CN117264987A

CN117264987A - Method for chemically controllable gene editing by engineering AcrIIA25.1 protein

Info

Publication number: CN117264987A
Application number: CN202311023039.0A
Authority: CN
Inventors: 宋国旭; 田勇; 张飞; 田春红; 高星
Original assignee: Institute of Biophysics of CAS
Current assignee: Institute of Biophysics of CAS
Priority date: 2022-02-11
Filing date: 2022-02-11
Publication date: 2023-12-22
Also published as: CN117487831A; CN114525293A; CN114525293B; CN117264988A; CN117286165A

Abstract

The invention discloses novel CRISPR-Cas9 inhibitor proteins AcrIIA25 and AcrIIA25.1 and a method for modifying and applying the novel CRISPR-Cas9 inhibitor proteins to chemically controllable gene editing. The inventors have discovered a novel family of type II-a Acr (AcrIIA 24-32) proteins from the mobile element of Streptococcus that can effectively inhibit type II-a Cas9 ortholog proteins in bacteria and eukaryotic cells. Among them, acriia25.1 can inhibit both DNA binding and DNA cleavage activity of SpyCas9, showing a novel and unique inhibition mechanism. Based on this, the inventors developed a novel chemically inducible iAcr system, creating an efficient method of chemically controllable gene editing. The invention has important application value.

Description

Method for chemically controllable gene editing by engineering AcrIIA25.1 protein

The invention is a divisional application of Chinese invention patent application with the application date of 2022, 02-11, the application number of CN 202210127749.7 and the invention name of novel CRISPR-Cas9 inhibitor and a method for modifying and applying the novel CRISPR-Cas9 inhibitor to chemically controllable gene editing.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a novel CRISPR-Cas9 inhibitor protein and a method for modifying the CRISPR-Cas9 inhibitor protein to be applied to chemically controllable gene editing.

Background

Clustered regularly interspaced short palindromic repeats (Clustered regularly interspaced short palindromic repeats, CRISPR) and their CRISPR-associated proteins (Cas) constitute a prokaryotic adaptive immune defense system against invasive genetic elements including phages and plasmids. CRISPR-Cas mediated defense processes can be divided into three phases: (1) adaptation phase: when a phage invades, a portion of the sequence information of the phage is inserted as a new spacer into the CRISPR locus of the bacterium, forming an immune memory; (2) expression stage: systematically expressing Cas genes and processing to form mature CRISPR RNA (crRNA); (3) interference stage: cas proteins recognize and destroy targeted nucleic acids under the guidance of crrnas, forming an immune defense. CRISPR-Cas systems are currently classified into 2 major classes (Class 1 and Class 2), 6 types, based on phylogenetic tree and mechanism of action. Class 1 systems (including I, III and type IV) use multi-subunit Cas protein complexes to target foreign nucleic acids; whereas Class 2 systems (including types II, V and VI) rely on a single effector protein (e.g., cas 9) to effect interference, and are therefore widely used. Up to now, various subtypes of Class 2CRISPR-Cas systems have been identified and developed as powerful gene editing tools, mainly including CRISPR-Cas9, cas12a (Cpf 1), cas13a (C2), etc. They play an increasingly important role in the fields of scientific research, agriculture, animal husbandry, clinical detection and treatment, etc. Among them, cas9 (SpyCas 9) from streptococcus pyogenes (Streptococcus pyogenes) is the most widely used Cas effector and has been developed as a powerful gene editing tool.

However, despite the long-term development of CRISPR gene editing technology in recent years, it still faces unsolved off-target problems, which hampers the application of CRISPR gene editing technology. To address this problem, some studies have further reduced off-target effects by engineering Cas proteins or guide RNAs to increase specificity of targeted recognition, or by expression of controlled Cas protein or guide RNA abundance versus time. In recent years, a series of proteins known as anti-CRISPR (Acr) have been found in prophages or phages that can inhibit the activity of a variety of CRISPR-Cas systems, and some Acr proteins (such as AcrIIA 4) have been demonstrated to reduce off-target effects in cells by inhibiting Cas9 activity. These properties make Acr a potential "on-off" tool for modulating CRISPR-based applications, so the exploration of Acr and its molecular mechanism research is of great importance in the field of gene editing.

Acr protein is a strategy that phages have evolved for the CRISPR-Cas system of antigenic organisms, i.e., the CRISPR-Cas system of prokaryotes is inactivated by encoding a protein (i.e., acr protein). The Acr proteins are found to be widely distributed in phages, prophages and invasive genetic elements, which 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 can widely inhibit I, II, III, V and type VI CRISPR-Cas systems. Since Acr proteins are of a wide variety and exhibit a high degree of sequence diversity, it is difficult to search for them by conventional sequence homology alignment methods. Scientists have used various bioinformatics and biochemistry methods to find and identify Acr proteins, including Guilt-by-association, self-targeting, machine-learning based bioinformatics methods, and biochemistry methods by Phage-first and High-throughput. Notably, 23 AcrIIA families have been discovered by a variety of methods. At present, only a small part of the molecular mechanism of AcrIIA protein is elucidated in detail. Studies show that these AcrIIA proteins mainly inhibit the third phase of CRISPR-Cas9 immunity-the interfering phase, and can be roughly divided into three strategies: (1) interfere with Cas and crRNA loading, such as AcrIIA15; (2) Preventing binding of Cas effect complex to targeting nucleic acid, such as AcrIIA4; (3) preventing Cas cleavage of the targeting nucleic acid, such as AcrIIA14. In view of the fact that Acr proteins can inhibit the CRISPR-Cas9 system by different strategies, and thus develop corresponding gene editing regulation tools, research on Acr molecular mechanisms has become a research hotspot in the field of gene editing.

Currently, some Acr proteins of these CRISPR-Cas systems are identified, and scientists believe that there are still broad Acr proteins that should be present and not found. This not only prevents us from understanding the manner of evolutionary competition between phage and host, but also prevents 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 proteins as effective gene editing tools to regulate the activity of Cas9 to address the safety issues of Cas9 applications. However, in the field of gene editing, the available Acr proteins and strategies for modulating Cas9 activity using Acr remain limited. Therefore, the novel CRISPR-Cas9 inhibitor protein and the method for modifying the Acr protein to be applied to controllable gene editing are found to have important significance in the field of gene editing.

Disclosure of Invention

The present invention aims to find new CRISPR-Cas9 inhibitor proteins and to establish a method of controlled gene editing based thereon.

