Cas9 protein with improved stability, kit and method for improving Cas9 protein stability

Abstract

The present invention discloses a Cas9 protein with improved stability, a kit, and a method for improving the stability of the Cas9 protein. The present invention finds that the Cas9 protein can be degraded by the autophagy-lysosome pathway mediated by a molecular chaperone, and amino acid sequence analysis finds that the Cas9 protein contains a motif that can bind to the molecular chaperone heat shock homologous protein 70. On this basis, the present invention constructs Cas9 mutant proteins respectively, and then obtains a highly stable Cas9 protein with combined mutations. The highly stable Cas9 protein constructed by the present invention will help expand the application of gene editing systems and effectively reduce the cost of gene editing.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and in particular to a Cas9 protein with improved stability, a kit, and a method for improving the stability of the Cas9 protein. Background Art

[0002] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a widely existing prokaryotic defense system in nature, used to defend against invading bacteriophages and foreign genetic materials. This defense system recognizes specific DNA sequences through a single-stranded guide RNA (gRNA) and guides CRISPR-associated (Cas) proteins with endonuclease activity to cut DNA double strands. The CRISPR / Cas system exists in a variety of bacteria and archaea, including three main types (type I, type II and type III) and 12 subtypes. Compared with type I and type III, the type II CRISPR / Cas system can rely on a single Cas protein to accurately target specific DNA sequences, so it is currently a widely used gene editing system. The CRISPR / Cas9 system is a member of the type II CRISPR / Cas system. Due to its simple operation and high editing efficiency, the system is currently the most widely used gene editing system in the world, and has brought many new possibilities to scientific research, especially providing strong technical support for disease treatment and crop improvement.

[0003] The CRISPR / Cas9 system consists of a Cas9 protein with DNA endonuclease activity and a guide RNA (gRNA) fused with a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). During gene editing, the gRNA guides the Cas9 protein to the complementary DNA sequence next to the adjacent protospacer-adjacent motif (PAM), and then the endonuclease activity of the Cas9 protein mediates specific double strand breaking (DSB). Insertion or deletion mutations caused by non-homologous end joining (NHEJ) repair triggered by DSB can lead to frameshifting of the target gene. Currently, plasmid DNA, mRNA or Cas9 / gRNA ribonucleoprotein complex (RNP) of the CRISPR / Cas9 system can be delivered to target cells through delivery systems such as viral vectors, nanoparticles or extracellular vesicles, and have been widely used in clinical / preclinical trials for the treatment of various genetic diseases. It is worth mentioning that on November 16, 2023, the Medicines and Healthcare products Regulatory Agency (MHRA) of the United Kingdom announced the approval of CASGEVY, a CRISPR / Cas9 gene editing therapy product jointly developed by Vertex Pharmaceuticals Incorporated and CRISPR Therapeutics. TM (exagamglogene autotemcel, Exa-cel) conditional marketing authorization. On December 8, 2023, the U.S. Food and Drug Administration (FDA) also approved the product for marketing. This is the world's first gene editing therapy approved for the treatment of sickle cell disease (SCD) and β-thalassemia (TDT), and it is also the first therapy approved for the use of CRISPR / Cas9 "gene scissors". The world's first approval of CRISPR / Cas9 gene editing therapy will bring major changes to the medical field, and indicates that CRISPR / Cas9 technology has a very broad application prospect.

[0004] Since the Cas9 / gRNA ribonucleoprotein (RNP) complex can achieve gene editing quickly and efficiently with high safety, its application is becoming more and more extensive. In the Cas9 / gRNA RNP complex, although the stability of gRNA can be improved by a variety of chemical modifications, a more stable Cas9 protein is still required for more efficient and durable use in gene editing. However, the degradation mechanism of Cas9 protein in cells has not yet been elucidated. Therefore, revealing the degradation mechanism of Cas9 protein in cells and constructing a highly stable Cas9 protein on this basis will help expand the application of gene editing systems and effectively reduce the cost of gene editing by reducing the amount of Cas9 protein in the Cas9 / gRNA RNP complex. Summary of the invention

[0005] In order to solve at least some of the problems in the prior art, the present invention constructs a highly stable Cas9 protein, thereby providing a new tool and new strategy for the CRISPR / Cas9 system in disease treatment, and can reduce the amount of Cas9 protein used and effectively reduce the cost of gene editing, thereby contributing to the further promotion and application of the CRISPR / Cas9 system. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, a Cas9 protein with improved stability is provided, which contains a mutation of at least one amino acid of the pentapeptide motif at positions 669-673 and / or 892-896.

