Cas12 protein and use thereof

Abstract

A Cas12 protein and the use thereof. The Cas12 protein has an amino acid sequence having at least 90% sequence identity with a sequence as set forth in SEQ ID NO: 1 or a homologous sequence thereof, and the amino acid sequence of the Cas12 protein is not SEQ ID NO: 1. The Cas12 protein has good thermal stability and trans-cleavage activity, which is beneficial for the storage and transportation of the Cas12 protein in practical applications, increases application scenarios of the Cas12 protein and a CRISPR system based on the Cas12 protein, and provides more choices for users. In addition, on the basis of the existing theoretical foundation of protein engineering, the Cas12 protein mutant with improved thermal stability can also serve as a template / starting sequence for subsequent protein engineering.

Description

A Cas12 protein and its application

[0001] This application claims priority to Chinese Patent Application No. 2023111716279, filed on September 11, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention belongs to the field of biotechnology, and specifically relates to a Cas12 protein and applications thereof. Background Art

[0003] CRISPR and CRISPR-associated genes (Cas) are the immune systems that bacteria use to defend against foreign viral infections. Cas nucleases, guided by guide RNA, can bind to substrate nucleic acids, thereby cutting the nucleic acids. The modular nature of CRISPR makes this technology widely used in genome engineering. In addition to applications related to gene editing (such as base editing, guide editing, epigenetic editing, transcriptional regulation editing, etc.), CRISPR technology can also be used for nucleic acid detection. CRISPR nucleic acid detection relies on the trans-cleavage activity of Cas nucleases, that is, it can non-specifically cut single-stranded nucleic acids after binding to substrate nucleic acids.

[0004] Summary of the Invention

[0005] The problem to be solved by the present invention is the defect that Cas12 protein has few options and strict use conditions in the prior art, and a Cas12 protein and its application are provided. The Cas12 protein of the present invention has excellent thermal stability and trans-cleavage activity, increases the application scenarios of Cas12 protein and CRISPR system based thereon, and provides users with more choices.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] A first aspect of the present invention provides a Cas12 protein, the Cas12 protein having an amino acid sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 1 or a homologous sequence thereof, and the amino acid sequence of the Cas12 protein is not SEQ ID NO: 1;

[0008] The Cas12 protein has:

[0009] (1) having a trans-cleavage activity not less than 50% of that of the Cas12 protein as shown in SEQ ID NO: 1, and a thermal stability greater than that of the Cas12 protein as shown in SEQ ID NO: 1; or

[0010] (2) having a thermal stability not lower than that of the Cas12 protein as shown in SEQ ID NO: 1, and having a trans-cleavage activity higher than that of the Cas12 protein as shown in SEQ ID NO: 1;

[0011] or,

[0012] The Cas12 protein has an amino acid sequence in which G146R / D156R / R182V / D235R / E292R / D350R / E795Q / F863V / R883K / R887K / D952N / C965Y / V1011A / Q1108L / A1113V / S1132T / S1214P mutations occur in the amino acid sequence shown in SEQ ID NO: 1.

[0013] In the present invention, the letter before the site represents the original amino acid residue, and the letter after the site represents the amino acid residue after substitution. " / " indicates that the amino acid substitutions before and after exist at the same time.

[0014] In some embodiments of the present invention, the amino acid sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO: 1 is mutated at at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: 1.

[0015] In some preferred embodiments of the present invention, the mutation occurs at a position corresponding to the REC1, REC2, PI, WED-III and / or Nuc domain of the Cas12 protein.

[0016] In some embodiments of the present invention, the mutations occur in C10, G49, L59, H200, H370, M456, Y549, C565, W602, M701, H733, R737, M746, P806, V936, S962, M957, I976, M1035, C1090, F304, T307, P342, V377, D384, R385, R386, V428, A451, V452, V463, V500, T814, V842, Y854, H873, S874, F884, Q888, D959, C965, A966 and I996 corresponding to the amino acid sequence shown in SEQ ID NO:1.

[0017] In some preferred embodiments of the present invention, the mutation occurs in S962 corresponding to the amino acid sequence shown in SEQ ID NO: 1, and one or more selected from C10, G49, L59, H200, H370, M456, Y549, C565, W602, M701, H733, R737, M746, P806, V936, M957, I976, M1035, C1090, F304, T307, P342, V377, D384, R385, R386, V428, A451, V452, V463, V500, T814, V842, Y854, H873, S874, F884, Q888, D959, C965, A966 and I996.

[0018] In some specific embodiments of the present invention, the mutation occurs at S962, C10, I976 and C1090 corresponding to the amino acid sequence shown in SEQ ID NO: 1.

[0019] In some examples of the present invention, the mutation is S962 / C10L / I976L / C1090D; wherein, S962 is mutated to any other natural amino acid G, A, V, L, I, P, F, Y, W, T, C, M, N, Q, D, E, K, R or H.

[0020] In some embodiments of the present invention, the mutation is selected from one or more of the following: C10L, G49K, L59K, H200E, H370K, M456I, Y549K, C565V, W602G, M701L, H733L, R737K, M746Y, P806A, V936F, S962X, M957L, I976L, M1035R, C1090D, F304I, T307F, P342T, V377D, D384K, R385I, R386K, V428K, A451E, V452K, V463K, V500R, T814D, V842S, Y854K, H873Y, S874K, F884D, Q888K, D959N, C965N, A966E, and I996V; where X is G, A, V, L, I, P, F, Y, W, T, C, M, N, Q, D, E, K, R, or H.