The invention firstly protects the application of the AcrIIA protein in inhibiting the editing, the regulation and/or the imaging of Cas9 genes 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 a 1) or a 2) or a 3) or a 4) as follows:

a1 Amino acid sequence is a protein shown in SEQ ID NO. 1;

a2 A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal of the protein shown in SEQ ID NO. 1;

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 a 1) or a 2) and has the function of inhibiting the activity of the Cas9 protein;

a4 A protein having 60% or more homology with the amino acid sequence defined in SEQ ID NO. 1 and having a function of inhibiting the activity of Cas9 protein;

the AcrIIA25 protein may be b 1) or b 2) or b 3) or b 4) as follows:

b1 Amino acid sequence is a protein shown as SEQ ID NO. 2;

b2 A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal of the protein shown in SEQ ID NO. 2;

b3 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in b 1) or b 2) and has the function of inhibiting the activity of the Cas9 protein;

b4 A protein having 60% or more homology with the amino acid sequence defined by SEQ ID NO. 2 and having a function of inhibiting the activity of Cas9 protein;

the acriia25.1 protein may be c 1) or c 2) or c 3) or c 4) as follows:

c1 Amino acid sequence is a protein shown in SEQ ID NO. 3;

c2 A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal 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 c 1) or c 2) and has the function of inhibiting the activity of the Cas9 protein;

c4 A protein having 60% or more homology with the amino acid sequence defined by SEQ ID NO 3 and having a function of inhibiting the activity of Cas9 protein;

the AcrIIA26 protein may be d 1) or d 2) or d 3) or d 4) as follows:

d1 Amino acid sequence is a protein shown as SEQ ID NO. 4;

d2 A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal 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 in d 1) or d 2) and has the function of inhibiting the activity of the Cas9 protein;

d4 A protein having 60% or more homology with the amino acid sequence defined by SEQ ID NO. 4 and having a function of inhibiting the activity of Cas9 protein;

the AcrIIA27 protein may be e 1) or e 2) or e 3) or e 4) as follows:

e1 Amino acid sequence is a protein shown as SEQ ID NO. 5;

e2 A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal 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 in the e 1) or the e 2) and has the function of inhibiting the activity of the Cas9 protein;

e4 A protein having 60% or more homology with the amino acid sequence defined by SEQ ID NO. 5 and having a function of inhibiting the activity of Cas9 protein;

the AcrIIA28 protein may be f 1) or f 2) or f 3) or f 4) as follows:

f1 Amino acid sequence is a protein shown as SEQ ID NO. 6;

f2 A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal of the protein shown in SEQ ID NO. 6;

f3 Protein obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in f 1) or f 2) and having the function of inhibiting the activity of the Cas9 protein;

f4 A protein having 60% or more homology with the amino acid sequence defined by SEQ ID NO. 6 and having a function of inhibiting the activity of Cas9 protein;

the AcrIIA29 protein may be g 1) or g 2) or g 3) or g 4) as follows:

g1 Amino acid sequence is a protein shown in SEQ ID NO. 7;

g2 A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal of the protein shown in SEQ ID NO. 7;

g3 Protein obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in g 1) or g 2) and having the function of inhibiting the activity of the Cas9 protein;

g4 A protein having 60% or more homology with the amino acid sequence defined by SEQ ID NO. 7 and having a function of inhibiting the activity of Cas9 protein;

the AcrIIA30 protein may be h 1) or h 2) or h 3) or h 4) as follows:

h1 Amino acid sequence is a protein shown in SEQ ID NO. 8;

h2 A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal of the protein shown in SEQ ID NO. 8;

h3 Protein obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in h 1) or h 2) and having the function of inhibiting the activity of the Cas9 protein;

h4 A protein having 60% or more homology with the amino acid sequence defined by SEQ ID NO. 8 and having a function of inhibiting the activity of Cas9 protein;

the AcrIIA31 protein may be i 1) or i 2) or i 3) or i 4) as follows:

i1 Amino acid sequence is a protein shown as SEQ ID NO. 9;

i2 A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal 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 the i 1) or the i 2) and has the function of inhibiting the activity of the Cas9 protein;

i4 A protein having 60% or more homology with the amino acid sequence defined by SEQ ID NO 9 and having a function of inhibiting the activity of Cas9 protein;

the acriia31.1 protein may be j 1) or j 2) or j 3) or j 4) as follows:

j1 Amino acid sequence is a protein shown as SEQ ID NO. 10;

j2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by SEQ ID NO. 10;

j3 Protein obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in j 1) or j 2) and having the function of inhibiting the activity of Cas9 protein;

j4 A protein having 60% or more homology with the amino acid sequence defined by SEQ ID NO 10 and having a function of inhibiting the activity of Cas9 protein;

the AcrIIA32 protein may be k 1) or k 2) or k 3) or k 4) as follows:

k1 Amino acid sequence is a protein shown as SEQ ID NO. 11;

k2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by SEQ ID NO. 11;

k3 Protein obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in k 1) or k 2) and having the function of inhibiting the activity of the Cas9 protein;

k4 A protein having 60% or more homology with the amino acid sequence defined by SEQ ID NO. 11 and having a function of inhibiting the activity of Cas9 protein;

The acriia32.1 protein may be l 1) or l 2) or l 3) or l 4) as follows:

l 1) the amino acid sequence is a protein shown in SEQ ID NO. 12;

l 2) a fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by SEQ ID NO. 12;

l 3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in the l 1) or the l 2) and has the function of inhibiting the activity of the Cas9 protein;

l 4) has 60% or more homology with the amino acid sequence defined by SEQ ID NO. 12, and has a function of inhibiting the activity of 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 a 1), b 1), c 1), d 1), e 1), f 1), g 1), h 1), i 1), j 1), k 1) or l 1), the proteins shown in SEQ ID NO. 1 to SEQ ID NO. 12 may be linked at their amino-or carboxy-terminal end to the tag shown in Table 1, respectively.

TABLE 1 sequence of tags

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein of the above a 3), b 3), c 3), d 3), e 3), f 3), g 3), h 3), i 3), j 3), k 3) or l 3), wherein the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of a 3), b 3), c 3), d 3), e 3), f 3), g 3), h 3), i 3), j 3), k 3) or l 3) may be synthesized artificially or may be obtained by synthesizing the coding gene and then biologically expressing the gene.

The gene encoding the protein of a 3), b 3), c 3), d 3), e 3), f 3), g 3), h 3), i 3), j 3), k 3) or l 3) above can be obtained by deleting one or several amino acid residues from the DNA sequence encoding the protein, and/or by making one or several base pair missense mutations, and/or by ligating the coding sequence of the tag shown in Table 1 at its 5 'and/or 3' end.

The term "homology" as used herein refers to sequence similarity to a natural 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-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 in percent (%), which can be used to evaluate the identity between related sequences.

The use of any of the AcrIIA proteins described above in inhibiting Cas9 protein activity is also within the scope of the invention.

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

The invention also protects a chemically induced iAcr system; the system employs the acriia25.1 and/or the acriia32.1 as chemically inducible anti-CRISPR proteins.

In the above system, the protein using AcrIIA25.1 as the chemically inducible anti-CRISPR can be specifically carried out using iA25.1 (Intin-AcrIIA 25.1 (S76)) in Table 4.

In the above system, the protein using AcrIIA32.1 as the chemically inducible anti-CRISPR can be specifically carried out using iA32.1 (Intin-AcrIIA 32.1 (T40)) in Table 4.

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

The use of any of the above systems for achieving chemically controllable gene editing is also within the scope of the present invention.

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

The method may be regulated by means of 4-hydroxy tamoxifen.

The application of any of the above AcrIIA proteins in the preparation of a chemically inducible iAcr system is also within the scope of the present invention.

The use of any of the AcrIIA proteins described above for chemically controllable gene editing is also within the scope of the invention.

In any of the above applications, the AcrIIA protein may specifically be the AcrIIA25.1, the AcrIIA32.1, ia25.1 (intel-AcrIIA 25.1 (S76)) or ia32.1 (intel-AcrIIA 32.1 (T40)) in table 4.