[0007] In certain embodiments, according to the Cas9 protein with improved stability described in the present invention, the pentapeptide motif at positions 669-673 and / or 892-896 is a binding domain of the molecular chaperone heat shock cognate protein 70.

[0008] In certain embodiments, according to the Cas9 protein with improved stability described in the present invention, the pentapeptide motifs at positions 669-673 and 892-896 comprise the sequences shown by IRDKQ and QRKFD, respectively.

[0009] In certain embodiments, according to the Cas9 protein with improved stability described in the present invention, the mutation includes at least one of K672A, Q673A, Q892A, and R893A.

[0010] The second aspect of the present invention provides an isolated nucleic acid molecule encoding the Cas9 protein with improved stability according to the present invention.

[0011] The third aspect of the present invention provides a vector comprising the nucleic acid molecule of the present invention.

[0012] The fourth aspect of the present invention provides a host cell comprising the vector of the present invention.

[0013] In a fifth aspect of the present invention, a gene editing tool is provided, which comprises the Cas9 protein with improved stability described in the present invention and at least one guide RNA.

[0014] In a sixth aspect of the present invention, a kit for gene editing, gene targeting or gene cutting is provided, which comprises the Cas9 protein with improved stability, nucleic acid molecule, vector, host cell or gene editing tool described in the present invention.

[0015] In a seventh aspect of the present invention, a method for improving the stability of Cas9 protein is provided, comprising the step of introducing at least one amino acid mutation in the pentapeptide motif at positions 669-673 and / or 892-896 of wild-type Cas9.

[0016] An eighth aspect of the present invention provides a gene editing method, which comprises the step of contacting the gene editing tool described in the present invention with cells in vitro.

[0017] The present invention discovered for the first time that Cas9 protein can be degraded by the autophagy-lysosome pathway mediated by molecular chaperones, and amino acid sequence analysis revealed that Cas9 protein contains a "KFERQ"-like pentapeptide motif that can bind to the molecular chaperone heat shock homologous protein 70 (HSC70). On this basis, the present invention constructed Cas9 proteins with mutations in the "KFERQ"-like pentapeptide motif, and then obtained a highly stable Cas9 protein (HSCas9) with combined mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Detect and analyze the degradation of Cas9 protein by the autophagy-lysosome pathway mediated by molecular chaperones.

[0019] Figure 2 Construct and screen highly stable Cas9 proteins.

[0020] Figure 3 Inhibitory effect of HSCas9 on HBV replication. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] Cas9 proteins with improved stability

[0025] In one aspect of the present invention, a Cas9 protein with improved stability is provided (sometimes also referred to herein as a "Cas9 mutant protein" or a "mutated Cas9 protein"). The term "improved stability" refers to the property that the Cas9 protein can maintain conformational stability without degradation under degradation conditions, particularly in the presence of the molecular chaperone heat shock homologous protein 70 (HSC70). Preferably, the amount of Cas9 mutant protein degraded is less than 20%, preferably less than 10%, further preferably less than 5%, and further preferably less than 1%, or even substantially no degradation occurs, compared to the amount of Cas9 protein before mutation or wild-type Cas9 protein present.

[0026] In the present invention, amino acid residues can be represented by single letters or three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).

[0027] In a preferred embodiment, the Cas9 mutant protein of the present invention has at least one (e.g., any 2 or more than 2) amino acid mutation in the pentapeptide motif at positions 669-673 and / or 892-896 compared to the amino acid sequence of the wild-type Cas9 protein. Further preferably, the mutation includes at least one of a non-K amino acid at position 672, a non-Q amino acid at position 673, a non-Q amino acid at position 892, and a non-R amino acid at position 893.

[0028] In a preferred embodiment, non-K amino acids include but are not limited to at least one of the following: A, N, V, G, L, Q, F, W, Y, D, S, E, M, T, C, P, H and I, preferably amino acids carrying a positive charge such as A or H, and also preferably A.