[0021] In some preferred embodiments of the present invention, the mutation is selected from any one of the following: C10L, G49K, L59K, H200E, H370K, M456I, Y549K, C565V, W602G, M701L, H733L, R737K, M746Y, P806A, V936F, S962G, S962A, S962V, S962L, S962I, S962P, S962F, S962Y, S962W, S962T, S962C, S962M, S962N, S962Q , S962D, S962E, S962K, S962R, S962H, M957L, I976L, M1035R, C1090D, F304I, T307F, P342T, V377D, D384K, R385I, R386K, V428K, A451E, V452K, V463K, V500R, T814D, V842S, Y854K, H873Y, S874K, F884D, Q888K, D959N, C965N, A966E, and I996V.

[0022] In other preferred embodiments of the present invention, the mutation is selected from any two of the following:

[0023] C10L, L59K, M746Y, V936F, S962X, M957L, I976L, C1090D, T307F, A451E, and A966E; where X is G, A, V, L, I, P, F, Y, W, T, C, M, N, Q, D, E, K, R, or H.

[0024] In other preferred embodiments of the present invention, the mutations are selected from at least three, at least four or at least five of the following: C10L, G49K, L59K, M456I, Y549K, M701L, H733L, M746Y, V936F, S962X, M957L, I976L, C1090D, T307F, R385I, A451E, V452K, V500R and A966E.

[0025] In some specific embodiments of the present invention, the mutation is selected from any one of the following:

[0026] S962K / C1090D、M957L / C1090D、I976L / C1090D、S962K / I976L、M957L / I976L、C10L / C1090D、C10L / S962K、C10L / M957L、C10L / V936F、C10L / S962K / C1090D、C10L / M957L / C1090D、C10L / V936F / C1090D、C10L / I976L / C1090D、C10L / S962K / I976L、C10L / Y549K / C1090D、C10L / M957L / I976L、C10L / H733L / C1090D、C10L / V936F / I976L、Y549K / S962K / I976L、G49K / S962K / I976L、S962K / I976L / C1090D、G49K / Y549K / C1090D、M456I / S962K / I976L、C10L / M957L / S962K / C1090D、C10L / V936F / M957L / C1090D、C10L / S962K / I976L / C1090D、C10L / M965L / I976L / C1090D、C10L / Y549K / M957L / C1090D、C10L / V936F / I976L / C1090D、C10L / M957L / S962K / I976L、C10L / Y549K / I976L / C1090D、C10L / H733L / I976L / C1090D、C10L / M456I / I976L / C1090D、C10L / S962G / I976L / C1090D、C10L / S962A / I976L / C1090D、C10L / S962V / I976L / C1090D、C10L / S962L / I976L / C1090D、C10L / S962I / I976L / C1090D、C10L / S962P / I976L / C1090D、C10L / S962F / I976L / C1090D、C10L / S962Y / I976L / C1090D、C10L / S962W / I976L / C1090D、C10L / S962T / I976L / C1090D、C10L / S962C / I976L / C1090D、C10L / S962M / I976L / C1090D、C10L / S962N / I976L / C1090D、C10L / S962Q / I976L / C1090D、C10L / S962D / I976L / C1090D、C10L / S962E / I976L / C1090D、<h2 style=";text-align:left;direction:ltr">C10L / S962R / I976L / C1090D, C10L / S962H / I976L / C1090D, Y549K / S962K / I976L / C1090D, G49K / Y549K / S962K / I976L, Y549K / M957L / S962K / I976L, M456I / Y549K / S962K / I976L, L59K / Y549K / S962K / I976 L, C10L / Y549K / S962K / I976L / C1090D, G49K / Y549K / S962K / I976L / C1090D, C10L / Y549K / M957L / S962K / I976L, G49K / Y549K / M957L / S962K / I976L, M456I / Y549K / S962K / I976L / C1090D, C10L / G49K / Y549 K / S962K / I976L / C1090D、C10L / G49K / Y549K / M957L / S962K / I976L、C10L / M456I / Y549K / S962K / I976 L / C1090D、G49K / M456I / Y549K / S962K / I976L / C1090D、C10L / M456I / Y549K / M957L / S962K / I976L、C10 L / G49K / L59K / M456I / Y549K / V936F / M957L / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / M957L / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / H733L / M957L / S962K / I976L / C1090D、 C10L / G49K / L59K / M456I / Y549K / V936F / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / V936F / M957L / S962K / I976L、C10L / G49K / M456I / Y549K / V936F / M957L / S962K / I976L / C1090D、 G49K / L59K / M456I / Y549K / V936F / M957L / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / H733L / V936F / S962K / I976L / C1090D、<h2 style=";text-align:left;direction:ltr">C10L / G49K / L59K / M456I / Y549K / M957L / S962K / I976L,T307F / M746Y,S962K / A966E,L59K / A451E,A451E / S962K,T307F / M701L / M746Y,T307F / M746Y / S962K、T307F / R385I / M746Y、T307F / V500R / M701L / M746Y、T307F / M701L / M746Y / A966E、T307F / M701L / M746Y / S962K、T307F / R385I / M746Y / A966E、 T307F / R385I / M701L / M746Y, T307F / R385I / V500R / M746Y, T307F / R385I / M746Y / S962K, T307F / M746Y / S962K / A966E, T307F / V500R / M701L / M746Y / A 966E, T307F / R385I / V500R / M701L / M746Y, T307F / R385I / M701L / M746Y / A966E, T307F / R385I / V500R / M746Y / A966E, T307F / V500R / M746Y / S962K / A96 6E、T307F / R385I / M701L / M746Y / S962K、T307F / R385I / V500R / M746Y / S96 2K、A451E / S962K / A966E、L59K / S962K / A966E、R385I / S962K / A966E、C10L / S962K / A966E, S962K / A966E / C1090D, L59K / A451E / S962K / A966E, R385I / A451E / S962K / A966E, C10L / A451E / S962K / A966E, L59K / R385I / S962K / A966 E、C10L / L59K / S962K / A966E、L59K / R385I / A451E / S962K / A966E、C10L / L59K / A451E / S962K / A966E、C10L / R385I / A451E / S962K / A966E、L59K / A451E / S962K / A966E / C1090D、C10L / L59K / R385I / S962K / A966E、C10L / L59K / R385I / A451E / S962K / A966E / C1090D、C10L / L59K / R385I / A451E / S962K / A966E、C10L / L59K / R385I / A451E / V936F / S962K / A966E, L59K / R385I / A451E / S962K / A966 E / C1090D, C10L / L59K / A451E / S962K / A966E / C1090D, C10L / L59K / R385I / A451E / M7 01L / S962K / A966E, C10L / L59K / R385I / A451E / V452K / S962K / A966E, C10L / L59K / R385I / A451E / V500R / S962K / A966E, and C10L / L59K / R385I / A451E / M957L / S962K / A966E.