The inventors of the present application found a broad distribution of nine novel families of type II-A Acr proteins (acrIIA 24-32) and three Aca (Acr-associated) (Aca 11-13) proteins in a Streptococcus moving element by the guilt-by-association method, using a broad distribution of acrIIA6 as a starting marker. Studies have found that AcrIIA24-32 is a specific inhibitor of the Streptococcus CRISPR-Cas9 system, and can effectively inhibit type II-A Cas9 proteins, including SpyCas9, streptococcus thermophilus (Streptococcus thermophilus) Cas9 (CR 1/St1Cas9 and CR3/St3Cas 9) in bacteria and human cells. Among these Acrs, acrIIA26, acrIIA27, acrIIA30, and AcrIIA31 strongly block Cas9 binding to DNA, while AcrIIA24 prevents DNA cleavage activity of Cas 9. Notably, acriia25.1 and acriia32.1 can inhibit DNA binding and DNA cleavage activity of SpyCas9, demonstrating a novel and unique inhibition mechanism. The inventors of the present application have also developed a variety of chemically inducible iAcr (intrinsic-CRISPR) systems based on acriia25.1 and acriia32.1, which are fusions consisting of Acr protein and 4-hydroxy tamoxifen (4-HT, 4-hydroxytamoxifen) reactive inteins. The iAcr system can effectively control CRISPR-Cas9 mediated gene editing activity after' translation in human cells, and has strong application prospect. The application expands the diversity of II-A type Acr proteins and Acr inhibition mechanisms, and a chemically controllable gene editing method 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 in the dark reading frame as numbers. Arrows indicate the protein sequence homology relationship (shown as a percentage) between acr loci. The Aca gene is shown in white reading frame. Other adjacent genes are shown in gray, and some known gene structural information (e.g., AP2 DNA binding domains) is annotated according to the NCBI website. Asterisks indicate the genes detected in the E.coli plasmid interference experiments. (B) Plasmid and escherichia coli plasmid interference experimental schematic diagrams for analysis and design of 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 in inhibiting Cas9 ortholog proteins (SpyCas 9, st1Cas9, and St3Cas 9). # indicates that it is below the detection standard. n=3. Values are shown as mean ± SEM.

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

FIG. 3 is an illustration of the acrIIA24-32 being a streptococcal CRISPR-Cas9 system specific inhibitor protein. A is a plaque assay schematic diagram. Whether AcrIIA24-32 has broad-spectrum inhibitory activity against a variety of II-A, II-B, II-C Cas9 homologous proteins was investigated. Coli harbors plasmids expressing Cas9, sgrnas and Acr, followed by infection with T4 phage. The T4 phage gene 23 was designed to be targeted by a variety of Cas9 homologous proteins (abbreviated by strain names). B is a plaque assay experiment using 10-fold serial dilutions of T4 phage (black circles) to evaluate the inhibitory effect of different Acr proteins on different types of Cas9 ortholog proteins, including type II-a (SpyCas 9, st1Cas9, st3Cas9, and SaCas 9), type II-B (FnCas 9), and type II-C (NmeCas 9) CRISPR-Cas9 systems.

FIG. 4 is a schematic representation of an AcrIIA24-32 inhibiting type II-A Cas9 homologous protein in vitro. A is a schematic diagram of a DNA cleavage experiment. The Cas9 RNP complex targets DNA substrates with or without Acr. B-D is plasmid DNA targeted for linearization using SpyCas9 (B), st3Cas9 (C), and St1Cas9 (D) RNP complexes in the presence or absence of Acr. Open arrows indicate uncleaved linearized plasmid DNA. Filled arrows indicate cleavage products. Gel images represent three independent replicates.

FIG. 5 is a schematic representation of the ability of AcrIIA24-32 to inhibit Cas 9-mediated gene editing in human cells. A is a schematic of T7E1 experiments to examine the inhibitory activity of Acr on Cas9 protein in HEK293T cells. Plasmids encoding Cas9, sgrnas and Acr were co-transfected into human cells and subsequently analyzed by T7E1 experiments. (B-G) is a T7E1 experimental representative gel image to show the inhibitory activity of Acrs on SpyCas9 (B), st3Cas9 (D) and St1Cas9 (F). The target sites for human genes AAVS1 (SpyCas 9 targeted) and DYRK1A (St 1Cas9 and St3Cas9 targeted) are shown at the top of each gel image, PAM is highlighted in underline. The subtype and serial number of Acr are shown above the gel; a, acrIIA; c, acrIIC. Open arrows indicate T7E1 undigested bands (unedited). Filled arrows represent T7E1 digested bands (edited). Editing efficiency is shown as "index (%)" at the bottom of each lane. In the presence of different Acrs, spyCas9 (C), st3Cas9 (E) and St1Cas9 (G) -mediated gene editing efficiencies were quantified. n=3, the values show mean ± SEM.

Fig. 6 is an illustration of Acrs employing multiple strategies to inactivate Cas9 in human cells. A is a schematic of a plasmid designed for human telomere localization fluorescence imaging to investigate the strategy of Acrs inhibition of the II-a Cas9 ortholog protein. Plasmids encoding Cas9 fluorescent protein, their respective telomere-targeting sgRNA plasmids, and plasmids encoding Acr protein (blue Fluorescent protein TagBFP marker) into the U20S cell line. S_ (d) Cas9- (mCherry) ₃ Representing the three plasmids used in this experiment, including Spy_dCA9- (mCherry) ₃ 、St1_dCas9-(mCherry) ₃ And St3_dCA9- (mCherry) ₃ . B is a sequence targeting the human telomeres by Cas9 homologous proteins (Nme, spy, st1 and St 3) in U2OS cells. C is Nme_dCA9- (sfGFP) ₃ 、Spy_dCas9-(mCherry) ₃ Representative images of U2OS cells co-transfected with Acr plasmid. Fluorescent channels are shown at the top of the figure, with different Acr proteins shown on the right side of each row. The scale bar represents 10 μm. D is the protein of Spy_dCAs9- (mCherry) in the presence of different Acr proteins ₃ Cells forming telomeres were quantified. By having Nme_dCA9- (sfGFP) ₃ And Spy_dCA9- (mCherry) ₃ The percentage of co-localized telomere imaged cells was calculated. n = number of cells scored under each condition. E-H is transfected St3_dCA9- (mCherry) ₃ (E) Or St1_dCA9- (mCherry) ₃ (G) Nme_dCA9- (sfGFP) ₃ And representative images of U2OS cells following different Acr plasmids. The scale bar represents 10 μm. St3_dCAs9- (mCherry) was quantified under each condition using the same method as in D ₃ (F) And St1_dCA9- (mCherry) ₃ (H) Cell proportion of telomere forming.

FIG. 7 shows that Acrs has no effect on the formation of Cas9-sgRNA RNP complex. A and B are EMSA experiments to examine the effect of AcrIIA25.1, acrIIA26, acrIIA27, acrIIA28, and AcrIIA32.1 on SpyCas9-sgRNA RNP complex formation. The assay was analyzed on a non-denaturing polyacrylamide gel and sgrnas were stained by SYBR gold. The order of addition of the different reaction components (Acrs, spyCas9 and sgrnas) is shown above the dashed box. C is the EMSA assay performed to analyze the effect of AcrIIA24 protein on St3Cas9 binding to sgRNA when AcrIIA24 is added before or after the addition of sgRNA. D is the detection of binding of St1Cas9 to sgRNA using EMSA experiments with or without AcrIIA30 or AcrIIA 31.

