by Typhoon7000 (GE). All assays were performed in triplicate.

12. PE-mediated BFP-to-GFP gene editing in HEK293T-BFP cells

First, the inventors integrated BFP expression genes at the AAVS1 locus in HEK293T cells, constructing HEK293T-BFP cell lines. Briefly, HEK293T cells were transfected with Cas9, AAVS1 targeted sgRNA plasmids and a donor vector containing homologous sequences and carrying BFP and puromycin resistance genes. HEK293T-BFP cells were selected by puromycin (2. mu.g/ml) treatment and flow cytometry (FACSAria III, BD).

To examine the inhibitory activity of intein-Acr hybrids on Cas9 in human cells, PE-mediated BFP-to-GFP gene editing was performed in HEK293T-BFP cells. The plasmid encoding prime editor-PE2 was purchased from Addge (# 132775). The BFP-targettingpegrna plasmid was constructed by synthesizing a DNA sequence containing the target, sgRNA scaffold, PBS and RT template, and integrated into a U6-sgRNA vector. HEK293T-BFP cells were cultured in 24-well plates and then transfected with PE2 (1. mu.g), BFP-targeted pegRNA (0.5. mu.g) and Acr (0.25. mu.g) plasmids per well using Lipofectamine LTX reagent (Invitrogen), with or without 4-HT (1. mu.M, Selleck S7827).

Second, experimental results

1. Discovery of nine novel II-A type Acr (AcrIIA24-32) protein families

By the guilt-by-association principle, an Acr-associated gene (Aca) is often present near the Acr gene, and the encoded protein has a helix-turn-helix (HTH) conserved domain for regulating the activity of the Acr. Information retrieval was performed on AcrIIA6 (access: AVO22749.1) using BLAST program (see A in FIG. 1). The Acr candidate gene is established by combining the amino acid sequence size (generally 50-200 amino acids) of the Acr protein and whether the Acr protein exists in a phage/prophage area or not. In addition, by using an escherichia coli plasmid interference experiment, a screening system (see a B in a figure 1) for Acr proteins of SpyCas9, St1Cas9 and St3Cas9 systems is designed, and candidate Acr proteins are rapidly and accurately identified. The experimental method is to clone the gene encoding the Cas9 protein into a bacterial expression plasmid with gRNA, and the gene is used as an exogenous CRISPR-Cas9 system in Escherichia coli. In addition, the gene coding the candidate Acr protein is cloned into a compatible bacterial expression plasmid to be used as an exogenous anti-CRISPR system for detection. A targeting plasmid (designated pT) was also constructed that could be recognized and targeted by the exogenous CRISPR-Cas9 system. Whether the Acr can inhibit the Cas9 protein is identified by calculating the transformation efficiency of the pT plasmid in Escherichia coli with the exogenous Cas9 and candidate Acr plasmids.

Nine novel protein families of type II-A Acr (AcrIIA24-32) and three aca proteins were found from the mobile element of Streptococcus (S.coli) by screening by E.coli plasmid interference experiments (see A and B in FIG. 1). Of the 11 Acr proteins examined (two Acr homologous proteins: AcrIIA25.1 and AcrIIA32.1 added), 5 Acr proteins (AcrIIA25, AcrIIA27, AcrIIA28, AcrIIA32 and AcrIIA32.1) exhibited robust inhibitory activity against SpyCas9 and St3Cas 9. In addition, AcrIIA25.1 and AcrIIA26 were able to significantly inhibit SpyCas9, whereas AcrIIA24 and AcrIIA29 inactivated St3Cas9 to varying degrees. Meanwhile, AcrIIA30 and AcrIIA31 were found to strongly inhibit the activity of St1Cas9 (see C in fig. 1). However, in the experiments, other Acr candidate proteins from adjacent genes had no inhibitory activity on SpyCas9, St1Cas9, and St3Cas9 (data not shown). These data indicate the discovery of nine novel families of type II-a Acr (AcrIIA24-32) proteins, and confirmation by plasmid intervention experiments that most of these Acr proteins are able to robustly inhibit Streptococcus-derived type II-a Cas9 protein in e.

To further determine the distribution of the AcrIIA24-32 homologous proteins, a comprehensive phylogenetic analysis was performed based on the BLAST results (see FIG. 2). The data show that the homologous proteins of AcrIIA28, AcrIIA30, and AcrIIA32 are rare and distributed in only a few streptococcal genomes or phages (see E, G and I in fig. 2). In contrast, homologous proteins of other Acr proteins are more widely distributed in streptococcal mobility elements. Whereas the AcrIIA26, AcrIIA27, AcrIIA29 and AcrIIA31 protein families are present in various strains of streptococcus, such as streptococcus salivarius (s.salivarius) and streptococcus pyogenes (s.pyogenes) (see C, D, F and H in fig. 2). Furthermore, AcrIIA24 and AcrIIA25 homologous proteins are present not only in the streptococcus genomic strain, but also in various streptococcal phages (see a and B in fig. 2). The data indicate that the AcrIIA24-32 protein family is predominantly distributed in streptococci, suggesting that AcrIIA24-32 has a potential role in the arms race between streptococcal phages and the host.