[0027] In some examples of the present invention, the mutation is selected from any one of the following:

[0028] <h2 style=";text-align:left;direction:ltr">S962K / C1090D、I976L / C1090D、S962K / I976L、C10L / S962K、C10L / S962K / C1090D、C10L / S962K / I976L、C10L / M957L / I976L、 Y549K / S962K / I976L、G49K / S962K / I976L、S962K / I976L / C1090D、G49K / Y549K / C1090D、M456I / S962K / I976L、C10L / M957L / S 962K / C1090D、C10L / S962K / I976L / C1090D、C10L / M965L / I976L / C1090D、C10L / Y549K / M957L / C1090D、C10L / V936F / I976L / C1090D、C10L / M957L / S962K / I976L、C10L / Y549K / I976L / C1090D、C10L / H733L / I976L / C1090D、C10L / M456I / I976L / C1090D、 Y549K / S962K / I976L / C1090D、G49K / Y549K / S962K / I976L、Y549K / M957L / S962K / I976L、M456I / Y549K / S962K / I976L、L59K / Y549K / S962K / I976L、C10L / Y549K / S962K / I9 76L / C1090D, G49K / Y549K / S962K / I976L / C1090D, C10L / Y549K / M957L / S962K / I976L, G49K / Y549K / M957L / S962K / I976L, M456I / Y549K / S962K / I976L / C1090D, C10L / G49K / Y549K / S962K / I976L / C1090D、C10L / G49K / Y549K / M957L / S962K / I976L、C10L / M456 I / Y549K / S962K / I976L / C1090D、G49K / M456I / Y549K / S962K / I976L / C1090D、C10L / M 456I / Y549K / M957L / S962K / I976L、C10L / G49K / L59K / M456I / Y549K / V936F / M957L / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / M957L / S962K / I976L / C1090D、<h2 style=";text-align:left;direction:ltr">C10L / G49K / L59K / M456I / Y549K / H733L / M957L / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / V936F / S962K / I976L / C1090D、C10L / G49K / L59 K / M456I / Y549K / V936F / M957L / S962K / I976L、C10L / G49K / M456I / Y549K / V9 36F / M957L / S962K / I976L / C1090D、G49K / L59K / M456I / Y549K / V936F / M957L / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / H733L / V936F / S962K / I976L / C1090D 、C10L / G49K / L59K / M456I / Y549K / M957L / S962K / I976L、T307F / M746Y、S962 K / A966E、L59K / A451E、A451E / S962K、T307F / M701L / M746Y、T307F / M746Y / S 962K, T307F / R385I / M746Y, T307F / V500R / M701L / M746Y, T307F / M701L / M746Y / A966E, T307F / M701L / M746Y / S962K, T307F / R385I / M746Y / A966E, T307 F / R385I / M701L / M746Y, T307F / R385I / V500R / M746Y, T307F / R385I / M746Y / S962K, T307F / M746Y / S962K / A966E, T307F / V500R / M701L / M746Y / A966E, T3 07F / R385I / V500R / M701L / M746Y、T307F / R385I / M701L / M746Y / A966E、T307F / R385I / V500R / M746Y / A966E、T307F / V500R / M746Y / S962K / A966E、T307F / R385I / M701L / M746Y / S962K, T307F / R385I / V500R / M746Y / S962K, A451E / S962K / A966E, L59K / S962K / A966E, R385I / S962K / A966E, C10L / S962K / A966ES962K / A966E / C1090D, L59K / A451E / S962K / A966E, R385I / A451E / S962K / A96 6E, C10L / A451E / S962K / A966E, L59K / R385I / S962K / A966E, C10L / L59K / S962 K / A966E, L59K / R385I / A451E / S962K / A966E, C10L / L59K / A451E / S962K / A966 E. C10L / R385I / A451E / S962K / A966E, L59K / A451E / S962K / A966E / C1090D, C1 0L / L59K / R385I / S962K / A966E, C10L / L59K / R385I / A451E / S962K / A966E / C10 90D, C10L / L59K / R385I / A451E / S962K / A966E, C10L / L59K / R385I / A451E / V93 6F / S962K / A966E, L59K / R385I / A451E / S962K / A966E / C1090D, C10L / L59K / A4 51E / S962K / A966E / C1090D, C10L / L59K / R385I / A451E / M701L / S962K / A966E, C10L / L59K / R385I / A451E / V452K / S962K / A966E, C10L / L59K / R385I / A451E / V500R / S962K / A966E, and C10L / L59K / R385I / A451E / M957L / S962K / A966E.

[0029] In some embodiments of the present invention, the mutation includes C10L, S962K and / or C1090D; for example, S962K.