[0029] In a preferred embodiment, non-Q amino acids include, but are not limited to, at least one of the following: A, N, V, G, L, F, W, Y, D, S, E, M, T, C, P, H, R and I, preferably amino acids carrying a positive charge such as A, R, H or K, and A is also preferred.

[0030] In a preferred embodiment, non-R amino acids include, but are not limited to, at least one of the following: A, N, V, G, L, Q, F, W, Y, D, S, E, M, T, C, P, H, I and K, preferably amino acids carrying a positive charge such as A or H, and also preferably A.

[0031] In a most preferred embodiment, the mutation includes at least one of K672A, Q673A, Q892A, and R893A.

[0032] The term "AxxxB" means that the amino acid A at position xxx is changed to amino acid B. Unless otherwise specified, the amino acid A at position xxx from the N-terminus is changed to amino acid B. For example, K672A means that K at position 672 is mutated to A. When multiple amino acid sites are mutated at the same time, K672A-Q673A or K672A / Q673A and the like can be used to express them. For example, K672A / Q673A means that K at position 672 is mutated to A and Q at position 673 is mutated to A at the same time.

[0033] In addition, fusion proteins comprising Cas9 mutant proteins are within the scope of protection of the present invention, for example, fusion proteins may contain additional proteins or polypeptides, detectable markers, tags or any combination thereof. Tags include but are not limited to HA tags, FLAG tags, SnoopTag, BCCP, green fluorescent protein tags, etc. Any other components suitable for detection, separation, expression, and transport can form fusion proteins with the Cas9 mutant proteins of the present invention. In addition, those skilled in the art will understand these various tag fusion technologies, as well as how to prepare and use fusion proteins containing them.

[0034] The Cas9 mutant protein, derivative of the protein or fusion protein comprising the same of the present invention is not limited by the preparation method thereof. For example, it can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0035] Nucleic acid molecules

[0036] In one aspect of the present invention, an isolated nucleic acid molecule is provided, the term "isolated nucleic acid" includes DNA, RNA, cDNA and vectors encoding them, wherein the DNA, RNA, cDNA and vectors do not contain other biological materials from which they can be derived or associated, such as cellular components. Typically, the isolated nucleic acid will be purified from other biological materials from which they can be derived or associated, such as cellular components.

[0037] In certain embodiments, the nucleotide sequence is codon optimized for expression in prokaryotes. In certain embodiments, the nucleotide sequence is codon optimized for expression in eukaryotic cells. Exemplary isolated nucleic acid molecules encoding mutant Cas9 proteins can be easily prepared by recombinant DNA technology or chemical synthesis methods, without particular limitation.

[0038] Carrier

[0039] The present invention also provides a vector comprising a Cas9 mutant protein, an isolated nucleic acid molecule or a polynucleotide. The vector should be understood as a broad term, which refers to an expression vector, a delivery vector or a vector system. The vector system for delivering genes in animals includes viral vectors (such as retroviruses, adeno-associated viruses and integrated phage viruses) and non-viral vectors (such as transposons and nanoparticles). The vector generally contains one or more expression cassettes (comprising one or more expression control sequences), wherein the expression control sequence is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence or mRNA, respectively.

[0040] Many different types of vectors are known. For example, phage, plasmid and viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors and herpes simplex vectors). The vector also includes a regulatory element operably linked to the nucleic acid molecule or polynucleotide. In certain embodiments, the regulatory element is selected from one or more of the following: an enhancer, a transposon, a promoter, a terminator, a leader sequence, a polyadenylation sequence, a marker gene.

[0041] The vector can be introduced into the desired cell by many conventional techniques. For example, the vector can be directly introduced into the cell using methods such as electroporation and microinjection, or the vector can be directly introduced into the cell using gene gun methods such as DNA particle bombardment.

[0042] In a preferred embodiment, the vector of the present invention comprises a combination of one or more vectors, the one or more vectors comprising:

[0043] (1) a first regulatory element, which is operably linked to a nucleotide sequence encoding a gRNA;

[0044] (2) a second regulatory element, which is operably linked to a polynucleotide encoding the Cas mutant protein;

[0045] Components (1) and (2) are located on the same or different carriers of the combination.