Fig. 8 is a graph showing multiple mechanisms of action of Acrs to inhibit Cas9 in vitro. a-F is an EMSA experiment performed by adding Acrs to the reaction system before or after adding target DNA to analyze different Acr proteins for Cas9 effects of RNP binding to DNA, including AcrIIA25.1 (A), acrIIA26 (B), acrIIA27 (C), acrIIA32.1 (D), acrIIA24 (E), acrIIA30, acrIIA31 (F), cas9 RNP (256 nM) and Acr gradients (0.125, 0.25, 0.5, 0.1, 0.2, 0.4, 0.8 and 1.6. Mu.M). Experiments were performed on non-denaturing gels and the targeted DNA was labeled with Cy 5. Experiments were performed in triplicate and representative picture presentations were selected. G is by adding additional Mg in EMSA ²⁺ To restore the cleavage activity of Cas9 on DNA to conduct 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 the different conditions shown in G. SpyCas9 RNP (500 nM), st1Cas9 RNP (500 nM), acrs (10 μm) and substrate DNA (50 nM) (non-target strand is labeled with Cy 3). Experiments were repeated 3 times and representative gel patterns are shown. J is a summary of the different inhibition mechanisms of the anti-CRISPR proteins identified in this study. AcrIIA26, acrIIA27, acrIIA30, and AcrIIA31 block binding of Cas9 to DNA, while AcrIIA24 inhibits DNA cleavage activity of Cas9. Notably, acriia25.1 and acriia32.1 can inhibit both DNA binding and DNA cleavage activity of Cas9.

Fig. 9 is a variety of mechanisms by which Acrs inhibits St3Cas 9. DNA cleavage assays were performed to analyze the effect of AcrIIA24, acrIIA25, and AcrIIA32.1 on St3Cas9 cleavage DNA activity under different conditions, as shown in G in fig. 8. St3Cas9 RNP (500 nM), acrs (10. Mu.M) and substrate DNA (50 nM) (target strand labeled with Cy 5). Experiments were repeated 3 times and representative gel patterns are shown.

FIG. 10 is the creation of a chemically inducible anti-CRISPR system to achieve chemically controllable gene editing. A is the schematic diagram of the iAcr system. Insertion of ligand-dependent inteins into Acr proteins inactivates Acr. Binding of 4-HT can trigger intein protein self-splicing and restore Acr activity to inhibit Cas9.B is a schematic diagram 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 BFPs (HEK 293T-BFP cells) were transfected with plasmids encoding guide editors, BFP-targeted pegrnas and intein-Acr hybrids in the presence or absence of 4-HT (1 μm). The percentage of GFP positive cells was calculated by flow cytometry 72 hours after transfection. PE can convert BFP to GFP by substituting CC for GT resulting in a single H66Y amino acid substitution. The target sequence and PAM sequence are shown in phantom and underlined, respectively. C is a comparison of the efficiency of conversion of BFP to GFP under different conditions. intein-Acr variants are recognized by residues replaced by intein. Wild-type Acrs included C1 (AcrIIC 1), A4 (AcrIIA 4), A5 (AcrIIA 5), a25.1 (AcrIIA 25.1) and a32.1 (AcrIIA 32.1) as controls. n=3, the values show mean ± SEM. . D is a representative gel image of the T7E1 assay to show the inhibitory activity of Acr and iAcr proteins on SpyCas9 in the presence or absence of 4-HT. HEK293T cells were transfected with Cas9 (1 μg), sgRNA (0.5 μg) and Acr (0.5 or 0.25 μg) plasmids. The targeting sequence is shown on top of each gel image, PAM is highlighted in underline. Editing efficiency is shown as "index (%)" at the bottom of each lane. E and F are representative gel images of T7E1 assays, used 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 μg), sgRNA (0.5 μg) and Acr (0.25 μg) plasmids. Targeting sequences for human EMX1 (targeted by SpyCas 9) and DYRK1A (targeted by St3Cas 9) are shown on top of each gel, PAM is highlighted in underline. Editing efficiency is shown as "index (%)" at the bottom of each lane.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

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

TABLE 2

Examples

1. Materials and methods

1. Microorganism

Coli strains (TOP 10 or Mach1-T1, biomed) were used for plasmid amplification and plasmid intervention experiments. Coli strain (T7 Express, biomed) was used for protein expression and plaque assay analysis. Coli is typically (unless otherwise indicated) cultivated in a Lysogenic Broth (LB) medium at 37 ℃ with the appropriate antibiotics (if needed): ampicillin (50. Mu.g/ml), kanamycin (50. Mu.g/ml) or chloramphenicol (25. Mu.g/ml).

2. Cell lines

HEK293T and HEK293T-BFP cells were placed in DMEM medium (Gibco) containing 10% (vol/vol) fetal bovine serum (FBS, gibco) at 37℃with 5% CO ₂ Culturing in an incubator.

U2OS cells were placed in McCoy's 5A (modified) medium (Gibco) containing 10% FBS at 37℃with 5% CO ₂ Culturing in an incubator.

3. Bioinformatics analysis

The BLASTP program was used to search the non-redundant protein database for AcrIIA6 (accession number: AVO 22749.1) homologous proteins to manually check whether the acr gene and the aca gene are possible from neighboring candidate genes. HHPred of MPI bioinformatics kit is used to identify DNA binding domains from neighboring genes, where aca candidates are screened. BLASTP search was performed using the aca gene to screen acr candidate genes and further verified by biochemical analysis.

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

4. Plasmid interference experiments in E.coli

The DNA sequence encoding the Acr protein was synthesized by bomad and ligated into the pBAD24 vector. Using CaCl ₂ The heat shock procedure transformed the plasmid into E.coli. Briefly, E.coli TOP10 or Mach1-T1 strain harboring Acr plasmid was cultured overnight in LB medium containing 0.2% arabinose, and then used as competent cells. Transformation was then performed with 25ng pT and 25ng Cas9 plasmid (with matched or mismatched spacers). After 2 hours of recovery in LB medium containing 0.2% arabinose, cells were inoculated 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 (tan 3500) and counted by ImageJ software. The inhibitory activity of each Acr was calculated by the conversion ratio between the pT-targeting and non-pT-targeting Cas9 plasmids.