2. AcrIIA24-32 is a streptococcal CRISPR-Cas9 system-specific arrestin

In order to further investigate whether AcrIIA24-32 has broad-spectrum inhibitory activity, plaque assay experiments were performed. A number of well-defined Cas9 homologous proteins were selected for validation, including type II-a (SpyCas9, St1Cas9, St3Cas9, and SaCas9), type II-B (FnCas9), and type II-C (NmeCas9) CRISPR systems (a in fig. 3). Coli carries a Cas9 expression plasmid capable of targeting the T4 phage gene 23, while the Cas9 plasmid not targeting the T4 phage is a control. Coli transformed with Cas9 expression plasmids were infected with 10-fold serial dilutions of T4 phage in the presence or absence of Acr (a in fig. 3). It was found that AcrIIA30 and AcrIIA31 can specifically inhibit St1Cas9, while other Acr proteins can strongly inhibit both SpyCas9 and St3Cas9 (fig. 3B).

Furthermore, AcrIIA24-32 showed no detectable inhibitory activity against SaCas9, NmeCas9 and FnCas 9. The data indicate that AcrIIA24-32 is an inhibitory protein specific for the streptococcal CRISPR-Cas9 system.

3. AcrIIA24-32 can inhibit II-A type Cas9 homologous protein in vitro

In plasmid interference experiments, some of the proteins in AcrIIA24-32 exhibited weak inhibitory activity on the CRISPR-Cas9 system of streptococci (e.g., AcrIIA29 weakly inhibits St3Cas 9). However, AcrIIA24-32 was able to restore T4 phage to nearly the same level as non-targeted controls in plaque assay experiments, indicating that these Acr proteins have robust inhibitory activity against both type II-a Cas9 homologs (SpyCas9, St1Cas9, and St3Cas9) (B in fig. 3).

To eliminate this difference and confirm the inhibitory activity of AcrIIA24-32 on the streptococcal CRISPR-Cas9 system, Cas9 and Acr proteins were purified and DNA cleavage experiments were performed in vitro. AcrIIA24-32 inhibited the activity of SpyCas9, St1Cas9, and St3Cas9 proteins, as determined by Cas9RNP targeting linearized plasmid DNA in the presence or absence of Acr (a in fig. 4). The results show that the results of the DNA cleavage experiments are substantially identical to the results of the plasmid interference experiments. SpyCas9 was able to be inhibited by AcrIIA25, AcrIIA25.1, AcrIIA26, AcrIIA27, AcrIIA28, AcrIIA32, and AcrIIA32.1 (fig. 4B). Other Acr proteins besides AcrIIA25.1 and AcrIIA30 could inhibit St3Cas9, while AcrIIA24, AcrIIA25, AcrIIA27, AcrIIA31, and AcrIIA32 showed stronger inhibitory activity against St3Cas9(C in fig. 4). Furthermore, only AcrIIA30 and AcrIIA31 could effectively inactivate St1Cas9 in vitro, which is consistent with the results of plasmid interference and plaque assay experiments (D in fig. 4). DNA cleavage experiments were also performed under different conditions, i.e. the apo-Cas9 and Acr protein were pre-incubated before introducing sgRNA and target DNA into the reaction (data not shown). No significant difference was found in the experimental results under these two reaction conditions, indicating that AcrIIA24-32 acts mainly on Cas9RNP and affects the downstream function of Cas9 RNP.

4. AcrIIA24-32 can inhibit Cas 9-mediated gene editing in human cells

Given the widespread use of various Cas9 orthologous proteins in eukaryotic cells, it was examined whether the corresponding Acrs could inhibit Cas9 protein in human cells. Plasmids encoding Cas9, genome-targeted sgrnas, and Acrs were co-transfected into HEK293T cells. Gene editing efficiency was analyzed 72 hours after transfection using T7 endonuclease 1(T7E1) (a in fig. 5). The human endogenous loci AAVS1 and DYRK1A were designed for gene editing of the type II-a Cas9 ortholog protein of streptococcus.

Strikingly, it was observed that multiple Acrs showed strong inhibitory effects on SpyCas9, St1Cas9, and St3Cas9 in human cells (see table 6). AcrIIA26, AcrIIA27, AcrIIA28, AcrIIA32 and AcrIIA32.1 proteins almost completely inhibited the activity of SpyCas9 at levels comparable to the known potent inhibitory protein AcrIIA5, whereas AcrIIA25 only weakly inhibited SpyCas9 (mean inhibitory activity 44%) (B and C in fig. 5). Furthermore, St3Cas 9-mediated gene editing could be inhibited by a variety of Acrs to varying degrees, including AcrIIA24 (mean 91%), AcrIIA25 (mean 56%), AcrIIA27 (mean 83%), AcrIIA28 (mean 84%), AcrIIA29 (mean 33%), AcrIIA32 (mean 98%) and AcrIIA32.1 (mean 93%) (D and E in fig. 5). It was also found that AcrIIA30, AcrIIA31, and AcrIIA31 homologous proteins (acriiaa 31.1) can effectively inhibit the activity of St1Cas9(F and G in fig. 5).