[0030] In some embodiments of the present invention, the homologous sequence is selected from Cas12a proteins derived from different species or Cas12 proteins derived from different subtypes.

[0031] In the present invention, the homologous sequence of the Cas12 protein is preferably selected from the amino acid sequence of AsCas12a, BbCas12a, BoCas12a, FnCas12a, HkCas12a, Lb4Cas12a, Lb5Cas12a, OsCas12a or TsCas12a.

[0032] In the present invention, the homologous sequence of the Cas12 protein is preferably selected from Cas12a protein, Cas12b protein, Cas12c protein, Cas12d protein, Cas12e protein, Cas12f protein, Cas12g protein, Cas12i protein, Cas12j protein, Cas12k protein, Cas12l protein and TnpB protein.

[0033] The second aspect of the present invention provides a fusion protein or conjugate, which comprises the Cas12 protein as described in the first aspect fused or conjugated to a protein active functional domain or a non-protein active functional domain.

[0034] In some embodiments of the present invention, the fusion or conjugation includes fusion or conjugation directly or through a linker.

[0035] In some embodiments of the present invention, the protein active functional domain is selected from a polypeptide and a protein domain.

[0036] In some embodiments of the present invention, the non-protein active functional domain is selected from the group consisting of a nucleic acid molecule, a small molecule cytotoxic agent, a small molecule immunomodulator, and a small molecule pathway regulator.

[0037] In some preferred embodiments of the present invention, the polypeptide is selected from the group consisting of an affinity tag, a reporter tag, and a fusion tag.

[0038] In some preferred embodiments of the present invention, the protein domain is selected from a cytosine deaminase domain, an adenosine deaminase domain, a translation regulatory domain, a transcription regulatory domain, a nuclease domain, an epigenetic regulatory domain, a guide editing domain, a recombinase domain, a transposase domain and a transcription factor domain.

[0039] The third aspect of the present invention provides a combination of Cas12 proteins, comprising one or more Cas12 proteins as described in the first aspect, or the fusion protein or conjugate as described in the second aspect;

[0040] Optionally, the combination further includes a Cas12 protein mutant, and the Cas12 protein mutant is not the Cas12 protein described in the first aspect.

[0041] In some specific embodiments of the present invention, the Cas12 protein mutant has K632R / F863V / W890A / Q1108L / S1132T / S1214P, F863V / F884L / W890A / D952N / C965Y / V1011A / Q1108L / A1113V / S1132T / S1214P, K632R / F863V / Q1108L / S1132T / S1214P, F863V / F884L / D952N / C965Y / V1011A / Q1108L / A1113V / S1132 T / S1214P, F863V / D952N / C965Y / V1011A / Q1108L / A1113V / S1132T / S1214P, D156R / D235R / E292R / D350R, D156R / G532R / K53 8V / Y542R / K595R, D156R / R883K / R887K, G532R / K595R, D156R / G532R / K538V / Y542R / K595R, D832A / D925A / D1180A, R1138A, Amino acid sequences of G146R / R182V / E795Q, G532R / K595R, G532R / K538V / Y542R, or G532R / K595R mutations.

[0042] The fourth aspect of the present invention provides a CRISPR-Cas12 system, comprising the Cas12 protein as described in the first aspect, or the fusion protein or conjugate as described in the second aspect, or the combination as described in the third aspect, and a guide polynucleotide;

[0043] The guide polynucleotide forms a CRISPR complex with the Cas12 protein or fusion protein or conjugate, and the guide polynucleotide is partially complementary to the target nucleic acid and guides the CRISPR complex to hybridize with the target nucleic acid.

[0044] The fifth aspect of the present invention provides a vector system or recombinant cell, comprising a nucleic acid encoding the Cas12 protein as described in the first aspect, or the fusion protein or conjugate as described in the second aspect, or the combination as described in the third aspect, and one or more vectors encoding a nucleic acid of a guide polynucleotide;

[0045] The guide polynucleotide forms a CRISPR complex with the Cas12 protein or fusion protein or conjugate, and the guide polynucleotide is partially complementary to the target nucleic acid and guides the CRISPR complex to hybridize with the target nucleic acid.

[0046] In some embodiments of the present invention, the vector is selected from the group consisting of a plasmid, a virus, a bacteriophage, an extracellular vesicle, a liposome, and a lipid nanoparticle.

[0047] In some embodiments of the present invention, the starting cell of the recombinant cell is a eukaryotic cell or a prokaryotic cell.

[0048] In the present invention, the eukaryotic cells are selected from plant cells, fungal cells, animal cells or human cells, such as CHO cells, insect cells or HEK293 cells.

[0049] In the present invention, the prokaryotic cells are selected from bacterial cells, such as Escherichia coli cells.

[0050] The sixth aspect of the present invention provides a nucleic acid molecule encoding the Cas12 protein as described in the first aspect, the fusion protein or conjugate as described in the second aspect, the combination as described in the third aspect, and / or a guide polynucleotide;

[0051] The guide polynucleotide forms a CRISPR complex with the Cas12 protein or fusion protein or conjugate, and the guide polynucleotide is partially complementary to the target nucleic acid and guides the CRISPR complex to hybridize with the target nucleic acid.

[0052] The seventh aspect of the present invention provides a composition comprising the Cas12 protein as described in the first aspect, the fusion protein or conjugate as described in the first aspect, the combination as described in the third aspect, the CRISPR-Cas12 system as described in the fourth aspect, the vector system or recombinant cell as described in the fifth aspect, and / or the nucleic acid molecule as described in the sixth aspect.