[0046] The first and second regulatory elements include a promoter (eg, a constitutive promoter or an inducible promoter), an enhancer, an internal ribosome entry site (IRES), and other expression control elements (eg, transcription termination signals, such as polyadenylation signals and poly-U sequences).

[0047] Host cells

[0048] The present invention also provides a host cell, which includes the above-mentioned vector. Suitable host cells for cloning or expressing DNA are prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include, but are not limited to, Escherichia coli, Bacillus subtilis, etc., for example, can be Escherichia coli cells (E.coli), such as Escherichia coli HMS174 (DE3) or BL21 (DE3). Commonly used eukaryotic host cells include, but are not limited to, yeast cells, insect cells, mammalian cells, etc.

[0049] The present invention also covers the progeny, clones, cell lines or cells derived from the cell lines of the above-mentioned cells, wherein the progeny, clones, cell lines or cells derived from the cell lines of the above-mentioned cells contain the nucleic acid molecules of the present invention or have been subjected to gene editing.

[0050] Gene editing tools

[0051] The present invention provides a gene editing tool, which is an engineered non-naturally occurring vector system, or a CRISPR-Cas9 system, which includes a Cas9 mutant protein or a nucleic acid molecule encoding the Cas mutant protein and a nucleic acid molecule encoding one or more guide RNAs (also called guide RNAs).

[0052] In certain embodiments, the nucleic acid sequence encoding the Cas9 mutant protein and the nucleic acid encoding one or more guide RNAs do not co-exist naturally. In certain embodiments, the nucleic acid molecule encoding the Cas9 mutant protein and the nucleic acid molecule encoding one or more guide RNAs are artificially synthesized.

[0053] Those skilled in the art will appreciate that the one or more guide RNAs can target one or more target sequences in a cell. The guide RNA is used to guide the Cas9 mutant protein to the target sequence position and then modify, edit or cut the target sequence, thereby changing or modifying the expression of the one or more gene products. The cells herein include one or more derived from animal, plant or microbial cells.

[0054] In certain embodiments, the Cas9 mutant protein directs cleavage of one or both strands at the location of the target sequence.

[0055] In a preferred embodiment, the engineered CRISPR system of the present invention comprises the above-mentioned Cas9 mutant protein and one or more guide RNAs, wherein the guide RNA includes a discontinuous repeat sequence (Direct repeat, DR) and a spacer sequence inserted therein capable of hybridizing with a target nucleic acid, and the Cas variant protein is capable of binding to the guide RNA and targeting a target nucleic acid sequence complementary to the spacer sequence.

[0056] Gene Editing Methods

[0057] On the one hand, the present invention provides an in vitro gene editing method for non-therapeutic and diagnostic purposes, such as for scientific research, or for screening for purposes such as antiviral candidate drugs. The in vitro method includes the step of contacting the gene editing tool with the cell in vitro. The in vitro gene editing method may also be referred to as a method for modifying a target nucleic acid in vitro, wherein the in vitro cell contains a target nucleic acid bound to the gRNA in the gene editing tool. The modification refers to a break in the target nucleic acid sequence, such as a single-strand / double-strand break in DNA, or a single-strand break in RNA. It is understood that the modification may also be the insertion of an exogenous nucleic acid into the break, or the modification may also be the introduction of another point mutation.

[0058] Reagent test kit

[0059] The present invention also provides a kit for nucleic acid detection, gene editing, gene targeting or gene cutting, which comprises the Cas9 protein with improved stability and instructions for illustrating the implementation steps of nucleic acid detection, gene editing, gene targeting or gene cutting.

[0060] The kit may further comprise other reagents capable of performing routine detection by various assay types, such as ELISA assay, immunoassay, protein chip or microarray, DNA / RNA chip or microarray, RT-PCR, third-generation sequencing, mass spectrometry, immunohistochemistry, flow cytometry, or cell screening.

[0061] In addition to the above components, the kit of the present invention may also include precautions related to regulating the manufacture, use or sale of diagnostic kits in a form specified by a governmental agency. In addition, the kit of the present invention may also be provided with detailed instructions for use, storage and troubleshooting. The kit may also be optionally arranged in a suitable robotically operated device preferably for high throughput settings.