5. Plaque assay experiments

Coli (T7 Express, biomed) cells were co-transformed with a plasmid expressing Cas9-sgRNA targeting phage T4 and a compatible plasmid encoding Acr protein. Both the Cas9 plasmid and the empty pBAD24 plasmid (without Acr) for the non-targeted phage T4 served as controls. Coli harboring Acr and Cas9 plasmids were cultured in LB medium containing 50 μg/ml ampicillin and 25 μg/ml chloramphenicol and grown overnight at 37 ℃. The following 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 the Cas9 protein. After two hours, acr protein expression was induced by the addition of 0.2% arabinose. After two more hours, 200. Mu.l of culture was incubated with 4ml of melted LB-agar (0.7%, supplemented with 10mM MgSO) ₄ ) Mix and pour over a solution containing 10mM MgSO ₄ And 0.2% arabinose solid LB-agar (1.5%, containing 1mM IPTG, 50. Mu.g/ml ampicillin and 25. Mu.g/ml chloromycetin)Plain) plate. Next, a 10-fold dilution of phage T4 was spotted on 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 routinely induced to express protein in LB medium containing 1mM IPTG and 50. Mu.g/ml kanamycin and incubated at 18℃for 16 hours. Cells were harvested and resuspended in lysis buffer (50 mM 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 protein eluted with 500mM imidazole. Amicon Ultra centrifugal filters (Millipore) were used to concentrate proteins and buffer was exchanged to storage buffer (20 mM HEPES-NaOH, pH 7.5, 5% (v/v) glycerol, 300mM NaCl and 1mM DTT). For Acr protein, after incubation with Tobacco Etch Virus (TEV) protease at 4 ℃ overnight, 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 DNA are shown in Table 3.

TABLE 3 Table 3

Note that: * Which represents fluorescein.

All sgrnas in the assay were prepared using an in vitro T7 transcription kit (Invitrogen) according to manufacturer's manual, and a linearized sgRNA plasmid was used to generate the transcription template. 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 and Cas9 protein (500 nM), sgRNA (500 nM), 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 sgRNA for 10 min at 37 ℃. Acr protein was then added and incubated for an additional 20 minutes at room temperature. Target DNA was added and incubated at 37 ℃ for 10 minutes. The reaction was stopped by adding 1. Mu.l Proteinase K (PK). The products were analyzed on a 1% agarose/1×TAE (Tris-acetate-EDTA) gel.

For FIGS. 8 and 9, fluorescently labeled substrate DNA was used for cleavage assays, prepared by annealing synthetic oligonucleotides of target and non-target strands labeled with Cy5 or Cy 3. The procedure for the cleavage assay is shown as G in figure 9. Briefly, cas9 protein (500 nM), and sgRNA (500 nM) were mixed at 37 ℃ for 10 min to form Cas9 RNP complexes, and the reaction system used 1 x binding buffer (150 mM KCl, 5mM EDTA, 5mM MgCl ₂ 1mM DTT, 5% (v/v) glycerol, 50. Mu.g/ml heparin, 0.01% Tween 20 and 100. Mu.g/ml BSA, 20mM Tris-HCl pH 7.6) to eliminate the lytic activity of Cas 9. Then, acr protein (10. Mu.M) and substrate DNA (50 nM) were added in different order and incubated at room temperature for 20 minutes, respectively. Subsequently, mgCl is added ₂ (10 mM) to restore DNA cleavage activity of Cas9, then incubated for an additional 20 minutes at room temperature. The reaction was stopped by addition of Gel Loading Buffer II (Invitrogen) and incubated for 6 min at 85 ℃. The products were analyzed on a 12% denaturing PAGE gel and visualized by Typhoon 7000 (GE).

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

The DNA sequence encoding the Acr protein (AcrIIA 4, acrIIA5, acrIIA25.1 or AcrIIA 32.1) was cloned into pcdna3.1 vector for expression in human cells.

The intein 37R3-2 sequence was synthesized and inserted into the position of the Acr protein to construct an 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 Table 4

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Note that: the intein 37R3-2 is underlined singly and the Acr is double underlined.

9. T7 endonuclease I (T7E 1) detection

The target sequences in the AAVS1, EMX1 and DYRK1A loci and the primer sequences for PCR amplification are shown in Table 5. For fig. 5, HEK293T cells cultured in 24 well plates were transfected with 1 μg Cas9 plasmid, 0.5 μg sgRNA plasmid and 0.5 μg Acr plasmid per well using Lipofectamine LTX reagent (Invitrogen) according to the manufacturer's protocol. For FIG. 10, HEK293T cells were transfected with 1 μg of Cas9 plasmid, 0.5 μg of sgRNA plasmid, 0.5 or 0.25 μg of Acr (iAcr) plasmid in 24 well plates, with or without 4-HT (1 μM, selleck S7827). After 72 hours of 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 before adding T7 endonuclease I (NEB), and incubated at 37 ℃. Samples were separated in a 3% agarose/1×TAE gel. The bands were quantified using ImageJ software.

The calculation formula of the genome editing efficiency of the mammalian cells is as follows:

TABLE 5 primers and target sequences used in T7E1 detection

10. Human cell telomere fluorescent imaging

For imaging, U2OS cells were cultured on a 15mm 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 300ng of the Acr plasmid. 24 hours after transfection, cells were fixed with 4% paraformaldehyde (Beyotime) and observed and imaged using a nikon a1r+ confocal microscope and 60 x oily objective.

Cell quantification cells from each condition were encoded by a single experimenter by a marker number. While another experimenter, who did not know these conditions, observed and scored the cells under a microscope. For quantification, only TagBFP and mCherry fluorescence were expressed simultaneously and nme_dcas9- (sfGFP) ₃ Assessing the presence or absence of co-localized s_ (d) Cas9- (mCherry) in cells forming green telomeres ₃ Red telomere case.

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

RNA EMSA was performed by incubating Cas9 protein (256 nM) and sgRNA (256 nM) in the order shown in the legend with or without Acrs (5 μm). The reaction was performed in 1 Xbinding buffer (150 mM KCl, 5mM EDTA, 5mM MgCl) ₂ 1mM 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 protein were added in different order and incubated at 37 ℃ for 10 min, respectively. Samples were analyzed on a 6% Tris-borate-EDTA (TBE) polyacrylamide gel and visualized using a Typhoon7000 (GE) SYBRGold (Invitrogen) stain.

The inventors performed DNA EMSA, briefly, cas9-sgRNA complexes were incubated in 1 x binding buffer for 10 min at 37 ℃ at concentrations shown in the legend of the figure. 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 Acr protein at different concentrations was added and incubated at room temperature for 20 minutes. 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 XTBE 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 the BFP expression gene at the AAVS1 locus in HEK293T cells, constructing HEK293T-BFP cell lines. Briefly, HEK293T cells were transfected with Cas9, AAVS 1-targeted sgRNA plasmids and a donor vector comprising a homologous sequence and harboring BFP and puromycin resistance genes. HEK293T-BFP cells were selected by puromycin (2. Mu.g/ml) treatment and flow cytometry (FACSariaIII, 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 Addgene (# 132775). The BFP-targeting pegRNA plasmid was constructed by synthesizing a DNA sequence containing the target, the sgRNA scaffold, PBS and RT templates, and integrated into the U6-sgRNA vector. HEK293T-BFP cells were cultured in 24 well plates and then transfected per well with PE2 (1. Mu.g), BFP-targeted pegRNA (0.5. Mu.g) and Acr (0.25. Mu.g) plasmids with or without 4-HT (1. Mu.M, selleck S7827) using Lipofectamine LTX reagent (Invitrogen).