[0053] The eighth aspect of the present invention provides a pharmaceutical composition, comprising the Cas12 protein as described in the first aspect, the fusion protein or conjugate as described in the second aspect, the combination as described in the third aspect, the CRISPR-Cas12 system as described in the fourth aspect, the vector system or recombinant cell as described in the fifth aspect, the nucleic acid molecule as described in the sixth aspect, and / or the composition as described in the seventh aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0054] The ninth aspect of the present invention provides a kit comprising the Cas12 protein as described in the first aspect, the fusion protein or conjugate as described in the second aspect, the combination as described in the third aspect, the CRISPR-Cas12 system as described in the fourth aspect, the vector system or recombinant cell as described in the fifth aspect, the nucleic acid molecule as described in the sixth aspect, the composition as described in the seventh aspect, and / or the pharmaceutical composition as described in the eighth aspect.

[0055] The tenth aspect of the present invention provides a Cas12 protein as described in the first aspect, a fusion protein or conjugate as described in the second aspect, a combination as described in the third aspect, a CRISPR-Cas12 system as described in the fourth aspect, a vector system or recombinant cell as described in the fifth aspect, a nucleic acid molecule as described in the sixth aspect, a composition as described in the seventh aspect, a pharmaceutical composition as described in the eighth aspect, or a kit as described in the ninth aspect for the preparation of a drug for in vivo or in vitro gene editing.

[0056] In some embodiments of the present invention, the drug is selected from diagnostic, preventive and / or therapeutic drugs or agents.

[0057] The eleventh aspect of the present invention provides a method for in vivo or in vitro gene editing, comprising administering to a subject in need thereof an effective amount of the Cas12 protein as described in the first aspect, the fusion protein or conjugate as described in the second aspect, the combination as described in the third aspect, the CRISPR-Cas12 system as described in the fourth aspect, the vector system or recombinant cell as described in the fifth aspect, the nucleic acid molecule as described in the sixth aspect, the composition as described in the seventh aspect, the pharmaceutical composition as described in the eighth aspect, or the kit as described in the ninth aspect.

[0058] The twelfth aspect of the present invention provides a method for in vivo or in vitro gene editing, comprising contacting the Cas12 protein as described in the first aspect, the fusion protein or conjugate as described in the second aspect, the combination as described in the third aspect, the CRISPR-Cas12 system as described in the fourth aspect, the vector system or recombinant cell as described in the fifth aspect, the nucleic acid molecule as described in the sixth aspect, the composition as described in the seventh aspect, the pharmaceutical composition as described in the eighth aspect, or the kit as described in the ninth aspect with the target cell.

[0059] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0060] In the present invention, the letters in the amino acid sequence represent the single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): Alanine: Ala-A, Arginine: Arg-R, Aspartic acid: Asp-D, Cysteine: Cys-C, Glutamine: Gln-Q, Glutamic acid: Glu-E, Histidine: His-H, Glycine: Gly-G, Asparagine: Asn-N, Tyrosine: Tyr-Y, Proline: Pro-P, Serine: Ser-S, Methionine: Met-M, Lysine: Lys-K, Valine: Val-V, Isoleucine: Ile-I, Phenylalanine: Phe-F, Leucine: Leu-L, Tryptophan: Trp-W, Threonine: Thr-T. In addition, any amino acid: Xaa-X. In some cases, X is any natural amino acid.

[0061] In the present invention, the numbers represented by the sites refer to the positions of the amino acid residues corresponding to the wild-type Cas12 protein amino acid sequence SEQ ID NO: 1.

[0062] Sequence identity

[0063] As used herein, the term "sequence identity" (identity or percent identity) is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a certain position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or a certain position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent sequence identity" (percent identity) between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% sequence identity. Typically, comparisons are made when two sequences are aligned to produce maximum sequence identity. Such comparisons can be performed using published and commercially available alignment algorithms and programs, such as, but not limited to, ClustalΩ, MAFFT, Probcons, T-Coffee, Probalign, BLAST, which can be reasonably selected by one of ordinary skill in the art. Those skilled in the art can determine appropriate parameters for aligning sequences, including, for example, any algorithms needed to achieve better alignment or optimal comparison over the entire length of the sequences being compared, as well as any algorithms needed to achieve better alignment or optimal comparison over a portion of the sequences being compared.

[0064] CRISPR-Cas12 system

[0065] As used herein, the terms "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated (Cas) (CRISPR-Cas) System" or "CRISPR System" are used interchangeably and have the meaning generally understood by those skilled in the art, which generally include transcripts or other elements related to the expression of CRISPR-associated ("Cas") genes, or transcripts or other elements capable of directing the activity of the Cas genes. Such transcripts or other elements may include sequences encoding Cas effector proteins and guide polynucleotides.

[0066] In some embodiments, the Cas12 protein described herein refers to a protein having an amino acid sequence comprising or having at least 50%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO: 1. When the CRISPR-Cas12 system includes a fusion protein or conjugate comprising the Cas12 protein and a protein domain, the percentage of sequence identity between the Cas12 portion of the fusion protein or conjugate and the reference sequence is calculated.

[0067] In the present invention, the CRISPR-Cas12 system comprises a Cas12 protein having at least 50% sequence identity with SEQ ID NO: 1 or a nucleic acid encoding the Cas12 protein, and a guide polynucleotide or a nucleic acid encoding the guide polynucleotide, wherein the guide polynucleotide comprises a guide sequence and a backbone sequence, and the guide sequence is engineered to hybridize with the target nucleic acid, and the guide polynucleotide is capable of forming a CRISPR complex with the Cas12 protein and guiding the sequence-specific binding of the CRISPR complex to the target nucleic acid.