[0062] In certain embodiments, the components of the kit of the present invention can be provided as dry powders. When reagents and / or components are provided as dry powders, the powder can be restored to its original state by adding a suitable solvent. It is expected that the solvent can also be arranged in another container. The container will usually include at least one vial, test tube, flask, bottle, syringe and / or other container means, wherein the solvent is optionally placed in equal parts. The kit can also include a means for a second container containing a sterile, pharmaceutically acceptable buffer and / or other solvent.

[0063] In certain embodiments, the components of the kit of the present invention may be provided in the form of a solution, such as an aqueous solution. In the case of being present in an aqueous solution state, the concentration or content of these components can be easily determined by those skilled in the art according to different needs. For example, for storage purposes, the concentration of the components may be present in a higher form, and when in working state or use, the concentration may be reduced to the working concentration by, for example, diluting the above-mentioned higher concentration solution.

[0064] In the case where there is more than one component in the kit, the kit will also typically include a second, third or other additional container in which the additional components may be placed separately. In addition, a combination of various components may be included in the container.

[0065] Methods to improve Cas9 protein stability

[0066] In one aspect of the present invention, a method for improving the stability of Cas9 protein is provided, which comprises the step of introducing at least one amino acid mutation in the pentapeptide motif at positions 669-673 and / or 892-896 of the wild-type Cas9 protein.

[0067] In a preferred embodiment, the introduced mutation includes at least one of a non-K amino acid at position 672, a non-Q amino acid at position 673, a non-Q amino acid at position 892, and a non-R amino acid at position 893.

[0068] In a preferred embodiment, non-K amino acids include but are not limited to: A, N, V, G, L, Q, F, W, Y, D, S, E, M, T, C, P, H and I, preferably positively charged amino acids such as A or H, and A is also preferred.

[0069] In a preferred embodiment, non-Q amino acids include but are not limited to: A, N, V, G, L, F, W, Y, D, S, E, M, T, C, P, H, R, I and K, preferably positively charged amino acids such as A, R, H or K, and A is also preferred.

[0070] In a preferred embodiment, non-R amino acids include but are not limited to: A, N, V, G, L, Q, F, W, Y, D, S, E, M, T, C, P, H and I, preferably positively charged amino acids such as A or H, and A is also preferred.

[0071] In a most preferred embodiment, the introduced mutation includes at least one of K672A, Q673A, Q892A, and R893A.

[0072] Those skilled in the art are familiar with how to introduce the desired mutations to obtain Cas9 mutant proteins, for example, they can be prepared by direct chemical synthesis methods, or they can be prepared by genetic engineering methods. A non-limiting example is, for example, the production of Cas9 mutant proteins by site-directed mutagenesis based on PCR. In certain embodiments, the step of introducing mutations includes centering on the site of mutation, dividing the DNA sequence design of Cas9 protein into two parts, designing two pairs of primers to amplify the two parts of DNA sequence respectively, and introducing the sequences that need to be mutated on the primers at the same time, and finally loading the two fragments onto the plasmid vector by cloning. The combination of mutations is achieved by splitting the DNA of Cas9 protein into multiple segments, and using PCR and cloning to achieve construction.

[0073] In addition, the present invention further found that highly stable mutant Cas9 protein can significantly enhance the inhibitory ability of the CRIPR / Cas9 system on hepatitis B virus (HBV) replication. Therefore, the present invention also provides the use of the mutant Cas9 protein in the preparation of a drug for HBV infection.

[0074] Example 1

[0075] The following shows that Cas9 protein can be degraded through the autophagy-lysosomal pathway mediated by molecular chaperones.

[0076] In order to explore the degradation mechanism of Cas9 protein in cells, the amino acid sequence of Cas9 protein was first analyzed. The results showed that the amino acid sequence of Cas9 protein contained four "KFERQ"-like pentapeptide motifs that may bind to HSC70 protein, namely 669 QUR 673 , 793 QILKE 797 , 892 QRKD 896 and 965 RdQ 969 ( Figure 1 A).

[0077] Then, protein co-immunoprecipitation (Co-IP) experiments confirmed that HSC70 and Cas9 proteins could bind to each other in cells ( Figure 1 B and 1C). At the same time, cell immunofluorescence experiments combined with confocal microscopy confirmed that HSC70 and Cas9 proteins can co-localize in cells ( Figure 1 D).