Thus, the results indicate that multiple Acrs from AcrIIA24-32 and their orthologous proteins can effectively inhibit streptococcal Cas9 homologous proteins in human cells (SpyCas9, St1Cas9, and St3Cas 9).

TABLE 6

Note: 20% < inhibitory activity < 50%, labeled "+"; 50% < inhibitory activity < 80%, labeled "+"; inhibitory activity > 80%, labeled "+++"; ND, not determined.

5. Acrs employs multiple strategies to inactivate Cas9 in human cells

To investigate the mechanism of inhibition of Cas9 by Acr protein, Cas9 protein-mediated telomere co-localization fluorescence imaging experiments were performed in the U20S cell line (a and B in fig. 6). AcrIIA24, AcrIIA25.1, AcrIIA26, AcrIIA27, AcrIIA30, AcrIIA31, and AcrIIA32.1 were selected, considering that these proteins have high activity in inhibiting Cas9 in human cells. The mechanism of action of Acrs can be assessed by imaging telomeric DNA with mCherry-labeled streptococcal Cas9 protein, while superfolder (sf) GFP-labeled NmeCas9 can be used as the telomere indicator signal.

First, the effect of potent inhibitors on the binding of SpyCas9 to DNA was studied, including AcrIIA25.1, AcrIIA26, AcrIIA27, and AcrIIA32.1, as well as AcrIIA4 and AcrIIA5 as controls. Spy _ dCas9- (mCherry) was observed₃And Nme _ dCas9- (sfGFP)₃In the case of co-expressing AcrIIA5Co-localization to telomeres in cells, whereas in cells expressing AcrIIA4, the band of Spy _ dCas9- (mCherry) was eliminated₃Red telomeres formed (C in fig. 6). However, in cells expressing AcrIIA25.1, AcrIIA26, AcrIIA27 or AcrIIA32.1 all resulted in Spy _ dCas9- (mCherry)₃The red telomeres formed are lost without affecting Nme _ dCas9- (sfGFP)₃Green telomeric points formed (C in fig. 6). Subsequently, Spy _ dCas9- (mCherry) in the presence of different Acr proteins was quantified₃Number of telomeric cells. 93.1% of the cells expressing AcrIIA5 were observed to be Spy _ dCas9- (mCherry)₃Red telomeres formed, whereas red telomeres were rarely observed in cells expressing AcrIIA25.1, AcrIIA26, AcrIIA27, AcrIIA32.1, or AcrIIA4 (D in fig. 6). These results demonstrate that AcrIIA26, AcrIIA27, and AcrIIA32.1 can block SpyCas9 binding to targeted DNA efficiently in human cells.

Subsequently, to explore the mechanism of action of the potent inhibitory proteins of St1Cas9 and St3Cas9, a similar fluorescence imaging system was established using St1Cas9 and St3Cas9 for targeting human telomeres in U2OS cells (a and B in fig. 6). St3_ dCas9- (mCherry) was observed₃Or St1_ dCas9- (mCherry)₃With Nme _ dCas9- (sfGFP)₃Co-localisation to telomeres in the cells (E and G in figure 6). AcrIIA24 for St3_ dCas9- (mCherry)₃There was no effect on telomere co-localization, whereas AcrIIA27, AcrIIA32.1, AcrIIA30 and AcrIIA31 could significantly eliminate red telomeres formed by St3Cas9 or St1Cas9(G and E in fig. 6). In addition, St3_ dCas9- (mCherry) was observed in 96.8% AcrIIA 5-expressing cells and 96.4% AcrIIA 24-expressing cells by quantitative experiments₃Red telomeres formed, whereas in cells expressing AcrIIA27 or AcrIIA32.1 no St3_ dCas9- (mCherry) was observed₃Red telomeres formed (F in fig. 6). In addition, St1_ dCas9- (mCherry) was observed in 72.4% AcrIIA 5-expressing cells, 3.4% AcrIIA 30-expressing cells and 0% AcrIIA 31-expressing cells₃Red telomeres formed (H in fig. 6).

The results show that Acrs from streptococcal mobile elements employ multiple inhibition strategies to inhibit Cas9 protein. AcrIIA26, AcrIIA27, AcrIIA30, AcrIIA31, and AcrIIA32.1 effectively block binding of Cas9 protein to DNA. However, AcrIIA24 did not prevent binding of Cas9 protein to DNA targets, suggesting that AcrIIA24 might specifically inhibit cleavage of substrate DNA by Cas9, with a mechanism of action similar to AcrIIC 1.