[0068] Cas12 mutants

[0069] In some embodiments, the Cas12 protein provided herein comprises one or more mutations compared to the wild-type Cas12 protein (SEQ ID NO: 1), such as a single amino acid insertion, a single amino acid deletion, a single amino acid substitution, or a combination thereof. In some instances, the Cas12 protein provided herein comprises a single amino acid substitution. In other instances, the Cas12 protein provided herein comprises multiple amino acid substitutions. In other instances, the Cas12 protein provided herein comprises multiple amino acid substitutions, including S962X in the multiple amino acid substitutions. In other instances, the Cas12 protein provided herein comprises multiple amino acid substitutions, including S962X in the multiple amino acid substitutions.

[0070] One type of modification or mutation includes replacing amino acid residues with similar biochemical properties with amino acids, i.e., conservative substitutions. Typically, conservative substitutions have little or no effect on the activity of the resulting protein or peptide. For example, conservative substitutions are amino acid substitutions in the Cas12 protein that do not substantially affect the binding of the Cas12 protein to the target nucleic acid molecule complementary to the guide sequence in the guide polynucleotide.

[0071] Protein domain

[0072] In some embodiments, the Cas12 protein is covalently linked or fused to a homologous or heterologous protein domain.

[0073] In some embodiments, the protein domain is selected from one or more of the following: a cytosine deaminase domain, an adenosine deaminase domain, a translation regulatory domain, a transcription regulatory domain, a nuclease domain, an epigenetic regulatory domain, a guide editing domain, a recombinase domain, a reverse transcriptase domain, a transposase domain, and a transcription factor domain.

[0074] Vector system

[0075] Another aspect of the present invention relates to a vector system comprising the CRISPR-Cas12 system described herein, wherein the vector system comprises one or more vectors comprising a polynucleotide sequence encoding the Cas12 protein and a polynucleotide sequence encoding the guide polynucleotide.

[0076] In some embodiments, the vector system comprises at least one plasmid or viral vector (e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus, or herpes simplex virus). In some embodiments, the polynucleotide sequence encoding the Cas12 protein and the polynucleotide sequence encoding the guide polynucleotide are located on the same vector. In some embodiments, the polynucleotide sequence encoding the Cas12 protein and the polynucleotide sequence encoding the guide polynucleotide are located on multiple vectors.

[0077] In some embodiments, the polynucleotide sequence encoding Cas12 protein and / or the polynucleotide sequence encoding guide polynucleotide are operably connected to a regulatory element. Regulatory elements include promoters, enhancers, internal ribosome entry sites (IRES) and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory elements include regulatory elements that make the nucleotide sequence constitutively expressed in many types of host cells, and regulatory elements (e.g., tissue-specific regulatory sequences) that make the nucleotide sequence expressed only in certain host cells. Tissue-specific promoters can be directly expressed mainly in desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas) or specific cell types (e.g., lymphocytes). Regulatory elements can also guide expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type-specific.

[0078] cell

[0079] Another aspect of the present invention relates to cells comprising CRISPR-Cas12 systems as described herein.Cells (e.g., cells that can be used to produce cell-free systems) can be eukaryotic or prokaryotic. Examples of such cells include, but are not limited to, bacteria, archaebacteria, plants, fungi, yeasts, insects, and mammalian cells, such as lactobacilli, lactococci, bacillus (e.g., bacillus subtilis), Escherichia (e.g., Escherichia coli), Clostridium, Saccharomyces, or Pichia (e.g., Saccharomyces cerevisiae or Pichia pastoris), Kluyveromyces lactis, Salmonella typhimurium, Drosophila cells, Caenorhabditis elegans cells, African clawed frog cells, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian cell lines (e.g., Hela cells, bone marrow cell lines, and lymphoid cell lines).

[0080] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0081] The reagents and raw materials used in the present invention are commercially available.

[0082] The positive progress effect of the present invention is:

[0083] The Cas12 protein of the present invention has excellent thermal stability and trans-cleavage activity, which is beneficial to the storage and transportation of the Cas12 protein in practical applications, increases the application scenarios of the Cas12 protein and the CRISPR system based thereon, and provides users with more choices; in addition, based on the existing theoretical basis of protein engineering, the Cas12 protein mutant with improved thermal stability involved in the present invention can also be used as a template / starting point sequence for subsequent protein engineering, because the starting point protein with high thermal stability can tolerate many amino acid mutations with high activity but reduced protein stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1a is a comparison of the stability (characterized by Tm) and activity of some mutants and wild-type (WT) (native sequence) Cas12a.

[0085] Figure 1b is a comparison of the relative trans-cleavage activities of some mutants and wild-type (WT) (native sequence) Cas12a at different temperatures.

[0086] Figure 1c: Comparison of the relative trans-cleavage activities of some mutants and wild-type (native sequence) Cas12a at different reaction times.

[0087] Figure 2 is a schematic diagram of the alignment of Cas12a protein sequences from different species. The sequences involved in the figure are shown in SEQ ID NO: 2-25.

[0088] FIG3 is a schematic diagram of the saturation mutagenesis study on site 962.

[0089] Figure 4a is a schematic diagram of the evaluation of the tolerance of MutA and MutB to nucleic acid mismatches. The sequences involved in the figure are shown in SEQ ID NOs: 26-42, respectively.

[0090] FIG4 b is a schematic diagram of the evaluation of the tolerance of mutants to different buffers.

[0091] FIG5 is a schematic diagram of the evaluation of the trans-cleavage activity of mutants on different substrates.

[0092] Figure 6a is a schematic diagram showing that mutation combinations a, b, and c can restore the proportion of soluble protein expressed by the inactivated mutant Comb17 in Escherichia coli.

[0093] Figure 6b is a schematic diagram showing that mutation combinations a, b, and c can restore the stability (Tm) of the inactivated mutant Comb17.