[0078] Further studies found that knocking down the expression of the HSC70 gene in HuH7 cells significantly increased the level of Cas9 protein in the cells ( Figure 1 E), while the level of Cas9 protein in cells was significantly reduced after overexpression of HSC70 ( Figure 1 F). Similarly, consistent results were obtained in HeLa-gHPV1 / Cas9 stably transfected cells ( Figure 1 G and 1H). The above results suggest that Cas9 protein may be degraded through the autophagy-lysosome pathway mediated by molecular chaperones.

[0079] To further confirm that Cas9 protein can be degraded by the chaperone-mediated autophagy-lysosome pathway, the present invention uses commercial chaperone-mediated autophagy-lysosome pathway agonist AR7 to treat HeLa-gHPV1 / Cas9 stably transfected cells. Since AR7 can activate the chaperone-mediated autophagy-lysosome pathway by specifically activating the transcriptional activity of the lysosomal associated protein 2A (LAMP2A) gene, the activation degree of AR7 is reflected by detecting the mRNA level of the LAMP2A gene in the cell.

[0080] The results showed that AR7 treatment activated the chaperone-mediated autophagy-lysosome pathway and significantly reduced the level of Cas9 protein ( Figure 1H and 1I). In addition, since the target protein degraded by the molecular chaperone-mediated autophagy-lysosomal pathway needs to rely on LAMP2A when passing through the lysosomal membrane, the present invention further detected the distribution of Cas9 and LAMP2A proteins in cells by confocal microscopy. The results showed that Cas9 and LAMP2A proteins can be co-localized in cells ( Figure 1 J). The above results indicate that Cas9 protein can be degraded via the autophagy-lysosome pathway mediated by molecular chaperones.

[0081] Figure 1(A) The "KFERQ"-like pentapeptide motif in Cas9 protein that potentially binds to HSC70 protein is shown. Co-IP experiments were performed using HSC70 antibody (B) and Flag antibody, the tag protein of Cas9-Flag fusion protein (C), and the mutual binding of Cas9 protein and HSC70 protein in HeLa-gHPV1 / Cas9 cells was analyzed. (D) Cell immunofluorescence experiment combined with confocal microscopy observed the distribution of Cas9 protein (green fluorescence) and HSC70 protein (red fluorescence) in HeLa-gHPV1 / Cas9 cells. (E) After co-transfection of PX458 plasmid and HSC70 siRNA or control siRNA (siNC) in HuH7 cells, the levels of HSC70 and Cas9 proteins in cells were detected by Western Blot, and α-tubulin was used as the intracellular reference protein. (F) PX458 plasmid and PCDH-HSC70-HA plasmid or vector control plasmid were co-transfected in HuH7 cells, and the levels of HSC70-HA and Cas9 proteins in cells were detected by Western Blot, with α-tubulin as the internal reference protein. (G) After HSC70 siRNA or control siRNA (siNC) was transfected in HeLa-gHPV1 / Cas9 cells, the levels of HSC70 and Cas9 proteins in cells were detected by Western Blot, with α-tubulin as the internal reference protein. (H) After PCDH-HSC70-HA plasmid or vector control plasmid was transfected in HeLa-gHPV1 / Cas9 cells, the levels of HSC70-HA and Cas9 proteins in cells were detected by Western Blot, with α-tubulin as the internal reference protein. (I) After HeLa-gHPV1 / Cas9 cells were treated with 20μmol / L AR7 for 24 hours, the level of intracellular Cas9 protein was detected by Western Blot, and α-tubulin was used as the intracellular reference protein. (J) The mRNA level of the LAMP2A gene in cells after the above AR7 drug treatment was detected by RT-qPCR. (K) Cell immunofluorescence experiment detected the distribution of Cas9 protein (green fluorescence) and LAMP2A protein (red fluorescence) in HeLa-gHPV1 / Cas9 cells.

[0082] Example 2

[0083] This example demonstrates the construction and screening of highly stable Cas9 proteins.