6. Acrs exhibit multiple mechanisms of action to inhibit Cas9

Gel migration Experiments (EMSA) were performed in vitro to further examine the mechanism of action of these actors on Cas 9. RNA EMSA was first performed to determine whether these Acrs affected Cas9-sgRNA RNP complex formation. AcrIIA25.1, AcrIIA26, AcrIIA27, AcrIIA28 and AcrIIA32.1 were found to have no effect on SpyCas9-sgRNARNP formation (A and B in FIG. 7). AcrIIA24, AcrIIA30, and AcrIIA31 also did not prevent binding of St3Cas9 or St1Cas9 to sgrnas (C and D in fig. 7).

Subsequently, DNAEMSA experiments were performed to investigate how Acrs acts on Cas9RNP to affect the downstream function of Cas9 RNP. Cas9 protein with catalytic activity and sgRNA are mixed to form a Cas9RNP complex, and 10mM EDTA is added into a reaction system to eliminate the cutting effect of Cas9 on DNA. One fluorescently labeled substrate DNA (Cy 5-labeled target strand) was designed to be targeted by SpyCas9, St1Cas9, and St3Cas 9. It was observed that AcrIIA25.1, AcrIIA26, AcrIIA27, and AcrIIA32.1 effectively abolished the binding of SpyCas9RNP to DNA only when added to the reaction system before the addition of the target DNA (a-D in fig. 8). It was also found that AcrIIA25.1 and AcrIIA32.1 can capture Cas9 complex bound to DNA, resulting in DNA hypermigration ("super-shift"), whereas AcrIIA26, AcrIIA27 cannot (a-D in fig. 8).

Next, the mechanism of inhibition by acriiaa 32.1 of St3Cas9 was investigated, with similar results as acriiaa 32.1 for SpyCas 9. It was also observed that, whether the AcrIIA24 protein was added before or after the target DNA was added, AcrIIA24 could capture the Cas9-DNA complex, resulting in DNA hypermigration ("super-shift") (E in fig. 8). The results indicate that AcrIIA24 should specifically inhibit the cleavage activity of St3Cas9 on DNA substrates. The inhibitory mechanism of St1Cas9 by AcrIIA30 and AcrIIA31 was also investigated, and it was found that AcrIIA30 and AcrIIA31 only effectively prevented binding of St1Cas 9RNP to DNA when added to the reaction system before addition of the target DNA (F in fig. 8). Like AcrIIA25.1 and AcrIIA32.1, AcrIIA30 can bind to DNA-binding Cas9 complex, resulting in DNA hypermigration ("super-shift"), whereas AcrIIA31 cannot (F in fig. 8).

Given some of the non-canonical behaviors of acriiaa 25.1, acriiaa 32.1, and AcrIIA30, it is speculated that these Acr proteins can not only block Cas9 from binding to DNA, but also inhibit Cas9 cleavage activity on DNA in Cas9-sgRNA-DNA-Acr quaternary complex. To validate the hypothesis, the EMSA was modified by adding additional Mg²⁺To restore the cleavage activity of Cas9 on DNA to perform DNA cleavage experiments (G in fig. 8). It was examined whether Acr protein affects DNA cleavage activity of Cas9 under different reaction conditions, i.e. Acrs is added before or after target DNA is added to the reaction system. Data show that by adding additional Mg in EMSA²⁺The DNA cleavage activities of SpyCas9, St1Cas9 and St3Cas9 were all effectively restored (H, I in fig. 8 and fig. 9). Compared to the AcrIIC1 protein, AcrIIA25.1, AcrIIA32.1, and control AcrIIA4, when added to the reaction system prior to the addition of target DNA, strongly inhibited the DNA cleavage activity of SpyCas 9. However, compared to the AcrIIA4 control, AcrIIA25.1 and AcrIIA32.1 added to the reaction system after addition of the target DNA also inactivated the DNA cleavage activity of SpyCas9 (H in fig. 8). In conjunction with EMSA and DNA cleavage assays, the data show that acriia25.1 and acriia32.1 can inhibit the DNA binding and DNA cleavage activity of SpyCas9, a novel inhibition mechanism different from other previously reported Acrs (J in fig. 8).

It was further investigated whether AcrIIA24, AcrIIA25 and acriiaa 32.1 inhibited the DNA cleavage activity of St3Cas 9. AcrIIA24, AcrIIA25, and AcrIIA32.1 were found to show strong inhibitory effects on DNA cleavage by St3Cas9, whether added before or after addition of target DNA (fig. 9). The results show that AcrIIA24 can effectively inhibit the DNA cleavage step of St3Cas9, while AcrIIA25 and AcrIIA32.1 inhibit the DNA binding and DNA cleavage activity of St3Cas 9. The inhibitory mechanism of AcrIIA30 and AcrIIA31 on the DNA cleavage activity of St1Cas9 was also investigated, particularly considering that AcrIIA30 can bind to Cas9-sgRNA-DNA ternary complex in EMSA. It was found that addition of AcrIIA30 and AcrIIA31 was only effective in inhibiting St1Cas 9-mediated DNA cleavage prior to addition of target DNA, indicating that both AcrIIA30 and AcrIIA31 could inhibit DNA binding of St1Cas9 without affecting the DNA cleavage activity of St1Cas9 (I and J in fig. 8). Taken together, the results indicate that these Acrs were found to exhibit multiple abilities to inhibit Cas9, the mechanisms of which include blocking DNA binding, DNA cleavage, or both.