[0094] Figure 6c is a schematic diagram showing that mutation combinations a, b, and c can restore the trans-cleavage activity of the inactivated mutant Comb17; NC is a blank control. DETAILED DESCRIPTION

[0095] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0096] Example 1: Determination of thermal stability (melting temperature) and activity of mutants (Figures 1a-1c)

[0097] The inventors obtained the wild-type sequence of LbCas12a protein (SEQ ID NO: 1) based on an artificial intelligence algorithm based on deep learning (https: / / doi.org / 10.48550 / arXiv.2307.12682), selected a series of mutation sites through artificial intelligence scoring, and performed saturation mutagenesis at some sites to screen for Cas12a protein mutants with good thermal stability and trans-cleavage activity.

[0098] SEQ ID NO: 1:

[0099] After obtaining the simulated Cas12a protein mutant, the stability and activity of the mutant were evaluated experimentally. Among them, the stability evaluation used Tm value as the evaluation index, and the determination method was Differential Scanning Fluorimetry (DSF) (for example, see G. Lucas et al., Thermal Shift and Stability Assays of Disease-Related Misfolded Proteins Using Differential Scanning Fluorimetry, Springer Protocols (2019)); Activity evaluation uses trans-cleavage activity as an evaluation indicator. After the Cas12a protein recognizes the substrate, its activated trans-cleavage activity is detected to cleave the single-stranded DNA probe to obtain the fluorescence intensity (for example, see Lin H, et al., (2022) Internet of medical things-enabled CRISPR diagnostics for rapid detection of SARSCoV-2 variants of concern. Front. Microbiol. 13: 1070940.).

[0100] The results are shown in Figures 1a-1c. As shown in Figure 1a, by comparing the height of the mutant column with the first wild-type column, many mutants have higher Tm values ​​than the wild-type. At the same time, by comparing the height of the asterisks of the mutants with the first wild-type asterisk, many mutants have higher trans-cleavage activity than the wild-type.

[0101] As shown in Figure 1b, different mutants have different activities relative to the wild type at different temperatures. For example, many mutants have higher trans-cleavage activity than the wild type at 42°C, 45°C and 50°C, some mutants have higher trans-cleavage activity than the wild type only at 42°C, 45°C or 50°C, and some mutants have higher trans-cleavage activity than the wild type at 42°C and 45°C, 42°C and 50°C, and 45°C and 50°C.

[0102] As shown in Figure 1c, the activity of different mutants relative to the wild type at different reaction temperatures varies. Many mutants have higher trans-cleavage activity than the wild type at 20 or 30 minutes of reaction.

[0103] Example 2: Comparison of Cas12a protein sequences

[0104] The inventors used ESPript 3.0 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi) to align the amino acid sequences of LbCas12a proteins according to the website instructions. The results are shown in Figure 2.

[0105] Through comparative studies, it was found that some mutations, such as I976L, are actually conserved sequences in Cas12a of different species. The inventors have demonstrated through a large number of experiments that this mutation can improve stability or activity. Therefore, those skilled in the art can reasonably expect that mutations at this site in different species may produce similar results.

[0106] Example 3: Saturation mutagenesis of site 962 confirmed that it is one of the important mutation sites

[0107] As shown in Figure 3, in the experiment of saturation mutation (replaced with 19 other amino acids) at site 962 of SEQ ID NO: 1 (Ser amino acid in the wild type), it was found that, except for Trp (W), these mutations improved the stability of the protein either in the wild-type background or in the C10L / I976L / C1090D background. Therefore, those skilled in the art have reason to believe that any mutation at site 962, as well as the combination of these mutations with other mutations, may improve the stability of the Cas12 protein.

[0108] The experimental method for saturation mutagenesis can be performed by those skilled in the art based on existing textbooks or technical manuals. For example, overlap extension PCR (overlap PCR) can be used to generate 19 mutants using primers for 19 other amino acid mutations. The primers can be obtained by designing and preparing primers according to conventional techniques in the art, or through services provided by suppliers.

[0109] Example 4: Evaluation of the Gain Effect of MutA and MutB

[0110] This example evaluates the gain of effect of two combined mutants, MutA and MutB.

[0111] MutA mutant: G49K / Y549K / S962K / I976L

[0112] MutB mutant: C10L / G49K / L59K / M456I / Y549K / S962K / I976L / C1090D.

[0113] The evaluation experimental plan is as follows:

[0114] The activity evaluation method described in Example 1 was used to observe the fluorescence change over 40 minutes.

[0115] The results are shown in Figures 4a and 4b. Figure 4a evaluates the tolerance of MutA and MutB to nucleic acid mismatches. The results show that the MutA and MutB mutants essentially maintain the wild-type tolerance to nucleic acid mismatches, i.e., do not introduce adverse effects. The following buffers were used in Figure 4b:

[0116] 1×NEBuffer 2.1: 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 100μg / mL BSA, pH 7.9, 25℃;

[0117] 1×NEBuffer 3.1: 100mM NaCl, 50mM Tris-HCl, 10mM MgCl2, 100μg / mL BSA, pH 7.9, 25℃;

[0118] CutSmart Buffer: 50mM potassium acetate, 50mM Tris-acetate, 10mM magnesium acetate, 100μg / mL BSA, pH 7.9, 25℃.

[0119] The results showed that, except for Lin buffer (see Lin H, et al., (2022) Internet of medical things-enabled CRISPR diagnostics for rapid detection of SARSCoV-2 variants of concern. Front. Microbiol. 13: 1070940.), the trans-cleavage activity of MutA and MutB mutants in various buffer systems was better than that of the wild type, indicating that mutations in MutA and MutB can give Cas12 protein better chemical tolerance.