[0084] In order to construct and screen highly stable Cas9 proteins, the four KFERQ-like motifs of Cas9 proteins were first mutated separately. Figure 2 As shown in A, 669QUR 673 , 793 QILKE 797 , 892 QRKD 896 and 965 RdQ 969 Mutated to 669 IRDAA 673 (Cas9M1), 793 AALKE 797 (Cas9M2), 892 AAKFD 896 (Cas9M3) and 965 RKD 969 (Cas9M4). Then, the expression plasmids of the Cas9 protein mutants and the wild-type Cas9 protein expression plasmids were transfected into HeLa cells, and the cell pellets were collected 48 hours after transfection and the total protein was extracted. The results showed that compared with the wild-type Cas9 protein, 669 QUR 673 and 892 QRKD 896 After the two pentapeptide motifs were mutated, the level of Cas9 protein in the cell was significantly higher than that of wild-type Cas9 protein ( Figure 2 B).

[0085] Subsequently, the two motifs were mutated together. The results showed that the level of Cas9 protein with two motifs mutated together in HeLa and HuH7 cells was significantly higher than that of Cas9 protein with single site mutation ( Figure 2 C), and the stability of the Cas9 protein with two combined mutations in HeLa and HuH7 cells was significantly higher than that of the wild-type Cas9 protein ( Figure 2 D-2G). It can be seen that 669 QUR 673 and 892 QRKD 896 After the two pentapeptide motifs are mutated, the intracellular level of Cas9 protein can be increased by enhancing its stability. Therefore, the present invention refers to the Cas9 protein with two sites mutated together as a highly stable Cas9 (HSCas9) protein. Then, the PX458-HSCas9 plasmid and the HSC70 expression plasmid PCDH-HSC70-HA were co-transfected into HeLa and HuH7 cells. Three days after transfection, the effect of HSC70 on the level of HSCas9 protein was detected by Western Blot experiment. The results showed that HSC70 had no significant effect on the level of HSCas9 protein ( Figure 2 H). This result indicates that HSC70 cannot promote 669 QUR673 and 892 QRKD 896 HSCas9 proteins with two pentapeptide motif mutations were degraded via the chaperone-mediated autophagy-lysosomal pathway.

[0086] Furthermore, the binding ability of Cas9 and HSCas9 to HSC70 was compared by Co-IP experiments. The results showed that the binding of HSCas9 protein to HSC70 protein was significantly weakened compared with that of Cas9 protein ( Figure 2 I). The above results show that 669 QUR 673 and 892 QRKD 896 The two pentapeptide motifs mainly mediate the binding of HSC70 to Cas9 protein, which in turn causes the degradation of Cas9 protein via the molecular chaperone-mediated autophagy-lysosome pathway. 669 QUR 673 and 892 QRKD 896 HSCas9 proteins with two pentapeptide motif mutations can increase the stability of the protein by antagonizing molecular chaperone-mediated autophagy-lysosomal degradation.

[0087] Figure 2(A) Schematic diagram of the mutation site of the KFERQ-like motif of Cas9 protein. (B) PX458 plasmid and expression plasmids with different KFERQ-like motif site mutations (Cas9M1, Cas9M2, Cas9M3 and Cas9M4) were transfected into HeLa cell lines, and cell pellets were collected 48 hours after transfection. The levels of different Cas9 proteins were detected by Western Blot experiments, and α-tubulin was used as the internal reference protein of the cells. Based on the T2A sequence between the coding sequence of Cas9 and EGFP protein in the PX458 plasmid, the EGFP protein can be used to indicate the transfection efficiency of different plasmids. (C) HeLa and HuH7 cell lines were transfected with PX458 plasmid and expression plasmids with different KFERQ-like motif site mutations (Cas9M1, Cas9M3 and HSCas9), and cell pellets were collected 48 hours after transfection. The levels of different Cas9 proteins were detected by Western Blot experiment. α-tubulin was used as the internal reference protein of the cells, and EGFP was used to indicate the transfection efficiency of the plasmids in each group of cells. EGFP protein can be used to indicate the transfection efficiency of different plasmids. (D) HeLa cells were transfected with PX458 plasmid and PX458-HSCas9 plasmid, and 48 hours after transfection, cycloheximide (CHX) was added for treatment for 0, 3, 6, 9, 12 and 15 hours, and the levels of Cas9 and HSCas9 proteins were detected by Western Blot experiment after cell pellets were collected. α-tubulin was used as the internal reference protein of the cells. (E) The stability of Cas9 and HSCas9 proteins was compared by gray value analysis of the protein bands in (D). (F) PX458 plasmid and PX458-HSCas9 plasmid were transfected into HuH7 cells, and CHX was added 48 hours after transfection for 0, 3, 6, 9, 12 and 15 hours, respectively. After collecting the cell pellet, the levels of Cas9 and HSCas9 proteins were detected by Western Blot experiment, and α-tubulin was used as the internal reference protein of the cells. (G) The stability of Cas9 and HSCas9 proteins was compared by gray value analysis of the protein bands in (F). (H) PX458-HSCas9 plasmid and HSC70 expression plasmid PCDH-HSC70-HA were co-transfected into Hela and HuH7 cells, and the level of HSCas9 protein was detected by Western Blot experiment 3 days after transfection, and α-tubulin was used as the internal reference protein of the cells. (I) HeLa cells were transfected with PX458 plasmid and PX458-HSCas9 plasmid respectively. After 48 hours of transfection, the cell pellet was collected and Co-IP experiment was performed with HSC70 antibody to compare and analyze the binding ability between HSCas9 and HSC70 protein. α-tubulin was used as the internal reference protein of the cell, and EGFP was used to indicate the transfection efficiency of the plasmid in each group of cells.