7. Establishment of chemically inducible iAcr (inductively anti-CRISPR) System to achieve chemically controllable Gene editing

Since acriiaa 25.1 and acriiaa 32.1 can inhibit DNA binding and DNA cleavage activity of Cas9, a strong potential for application is shown to be useful for modulating Cas 9-mediated genome editing. These two proteins were selected as candidate proteins for the development of chemically inducible anti-CRISPR. Also using AcrIIA4 and AcrIIA5 as controls, considering that AcrIIA4 can only block binding of Cas9 to DNA, whereas AcrIIA5 specifically inhibits DNA cleavage of Cas9, intein-Acr hybrids were designed by fusion of the Acr protein with ligand-dependent intein (intein)37R3-2, insertion of the intein into the Acr protein resulted in inactivation of Acr, while binding of 4-hydroxytrypamoxigen (4-HT) to the intein triggered the intein self-splicing and restored Acr activity to inhibit Cas9 (a in fig. 10).

The 4-HT-responsive intein was inserted into Acrs by replacing a single residue of Acrs (cysteine, alanine, serine or threonine) because intein protein splicing leaves a single cysteine residue and replacing these residues would minimize the effect of the resulting cysteine point mutation (table 4). To examine the effect of intein-Acr hybrids on Cas9 in human cells, a BFP-to-GFP reporter system was designed, using guided editing (PE) (fig. 10, B). Functional HEK293T cells are established, namely blue fluorescent protein is integrated at the AAVS1 site (HEK293T-BFP cells), and flow cytometry analysis shows that the expression ratio of BFP in the HEK293T-BFP cells is high. PE can convert BFP to GFP by replacing CC with GT, resulting in a single H66Y amino acid substitution (B in fig. 10). HEK293T-BFP cells were transfected with plasmids encoding PE, BFP-targeted pegRNA and intein-Acr hybrids in the presence or absence of 4-HT (1 μ M) (B in fig. 10). The effect of intein-Acr hybrids on Cas9 can be calculated by comparing the efficiency of BFP editing to GFP under each condition.

The results show that 4-HT treatment had no significant effect on the activity of PE and Wild Type (WT) Acrs in human cells (C in FIG. 10). Of the 10 intein-Acr variants, the activities of AcrIIA4(T28 and A58), AcrIIA5(S87), AcrIIA25.1(S59) were not regulated by 4-HT. Although AcrIIA5(a68), AcrIIA25.1(S30), and AcrIIA32.1(T24 and a73) can switch to an active state in response to 4-HT to inhibit Cas9, these four intein-Acr variants are weakly 4-HT dependent (on average 1.8-fold modulation). Only two intein-Acr variants, acriiaa 25.1(S76) and acriiaa 32.1(T40), could effectively inhibit PE-mediated BFP-to-GFP editing in the presence of 4-HT, showing 4-HT dependent modulation (3.3 and 3.6 fold change, respectively). We then named intein-AcrIIA25.1(S76) and intein-AcrIIA32.1(T40) as iAcrIIA25.1 (abbreviated as iA25.1) and iAcrIIA32.1 (abbreviated as iA32.1), respectively.

To further examine whether ia25.1 and ia32.1 possess 4-HT dependent activity to inhibit Cas 9-mediated gene editing, T7E1 experiments were performed in HEK293T cells. The human endogenous loci AAVS1 and EMX1 were designed for targeted editing of SpyCas 9. Consistent with the results of the PE-mediated BFP-to-GFP assay, 4-HT treatment had no effect on the activity of SpyCas9 and WTAcrs (D and E in fig. 10). Strikingly, the inhibitory activity of ia25.1 and ia32.1 was observed to be 4-HT dependent. Both proteins had a slight effect on SpyCas9 activity in the absence of 4-HT, but in the presence of 4-HT they switched to a high activity state to inhibit Cas9(D and E in fig. 10). To further examine the potential applications of iAcr, a T7E1 assay was performed to investigate whether ia32.1 can be activated by 4-HT to inhibit St3Cas9 mediated gene editing in human cells. As expected, the data show that ia32.1 with 4-HT triggering activity can inhibit St3Cas9 mediated gene editing in human cells (F in fig. 10).