2. Experimental results

1. Discovery of nine novel II-A type Acr (AcrIIA 24-32) protein families

By the rule of gurlt-by-association, i.e., the presence of a cognate gene (Aca) in the vicinity of the Acr gene, the encoded protein possesses a helix-helix (HTH) conserved domain for modulating the activity of Acr. Information retrieval was performed on AcrIIA6 (access: AVO 22749.1) using BLAST program (see a in fig. 1). The Acr candidate gene is established by combining with other characteristics such as the amino acid sequence size (generally 50-200 amino acids) of the Acr protein, whether the Acr protein exists in a phage/prophage region and the like. In addition, a screening system (see B in fig. 1) for Acr proteins of SpyCas9, st1Cas9 and St3Cas9 systems was designed using an escherichia coli plasmid interference experiment to rapidly and accurately identify candidate Acr proteins. The experimental approach was to clone the gene encoding Cas9 protein into bacterial expression plasmids with grnas, used as exogenous CRISPR-Cas9 system in e. And cloning the gene encoding the candidate Acr protein 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 Acr inhibits Cas9 protein was identified by calculating the transformation efficiency of pT plasmid in escherichia coli harboring exogenous Cas9 and candidate Acr plasmid.

By screening with E.coli plasmid interference experiments, nine novel families of type II-A Acr (AcrIIA 24-32) proteins and three aca proteins were found from the mobile element of Streptococcus (Streptococcus) (see FIGS. 1A and B). Of the 11 Acr proteins tested (two Acr homologous proteins: acrIIA25.1 and AcrIIA32.1 were added), 5 Acr proteins (AcrIIA 25, acrIIA27, acrIIA28, acrIIA32 and AcrIIA 32.1) exhibited robust inhibitory activity against SpyCas9 and St3Cas9. In addition, acrIIA25.1 and AcrIIA26 are able to significantly inhibit SpyCas9, whereas AcrIIA24 and AcrIIA29 inactivate 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 demonstrate the discovery of nine novel families of type II-a Acr (AcrIIA 24-32) proteins, and the confirmation of this by plasmid interference experiments that most of the Acr proteins are able to robustly inhibit the Streptococcus-derived type II-a Cas9 protein in e.

To further determine the distribution of AcrIIA24-32 homologous proteins, comprehensive phylogenetic analyses were performed based on BLAST results (see fig. 2). The data show that the homologous proteins of AcrIIA28, acrIIA30 and AcrIIA32 are few and distributed over only a few streptococcal genomes or phages (see E, G and I in fig. 2). In contrast, other proteins homologous to Acr proteins are more widely distributed in streptococcal mobile elements. While the AcrIIA26, acrIIA27, acrIIA29 and AcrIIA31 protein families exist in various strains of streptococcus, such as streptococcus salivarius (s. Salivarius) and streptococcus pyogenes (s. Pyogens) (see C, D, F and H in fig. 2). Furthermore, acrIIA24 and AcrIIA25 homologous proteins are present not only in the streptococcus genome strain but also in various streptococcus phages (see a and B in fig. 2). The data indicate that the AcrIIA24-32 protein family is mainly distributed in streptococcus, indicating that AcrIIA24-32 has potential roles in the army competition between streptococcus phage and host.

2. AcrIIA24-32 is a streptococcus CRISPR-Cas9 system specific inhibitor protein

To further investigate whether AcrIIA24-32 possesses a broad spectrum of inhibitory activity, plaque assay experiments were performed. A number of well-defined Cas9 homologous proteins were selected for validation, including type II-a (SpyCas 9, st1Cas9, st3Cas9 and SaCas 9), type II-B (FnCas 9) and type II-C (NmeCas 9) CRISPR systems (a in fig. 3). Coli carries a Cas9 expression plasmid capable of targeting the T4 phage gene 23, whereas Cas9 plasmid not targeting the T4 phage is a control. Coli infection of these transformed Cas9 expression plasmids with 10-fold serial dilutions of T4 phage in the presence or absence of Acr (a in fig. 3). AcrIIA30 and AcrIIA31 were found to specifically inhibit St1Cas9, while other Acr proteins could strongly inhibit both SpyCas9 and St3Cas9 (B in fig. 3).

Furthermore, acrIIA24-32 showed no detectable inhibitory activity on SaCas9, nmcas 9 and FnCas 9. The data indicate that AcrIIA24-32 is a streptococcus CRISPR-Cas9 system specific inhibitor protein.

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

In plasmid intervention experiments, some of the proteins in AcrIIA24-32 exhibited weak inhibitory activity against the CRISPR-Cas9 system of streptococci (e.g. AcrIIA29 weakly inhibited St3Cas 9). However, in plaque assay experiments AcrIIA24-32 was able to restore T4 phage to almost the same level as the non-targeted control, indicating that these Acr proteins had robust inhibitory activity against all type II-a Cas9 homologs (SpyCas 9, st1Cas9, and St3Cas 9) (B in fig. 3).

To eliminate this difference and confirm the inhibitory activity of AcrIIA24-32 on the streptococcus CRISPR-Cas9 system, cas9 and Acr proteins were purified and DNA cleavage experiments were performed in vitro. AcrIIA24-32 inhibited activity of SpyCas9, st1Cas9, and St3Cas9 proteins, as determined by targeting linearized plasmid DNA by Cas9 RNP in the presence or absence of Acr (a in fig. 4). The results showed that the results of the DNA cleavage experiments were substantially identical to those of the plasmid intervention experiments. SpyCas9 was able to be inhibited by AcrIIA25, acrIIA25.1, acrIIA26, acrIIA27, acrIIA28, acrIIA32, and AcrIIA32.1 (B in fig. 4). Other Acr proteins besides AcrIIA25.1 and AcrIIA30 can inhibit St3Cas9, while AcrIIA24, acrIIA25, acrIIA27, acrIIA31 and AcrIIA32 show stronger inhibitory activity against St3Cas9 (C in fig. 4). Furthermore, only AcrIIA30 and AcrIIA31 can effectively inactivate St1Cas9 in vitro, consistent with the results of plasmid intervention and plaque assay experiments (D in fig. 4). DNA cleavage experiments were also performed under different conditions, i.e. pre-incubation of apo-Cas9 and Acr proteins prior to introducing the sgrnas and target DNA into the reaction (data not shown). The experimental results were found to be not significantly different under these two reaction conditions, indicating that AcrIIA24-32 acts primarily on Cas9 RNP and affects downstream functions of Cas9 RNP.

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

In view of the wide range of applications 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 using T7 endonuclease 1 (T7E 1) 72 hours after transfection (FIG. 5A). The human endogenous loci AAVS1 and DYRK1A are designed for gene editing of type II-a Cas9 ortholog proteins of streptococcus.

Remarkably, it was observed that a plurality of Acrs showed strong inhibition of SpyCas9, st1Cas9 and St3Cas9 in human cells (see table 6). AcrIIA25.1, acrIIA26, acrIIA27, acrIIA28, acrIIA32 and AcrIIA32.1 proteins almost completely inhibited the activity of SpyCas9, the level of inhibition of these proteins was comparable to that of the known potent inhibitor protein AcrIIA5, whereas AcrIIA25 inhibited SpyCas9 only weakly (average inhibitory activity was 44%) (B and C in fig. 5). Furthermore, st3Cas 9-mediated gene editing can be inhibited to varying degrees by a variety of Acrs, including AcrIIA24 (average 91%), acrIIA25 (average 56%), acrIIA27 (average 83%), acrIIA28 (average 84%), acrIIA29 (average 33%), acrIIA32 (average 98%), and AcrIIA32.1 (average 93%) (D and E in fig. 5). It was also found that AcrIIA30, acrIIA31 and AcrIIA31 homologous proteins (AcrIIA 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 orthologues can effectively inhibit streptococcal Cas9 homologous proteins (SpyCas 9, st1Cas9, and St3Cas 9) in human cells.