[0088] Example 3

[0089] The following shows that HSCas9 can enhance the inhibitory effect of the CRISPR / Cas9 system on HBV replication in HuH7 cells.

[0090] The present invention first constructs an expression plasmid (abbreviated as pU6-t-g1-t-g2-t) capable of simultaneously expressing gHBV1 and gHBV2 through a tandem expression frame of gRNA. Then, the pBB4.5-HBV1.3 plasmid, the pU6-t-g1-t-g2-t plasmid and the PX458 / PX458-HSCas9 plasmid are co-transfected into HuH7 cells, and the cell culture supernatant and cell pellet are collected 4 days after transfection.

[0091] The results showed that compared with Cas9 protein, the ability of HSCas9 protein-mediated CRISPR / Cas9 system to reduce HBsAg and HBeAg levels in cell culture supernatant was significantly enhanced ( Figure 3 ), the above results indicate that HSCas9 can specifically enhance the inhibitory effect of CRISPR / Cas9 system on HBV replication in HuH7 cell line.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, a person skilled in the art should understand that the technical solutions described in the above embodiments can still be modified, or some of the technical features can be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Cas9 protein with improved stability, characterized in that It contains at least one amino acid mutation in the pentapeptide motif of the wild-type Cas9 protein sequence, such as IRDKQ and / or QRKFD, wherein the mutation is selected from at least one of the mutations of the K amino acid and the Q amino acid in IRDKQ to the A amino acid, and the mutation of the Q amino acid and the R amino acid in QRKFD to the A amino acid, respectively. The wild-type Cas9 protein is expressed by the PX458 plasmid.

2. A nucleic acid molecule, characterized in that It contains: (1) a polynucleotide sequence encoding the Cas9 protein with improved stability according to claim 1; or (2) A polynucleotide complementary to the polynucleotide sequence described in (1).

3. A carrier, characterized in that It comprises the nucleic acid molecule according to claim 2.

4. A host cell, characterized in that It comprises the vector according to claim 3.

5. A gene editing tool, characterized in that: It comprises the Cas9 protein with improved stability according to claim 1 and at least one guide RNA.

6. A kit, characterized in that It comprises the Cas9 protein with improved stability as described in claim 1, and the kit is used for nucleic acid detection, gene editing, gene targeting or gene cutting.

7. A method for improving the stability of Cas9 protein, characterized in that: The method comprises the step of introducing at least one amino acid mutation in the pentapeptide motif of the sequence of the wild-type Cas9 protein, such as IRDKQ and / or QRKFD, wherein the mutation is selected from at least one of the mutations of the K amino acid and the Q amino acid in IRDKQ to the A amino acid, and the mutation of the Q amino acid and the R amino acid in QRKFD to the A amino acid, and the wild-type Cas9 protein is expressed by the PX458 plasmid.

8. A gene editing method, characterized in that: The method comprises the step of contacting the gene editing tool described in claim 5 with cells in vitro.

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