Thus, the results indicate that these iAcrs exhibit potent 4-HT dependent regulation to post-translationally control CRISPR-Cas 9-mediated genome editing in human cells.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> institute of biophysics of Chinese academy of sciences

<120> novel CRISPR-Cas9 arrestin and method for applying CRISPR-Cas9 arrestin to chemically controllable gene editing through modification

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<170> PatentIn version 3.5

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Met Lys Lys Ala Gln Gln Leu Leu Lys Glu Ile Lys Thr Asn Asn Val

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Thr Asn Asn Ile Met Asp Ile Tyr Gly Tyr Asp Asn Glu Asn Gly His

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Phe Tyr Gly Val Tyr Gly Asp Val Val Asp Gly Gln Ile Asp Ser Arg

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Tyr Phe Ser Asp Asp Ala Ile Leu Asn Ala Ile Asp Lys Leu Leu Phe

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Leu Gly Asp Pro Ile Lys Arg Thr Asp Leu Pro Ser Asp Ala Asp Phe

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Lys Arg Thr Phe Phe Phe Glu Glu

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Met Lys Asn Arg Leu Leu Gly Ser Arg Tyr Thr Asp Ala Ile Lys Asn

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Asp Cys Gly Thr Ala Asn Lys Met Ser Asn Ile Tyr Asn Lys Leu Asn

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Lys Asp Ser Leu Arg Glu Ile His Ser Ala Leu Tyr Gly Leu Leu Thr

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Ala Gly Tyr Asp Ile Ser Asn Met Arg Asn Ile Glu Glu Leu Glu Lys

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Tyr Val Asn Leu Lys Lys Ser Arg Gly Gln Leu Leu Asn Val Ser Ser

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Asp Asp Ile Lys Leu Tyr His Lys Leu Phe Val Ile Arg Phe Gly Lys

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Met Lys Asn Gly His Met Ile Leu Gly Gln Arg Trp Thr Asn Ala Ile

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Arg Asn Glu Thr Gly Thr Ser Ser Lys Met Phe Asn Leu Ser Lys Arg

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Leu Tyr Asp Phe Lys Asp Asn Asn Leu Arg Glu Ile His Glu Ala Leu

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Tyr Gly Leu Leu Arg Ala Gly Tyr Asp Ile Ser Asn Met Arg Asp Val

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Glu Glu Leu Ala Lys Tyr Val Asp Val Lys Lys Ser His Gly Lys Leu

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Leu Asp Val Thr Arg Asp Asp Ile Glu Leu Tyr His Arg Leu Phe Val

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Ala Arg Phe Gly Lys

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Met Lys Lys Leu Tyr Ile Gln Thr Asn Gln Phe Ala Asn Gly Glu Leu

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Gln Val Glu Asn Thr Ser Tyr Glu Leu Cys Asp Thr Phe Lys Glu Leu

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Tyr Ser Val Ala Ser Asn Leu Val Asp Glu Asn Thr Leu Asn Phe Val

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Glu Asp Asn Phe Ile Glu Gln Asn Tyr Lys Asp Glu Tyr Asn Gly Val

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Tyr Glu Asn Asp Gly Asp Thr Gly Glu Phe Val Gly Gln Val Phe Glu

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Asn Lys Val Thr Glu Glu Gln Phe Lys Glu Leu Leu Glu Gln Leu Glu

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Ile Thr Tyr Thr Glu Phe Asp Pro Glu Glu Glu Leu Ala Lys Cys Ile

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Ala Asn Lys Asn Arg Lys Ser Glu Phe Tyr Gly Asn Gly Leu Lys Val

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Ile Ala Glu Tyr Leu Glu Ser Ile Ser His Glu Asp Ala Leu Ala Val

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Val Thr Tyr Tyr Tyr Phe Tyr Phe Gly Phe Gly Tyr Glu Asp Gln Leu

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Ile Ser Asp Ile Lys Asp Asp Gln Glu Asp Gly Val Lys Phe Glu His

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Val Glu Arg Ser Glu Thr Ile

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Met Lys Thr Phe Asn Ile Ile Val Ser Glu Ser Ala Asn Leu Lys Glu

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Asn Arg Arg Glu Ala Phe Lys Lys Ala Arg Glu Glu Tyr Ser Phe Ser

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Ser Lys Trp Lys Phe Asn Met Arg Asp Leu Thr Ala Ile Asp Asn Thr

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His Arg Arg Ala Trp Gly Arg Arg Tyr Leu Arg Val Glu Glu Ala

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Arg Ser Gln His Ala Lys Glu Ala Arg Ile Pro Gly Trp Ser Asp Lys

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Tyr Asn Lys Leu Glu Lys Lys Met Leu Ser Asp Phe Glu Glu Val Thr

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Gly Ile Lys Tyr Asp Thr Leu Glu Ser Glu Leu Ile Trp Asp Asn Leu

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Asp Arg Tyr Lys Asn Asp Pro Glu Lys Ile Gly Gly Met Arg Leu Glu

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Val Ile Lys Thr Asn Trp Asn Asn Gln Gln Ile Leu Val Gln Asn Ser

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Thr Glu Lys Glu Ile Thr Lys Tyr Phe Asn Ser Tyr Pro Phe Ala Ile

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Lys Leu Asn Trp Ile Lys Pro His Lys Glu Met Phe Ile Val Asn Phe

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Asp Thr Thr Ser Asn Lys Thr Phe Arg Lys Tyr Pro Tyr Asp Leu Lys