TABLE 6

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Note that: 20% < inhibitory activity <50%, labeled "+";50% < inhibitory activity <80%, labeled "++"; the inhibition activity is more than 80 percent, sign "+ ++"; 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 to inhibit Cas9 in human cells. The mechanism of action of Acrs can be assessed by imaging of telomeric DNA with mCherry-labeled streptococcal Cas9 protein, while super-folder, sf GFP-labeled nmercas 9 can be used as a telomere indicator signal.

First, the effect of potent inhibitory proteins on binding of SpyCas9 to DNA was studied, including AcrIIA25.1, acrIIA26, acrIIA27, and AcrIIA32.1, and AcrIIA4 and AcrIIA5 as controls. Spy_dCA9- (mCherry) was observed ₃ And Nme/u dCAS9- & lt- & gtsfGFP) ₃ Is capable of co-localization to telomeres in cells co-expressing AcrIIA5, whereas in cells expressing AcrIIA4, the expression of Spy_dCA9- (mCherry) is eliminated ₃ Red end-particles formed (C in fig. 6). However, spy_dCA9- (mCherry) was caused in cells expressing either AcrIIA25.1, acrIIA26, acrIIA27 or AcrIIA32.1 ₃ The red end-point formed was lost without affecting nme_dcas9- (sfGFP) ₃ Green end particle sites formed (C in fig. 6). Subsequently, spy_dCAs9- (mCherry) in the presence of different Acr proteins was quantified ₃ Cell number forming telomeres. The presence of Spy_dCA9- (mCherry) was observed in 93.1% of cells in AcrIIA5 expressing cells ₃ Red 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 AcrIIA25.1, acrIIA26, acrIIA27, and AcrIIA32.1 can block binding of SpyCas9 to the targeting DNA efficiently in human cells.

Subsequently, to explore the mechanism of action of potent inhibitory proteins of St1Cas9 and St3Cas9, similar fluorescence imaging systems were established using St1Cas9 and St3Cas9 for targeting human telomeres in U2OS cells (a and B in fig. 6). St3_dCAs9- (mCherry) was observed ₃ Or St1_dCA9- (mCherry) ₃ With Nme_dCA9- (sfGFP) ₃ Co-localize to telomeres in cells (E and G in fig. 6). AcrIIA24 pair St3_dCAs9- (mCherry) ₃ There was no effect on co-localization of telomeres, 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_dCA9- (mCherry) was observed in 96.8% of cells expressing AcrIIA5 and 96.4% of cells expressing AcrIIA24 by quantitative experiments ₃ Red end-points formed, whereas in cells expressing AcrIIA27 or AcrIIA32.1 no St3 dCas9- (mCherry) was observed ₃ Red end-particles formed (F in fig. 6). In addition, st1_dCA9- (mCherry) was observed in 72.4% of cells expressing AcrIIA5, 3.4% of cells expressing AcrIIA30 and 0% of cells expressing AcrIIA31 ₃ Red end-particles formed (H in fig. 6).

The results indicate that Acrs from streptococcal mobile elements employ a variety of inhibition strategies to inhibit Cas9 protein. AcrIIA25.1, acrIIA26, acrIIA27, acrIIA30, acrIIA31, and AcrIIA32.1 are effective in blocking Cas9 protein binding to DNA. However, acrIIA24 does not prevent binding of Cas9 protein to DNA targets, suggesting that AcrIIA24 may specifically inhibit cleavage of substrate DNA by Cas9, with a mechanism of action similar to AcrIIC1.

6. Acrs exhibit multiple mechanisms of action to inhibit Cas9

Gel migration experiments (electrophoretic mobility shift assays, EMSA) were performed in vitro to further examine the mechanism of action of these Acrs on Cas 9. RNA EMSA was first performed to determine whether these Acrs affected the formation of Cas9-sgRNA RNP complex. AcrIIA25.1, acrIIA26, acrIIA27, acrIIA28 and AcrIIA32.1 were found to have no effect on the formation of SpyCas9-sgRNA RNP (a and B in fig. 7). Nor AcrIIA24, acrIIA30, and AcrIIA31 prevents binding of St3Cas9 or St1Cas9 to sgrnas (C and D in fig. 7).

Subsequently, DNA EMSA experiments were performed to investigate how Acrs acted on Cas9 RNP to affect downstream functions of Cas9 RNP. The Cas9 protein with catalytic activity is mixed with sgRNA to form a Cas9 RNP complex, and 10mM EDTA is added into a reaction system to eliminate the cleavage effect of Cas9 on DNA. One fluorescently labeled substrate DNA (Cy 5-labeled target strand) design can be targeted by SpyCas9, st1Cas9, and St3Cas 9. It was observed that AcrIIA25.1, acrIIA26, acrIIA27, and AcrIIA32.1 effectively abrogated binding of SpyCas9 RNP to DNA only when added to the reflection system prior to the addition of 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 inhibition mechanism of acriia32.1 on St3Cas9 was studied, obtaining similar results as acriia32.1 on SpyCas 9. It was also observed that AcrIIA24 can capture Cas9-DNA complex, resulting in DNA hypermigration ("super-shift"), whether the AcrIIA24 protein was added before or after the addition of the target DNA (E in fig. 8). This result suggests that AcrIIA24 should specifically inhibit the cleavage activity of St3Cas9 on DNA substrates. The inhibition mechanism of St1Cas9 by AcrIIA30 and AcrIIA31 was also studied, and it was found that AcrIIA30 and AcrIIA31 effectively prevented binding of St1Cas9 RNP to DNA only when added to the reflection system prior to the addition of the target DNA (F in fig. 8). Like AcrIIA25.1 and AcrIIA32.1, acrIIA30 can bind to the Cas9 complex bound to DNA, resulting in DNA hypermigration ("super-shift"), whereas AcrIIA31 cannot (F in fig. 8).

Given some unusual behavior of AcrIIA25.1, acrIIA32.1, and AcrIIA30, these Acr proteins are speculated to not only block Cas9 binding to DNA, but also to inhibit the cleavage activity of Cas9 on DNA in Cas9-sgRNA-DNA-Acr quaternary complexes. To verify the hypothesis, by adding additional Mg in EMSA ²⁺ To restore the cleavage activity of Cas9 on DNA for DNA cleavage experiments (G in fig. 8). It was examined whether Acr protein would affect the DNA cleavage activity of Cas9 under different reaction conditions, i.e., adding Acrs before or after adding target DNA in the reaction system. The data shows that by adding additional Mg in EMSA ²⁺ DNA cleavage activity was effectively restored for all of SpyCas9, st1Cas9, and St3Cas9 (H, I in fig. 8 and fig. 9). Compared with AcrIIC1 protein, acrIIA25.1, acrIIA32.1 and control AcrIIA4 can strongly inhibit the cleavage activity of SpyCas9 on DNA when added to the reaction system prior to the addition of target DNA. However, the AcrIIA25.1 and AcrIIA32.1 can also inactivate the DNA cleavage activity of SpyCas9 by adding them to the reaction system after adding the target DNA, as compared to the AcrIIA4 control (H in fig. 8). In combination with EMSA and DNA cleavage assays, the data show that acriia25.1 and acriia32.1 can inhibit DNA binding and DNA cleavage activity of SpyCas9, a novel inhibition mechanism different from other previously reported Acrs (J in fig. 8).

Whether AcrIIA24, acrIIA25, and AcrIIA32.1 inhibited the DNA cleavage activity of St3Cas9 was further studied. AcrIIA24, acrIIA25 and AcrIIA32.1 were found to show strong inhibition of DNA cleavage by St3Cas9, whether added before or after addition of target DNA (fig. 9). The results indicate 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 mechanism of inhibition of the DNA cleavage activity of St1Cas9 by AcrIIA30 and AcrIIA31 was also studied, especially considering that AcrIIA30 can bind to Cas9-sgRNA-DNA ternary complex in EMSA. It was found that St1Cas 9-mediated DNA cleavage can be effectively inhibited only by adding AcrIIA30 and AcrIIA31 prior to the addition of the target DNA, indicating that both AcrIIA30 and AcrIIA31 can inhibit DNA binding of St1Cas9 without affecting the DNA cleavage activity of St1Cas9 (I and J in fig. 8). Taken together, the results demonstrate that these Acrs found exhibit a variety of abilities to inhibit Cas9, the mechanism of which includes blocking DNA binding, DNA cleavage, or both.

7. Establishment of chemically inducible anti-CRISPR (chemical induced anti-CRISPR) system to achieve chemically controllable gene editing

Since acriia25.1 and acriia32.1 can inhibit DNA binding and DNA cleavage activity of Cas9, it shows a strong application potential that can be used to modulate 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, given that AcrIIA4 can only block binding of Cas9 to DNA, while AcrIIA5 specifically inhibits DNA cleavage of Cas9, an intein-Acr hybrid was designed by fusion of Acr protein with ligand-dependent intein (intein) 37R3-2, insertion of an intein into Acr protein would result in Acr inactivation, while binding of 4-hydroxytamoxifen (4-hydroxytamoxifen, 4-HT) to the intein would trigger self-splicing of the intein protein and restore Acr activity to inhibit Cas9 (a in fig. 10).

The 4-HT responsive inteins were inserted into Acrs by replacing individual residues of Acrs (cysteine, alanine, serine or threonine) because intein protein splicing left individual cysteine residues, and replacement of these residues would minimize the effect of the resulting cysteine point mutations (Table 4). To examine the effect of intein-Acr hybrids on Cas9 in human cells, a BFP-to-GFP reporting system was designed, using guided editing (PE) (B in fig. 10). Functional HEK293T cells are established, namely blue fluorescent protein (HEK 293T-BFP cells) is integrated at an AAVS1 site, and flow cytometry analysis shows that the expression proportion 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. Mu.M) (FIG. 10B). The effect of intein-Acr hybrids on Cas9 can be calculated by comparing the efficiency of editing of BFP to GFP under each condition.

The results showed that 4-HT treatment had no significant effect on PE and wild-type (WT) Acrs activity in human cells (FIG. 10C). Of the 10 intein-Acr variants, the activity of AcrIIA4 (T28 and a 58), acrIIA5 (S87), acrIIA25.1 (S59) was not regulated by 4-HT. Although AcrIIA5 (a 68), acrIIA25.1 (S30) and AcrIIA32.1 (T24 and a 73) can switch to the activated state in response to 4-HT to inhibit Cas9, these four intein-Acr variants are weakly 4-HT dependent (1.8 fold modulation on average). Only two intein-Acr variants, acrIIA25.1 (S76) and AcrIIA32.1 (T40), were effective in inhibiting PE-mediated BFP-to-GFP editing in the presence of 4-HT, exhibiting 4-HT dependent regulation (3.3 and 3.6 fold changes, respectively). We then refer to intein-AcrIIA25.1 (S76) and intein-AcrIIA32.1 (T40) as iAcrIIA25.1 (abbreviated iA 25.1) and iAcrIIA32.1 (abbreviated iA 32.1), respectively.

To further examine whether ia25.1 and ia32.1 possess 4-HT dependent activity to inhibit Cas9 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 WT Acrs (D and E in FIG. 10). Remarkably, inhibitory activity of iA25.1 and iA32.1 was observed to be 4-HT dependent. These two proteins have a slight effect on SpyCas9 activity in the absence of 4-HT, but in the presence of 4-HT they switch to a highly active state to inhibit Cas9 (D and E in fig. 10). To further examine the potential application of iAcr, T7E1 analysis was performed to investigate whether ia32.1 could be activated by 4-HT to inhibit St3Cas 9-mediated gene editing in human cells. As expected, the data show that ia32.1 with 4-HT triggering activity can inhibit St3Cas 9-mediated gene editing in human cells (F in fig. 10).

Thus, the results indicate that these icacrs exhibit potent 4-HT-dependent modulation for post-translational control of CRISPR-Cas 9-mediated genome editing in human cells.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present 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 respect to specific embodiments, 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 application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

Application of acriia protein in inhibiting Cas9 gene editing, regulation and/or imaging in cells or eukaryotes;

the AcrIIA protein is AcrIIA25 or AcrIIA25.1 protein;

the AcrIIA25 protein is b 1) or b 2) as follows:

b1 Amino acid sequence is a protein shown as SEQ ID NO. 2;

b2 A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal of the protein shown in SEQ ID NO. 2;

the acriia25.1 protein is c 1) or c 2) as follows:

c1 Amino acid sequence is a protein shown in SEQ ID NO. 3;

c2 A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal of the protein shown in SEQ ID NO. 3;

the Cas9 protein is SpyCas9 protein or/and St3Cas9 protein.
2. The use according to claim 1, characterized in that: the cells are bacterial cells.
3. Use of an AcrIIA protein as claimed in claim 1 for inhibiting Cas9 protein activity; the Cas9 protein is SpyCas9 protein or/and St3Cas9 protein.
4. A use according to claim 3, characterized in that: the inhibition of Cas9 protein activity is manifested by inhibition of Cas9 protein binding to DNA and/or inhibition of Cas9 protein cleavage activity to substrate DNA.
5. A chemically inducible iAcr product, characterized in that: the product comprises acriia25.1, a ligand-dependent intein and 4-hydroxy tamoxifen as in claim 1; wherein the acriia25.1 is a chemically inducible anti-CRISPR protein; the iAcr is an incocable anti-CRISPR.
6. Use of the product of claim 5 for achieving chemically controllable gene editing; the chemistry is controlled by 4-hydroxy tamoxifen.
7. A method of chemically controllable gene editing, characterized by: the method adopts the chemically induced iAcr product of claim 5 for gene editing; the method relies on the regulation of 4-hydroxy tamoxifen.
8. Use of an AcrIIA protein as claimed in claim 1 in the preparation of a chemically inducible iAcr system; the iAcr is an incocable anti-CRISPR; the chemical induction is 4-hydroxy tamoxifen induction.
9. Use of an AcrIIA protein as claimed in claim 1 for chemically controllable gene editing; the chemistry is controlled by 4-hydroxy tamoxifen.