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Asn Leu Tyr Phe Leu Val Asp Lys Asn Arg Asp Lys Met Ser Gln Phe

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Ala Glu Phe Leu Ile Ile Cys Gly Arg Lys Ser His Phe Gly Gly Ser

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Arg Val Leu Tyr Glu Val Glu Gly Lys Lys Tyr Gln Ile Ile Phe Ser

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Ile Lys Arg Pro Ser Glu Leu Gly Pro Thr Ile Arg Leu Ile Asn Val

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Val Glu Thr Asp Thr Tyr Arg Asp Asp Leu Val Pro Lys Ile Ser Glu

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Glu Glu Ser Ile Leu Arg Ser Glu Asp Leu Asp Leu Lys Gly Lys Arg

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Val Ser Ile Lys Asp Ser Glu Leu Leu Glu Leu Met Ser Ile Ile Asp

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Ile Arg Asn His Ala Thr Gly Ser Asp Leu Asn Glu Leu Glu Glu Leu

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Trp Glu Glu Val Ala Asp Met Leu Asp Phe Trp Arg Asp Lys Asp Gly

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Asp Leu Leu Ile Glu Gly Arg Gly Met Lys Pro Ile Asp Gly Val Glu

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Tyr Val Gly Tyr Ala Asp Asn Gly Val Ile Trp Glu Arg

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Gln Arg Leu Lys Ile Ala Glu Lys Glu Tyr Asn Phe Lys Ala Gly Val

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Lys Glu Asp Leu Glu Ile Lys Asn Thr Thr Asp Lys Glu Ile Leu Asp

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Tyr Val Arg Asn Glu Leu Ser Lys Ile Asp Ser Lys Lys Gln Ala Asp

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Lys Asn Trp Ser Glu Lys Asn Arg Glu His Arg Asn Tyr Leu Ser Lys

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Arg Ser Ser Ala Arg Ser Phe Ile Asn Asn Asn Ala Thr His Glu Asp

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Leu Leu Glu Leu Lys Lys Ile Ile Glu Glu Lys Leu Lys

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Ser Val Leu Asp Tyr Ile Gln Glu Gln Leu Lys Val Ile Val Lys

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Ala Leu Ile Asn Lys Lys Arg Ser Ile Glu Asn Leu Thr Ile Glu Thr

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Met Leu Ile Asn Thr Ser Arg Val Glu Met Val Leu Met Asn Lys Ala

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Ile Ser Ala Tyr Arg Leu Ala Lys Glu Ile Gly Ile Gln Glu Ser Ser

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Ile Ser Leu Leu Arg Asn Gly Lys Lys Asp Leu Asp Lys Leu Ser Leu

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Glu Val Ala Met Arg Val Gln Ala Trp Ile Asp Ala Gly Asn Tyr Ser

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Phe Ser Tyr Asp Tyr Ser Glu Leu Ile Glu Lys Leu Glu Ala Asp Ile

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Glu Lys Gly Leu Ala Asp Glu Tyr Ile Tyr Ile Val Arg Gly Gly Tyr

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Asn Glu Val Met Glu Lys Cys Met Ile Ile Asp Tyr Tyr Tyr Asp Pro

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Glu Glu Ile Ala Glu Gly Asp Ile Ala Glu Lys Val Leu Thr Ser Ser

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Ala Leu Ala Glu Met Glu Lys Asp Asn Glu Ile Phe

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Met Thr Asp Glu Leu Thr Ala Arg Gln Arg Ala Asp Lys Lys Trp Asn

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Claims (10)

1. Use of an AcrIIA protein in inhibiting Cas9 gene editing, regulation, and/or imaging in bacteria, cells, or eukaryotes;

   the AcrIIA protein is AcrIIA24 protein, AcrIIA25 protein, AcrIIA25.1 protein, AcrIIA26 protein, AcrIIA27 protein, AcrIIA28 protein, AcrIIA29 protein, AcrIIA30 protein, AcrIIA31 protein, AcrIIA31.1 protein, AcrIIA32 protein or AcrIIA32.1;

   the AcrIIA24 protein is a1) or a2) or a3) or a4) as follows:

   a1) the amino acid sequence is protein shown as SEQ ID NO. 1;

   a2) 1, the N end or/and the C end of the protein shown in SEQ ID NO. 1 is connected with a label to obtain fusion protein;

   a3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a1) or a2) and has the function of inhibiting the activity of the Cas9 protein;

   a4) 1, and has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO, and has the function of inhibiting the activity of the Cas9 protein;

   the AcrIIA25 protein is b1) or b2) or b3) or b4) as follows:

   b1) the amino acid sequence is protein shown as SEQ ID NO. 2;

   b2) 2, the N end or/and the C end of the protein shown in SEQ ID NO. 2 is connected with a label to obtain fusion protein;

   b3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by b1) or b2) and has the function of inhibiting the activity of the Cas9 protein;

   b4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 2 and has the function of inhibiting the activity of Cas9 protein;

   the AcrIIA25.1 protein is c1) or c2) or c3) or c4) as follows:

   c1) the amino acid sequence is protein shown as SEQ ID NO. 3;

   c2) a fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 3;

   c3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in c1) or c2) and has the function of inhibiting the activity of the Cas9 protein;

   c4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 3 and has the function of inhibiting the activity of Cas9 protein;

   the AcrIIA26 protein is d1) or d2) or d3) or d4) as follows:

   d1) the amino acid sequence is protein shown as SEQ ID NO. 4;

   d2) fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 4;

   d3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by d1) or d2) and has the function of inhibiting the activity of the Cas9 protein;

   d4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 4 and has the function of inhibiting the activity of Cas9 protein;

   the AcrIIA27 protein is e1) or e2) or e3) or e 4):

   e1) the amino acid sequence is the protein shown in SEQ ID NO. 5;

   e2) a fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 5;

   e3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by e1) or e2) and has the function of inhibiting the activity of the Cas9 protein;

   e4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 5 and has the function of inhibiting the activity of Cas9 protein;

   the AcrIIA28 protein is f1) or f2) or f3) or f 4):

   f1) the amino acid sequence is the protein shown in SEQ ID NO. 6;

   f2) fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 6;

   f3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by f1) or f2) and has the function of inhibiting the activity of the Cas9 protein;

   f4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 6 and has the function of inhibiting the activity of Cas9 protein;

   the AcrIIA29 protein is g1) or g2) or g3) or g 4):

   g1) the amino acid sequence is the protein shown in SEQ ID NO. 7;

   g2) fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 7;

   g3) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in g1) or g2) and has the function of inhibiting the activity of Cas9 protein;

   g4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 7 and has the function of inhibiting the activity of Cas9 protein;

   the AcrIIA30 protein is h1) or h2) or h3) or h 4):

   h1) the amino acid sequence is the protein shown in SEQ ID NO. 8;

   h2) fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 8;

   h3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by h1) or h2) and has the function of inhibiting the activity of the Cas9 protein;

   h4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 8 and has the function of inhibiting the activity of Cas9 protein;

   the AcrIIA31 protein is i1) or i2) or i3) or i4) as follows:

   i1) the amino acid sequence is protein shown as SEQ ID NO. 9;

   i2) fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 9;

   i3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in i1) or i2) and has the function of inhibiting the activity of the Cas9 protein;

   i4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 9 and has the function of inhibiting the activity of Cas9 protein;

   the AcrIIA31.1 protein is j1) or j2) or j3) or j4) as follows:

   j1) the amino acid sequence is protein shown as SEQ ID NO. 10;

   j2) 10 in SEQ ID NO, and the N end or/and the C end of the protein is connected with a label to obtain a fusion protein;

   j3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in j1) or j2) and has the function of inhibiting the activity of the Cas9 protein;

   j4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 10 and has the function of inhibiting the activity of Cas9 protein;

   the AcrIIA32 protein is k1) or k2) or k3) or k 4):

   k1) the amino acid sequence is protein shown as SEQ ID NO. 11;

   k2) a fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID NO. 11;

   k3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in k1) or k2) and has the function of inhibiting the activity of the Cas9 protein;

   k4) a protein which has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 11 and has the function of inhibiting the activity of Cas9 protein;

   the AcrIIA32.1 protein is l1) or l2) or l3) or l4) as follows:

   l1) the amino acid sequence is the protein shown in SEQ ID NO. 12;

   l2) fusion protein obtained by attaching labels to the N-terminal or/and C-terminal of the protein shown in SEQ ID NO. 12;

   l3) is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in l1) or l2) and has the function of inhibiting the activity of the Cas9 protein;

   l4) has 60 percent or more than 60 percent of homology with the amino acid sequence defined by SEQ ID NO. 12 and has the function of inhibiting the activity of the Cas9 protein.
2. 2. Use of the AcrIIA protein of claim 1 to inhibit the activity of Cas9 protein.
3. 3. Use according to claim 2, characterized in that: the inhibition of the activity of the Cas9 protein is shown as inhibition of binding of the Cas9 protein to DNA and/or inhibition of cleavage activity of the Cas9 protein to substrate DNA.
4. 4. A system of chemically-induced icacr, characterized by: the use of said AcrIIA25.1 and/or said AcrIIA32.1 as a protein of chemically inducible anti-CRISPR as claimed in claim 1.
5. 5. The system of claim 4, wherein: the system relies on 4-hydroxy tamoxifen for regulation.
6. 6. Use of the system of claim 4 or 5 for effecting chemically controllable gene editing.
7. 7. A method of chemically controlled gene editing, comprising: the method employs the system of chemically inducible iAcr of claim 4 or 5 for gene editing.
8. 8. The method of claim 7, wherein: the method relies on 4-hydroxy tamoxifen for modulation.
9. 9. Use of the AcrIIA protein of claim 1 in the preparation of a chemically-inducible iAcr system.
10. 10. Use of the AcrIIA protein of claim 1 for chemically controlled gene editing.

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