[0120] Example 5: Evaluation of trans-cleavage activity of mutants on different substrates

[0121] The trans-cleavage activity of the mutants was tested on different substrates (target 1, target 2, and target 3) using the experimental conditions for trans-cleavage activity described in Example 4. The results are shown in Figure 5. The experiments show that the mutants described in the present invention have better trans-cleavage activity than the wild type (WT) when cleaving different substrates (e.g., target 1, target 2, and target 3), indicating that the effects of these mutations are broadly applicable and not specific to a specific nucleic acid sequence. The experimental conditions for trans-cleavage activity are the same as those described in Example 4.

[0122] Example 6: Mutations in the present invention can “restore” the stability and activity of the inactivated Cas12 mutant (Comb17)

[0123] This example is used to verify that mutations can "restore" the stability and activity of the inactivated Cas12 mutant (Comb17). The mutation combinations used are as follows:

[0124] Mutation combination a: G49K / S962K / I976L

[0125] Mutation combination b: G49K / Y549K / S962K / I976L

[0126] Mutation combination c: G49K / M456I / Y549K / S962K / I976L / C1090D

[0127] Com17 is:

[0128] G146R / D156R / R182V / D235R / E292R / D350R / E795Q / F863V / R883K / R887K / D952N / C965Y / V1011A / Q1108L / A1113V / S1132T / S1214P

[0129] The results are shown in Figures 6a-6c. Figure 6a shows that mutation combinations a, b, and c can restore the proportion of soluble protein expressed in the inactivated mutant Comb17 in E. coli (when analyzing the soluble protein ratio, the plasmid containing the mutant is transformed into the E. coli expression strain, and after induction of expression, the bacteria are lysed by ultrasound; the obtained whole bacterial lysate is centrifuged, the supernatant is taken out and marked as S, and the precipitate is resuspended with a volume of PBS buffer equal to the supernatant and marked as P; the S and P of each mutant are subjected to protein gel analysis to obtain the proportion of soluble protein, which is calculated as: S / [S+P]. The position indicated by the arrow in the figure is the target protein, and only this part is used for calculation); Figure 6b shows that mutation combinations a, b, and c can restore the stability of the inactivated mutant Comb17 (T m ); Figure 6c shows that the mutation combinations a, b, and c can restore the trans-cleavage activity of the inactivated mutant Comb17.

[0130] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A Cas12 protein, characterized in that The Cas12 protein has an amino acid sequence that has at least 90% sequence identity to the sequence shown in SEQ ID NO: 1 or a homologous sequence thereof, and the amino acid sequence of the Cas12 protein is not SEQ ID NO: 1; The Cas12 protein has: (1) having a trans-cleavage activity not less than 50% of the Cas12 protein as shown in the amino acid sequence of SEQ ID NO: 1, and having a thermal stability higher than the Cas12 protein as shown in the amino acid sequence of SEQ ID NO: 1; or, (2) having a thermal stability not lower than that of the Cas12 protein as shown in SEQ ID NO: 1, and having a trans-cleavage activity higher than that of the Cas12 protein as shown in SEQ ID NO: 1; or, The Cas12 protein has an amino acid sequence in which G146R / D156R / R182V / D235R / E292R / D350R / E795Q / F863V / R883K / R887K / D952N / C965Y / V1011A / Q1108L / A1113V / S1132T / S1214P mutations occur in the amino acid sequence shown in SEQ ID NO:

1.

2. The Cas12 protein according to claim 1, characterized in that The amino acid sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO: 1 is mutated at at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: 1; Preferably, the mutation occurs at a position corresponding to the REC1, REC2, PI, WED-III and / or Nuc domains of the Cas12 protein.

3. The Cas12 protein as claimed in claim 2, characterized in that The mutations occur in C10, G49, L59, H200, H370, M456, Y549, C565, W602, M701, H733, R737, M746, P806, V936, S962, M957, I976, M1035, C1090, F304, T307, P342, V377, D384, R385, R386, V428, A451, V452, V463, V500, T814, V842, Y854, H873, S874, F884, Q888, D959, C965, A966 and I996 corresponding to the amino acid sequence shown in SEQ ID NO: 1; Preferably, the mutation occurs in S962 corresponding to the amino acid sequence as shown in SEQ ID NO: 1, and one or more selected from C10, G49, L59, H200, H370, M456, Y549, C565, W602, M701, H733, R737, M746, P806, V936, M957, I976, M1035, C1090, F304, T307, P342, V377, D384, R385, R386, V428, A451, V452, V463, V500, T814, V842, Y854, H873, S874, F884, Q888, D959, C965, A966 and I996; More preferably, the mutation occurs in S962, C10, I976 and C1090 corresponding to the amino acid sequence shown in SEQ ID NO: 1; For example, the mutation is S962 / C10L / I976L / C1090D; wherein S962 is mutated to any other natural amino acid G, A, V, L, I, P, F, Y, W, T, C, M, N, Q, D, E, K, R or H.

4. The Cas12 protein according to any one of claims 1 to 3, characterized in that The mutation is selected from one or more of the following: C10L, G49K, L59K, H200E, H370K, M456I, Y549K, C565V, W602G, M701L, H733L, R737K, M746Y, P806A, V936F, S962X, M957L, I976L, M1035R, C1090D, F304I, T307F, P342T, V377D, D384K, R385I, R386K, V428K, A451E, V452K, V463K, V500R, T814D, V842S, Y854K, H873Y, S874K, F884D, Q888K, D959N, C965N, A966E, and I996V; wherein X is G, A, V, L, I, P, F, Y, W, T, C, M, N, Q, D, E, K, R, or H; Preferably, the mutation is selected from any one of the following: C10L, G49K, L59K, H200E, H370K, M456I, Y549K, C565V, W602G, M701L, H733L, R737K, M746Y, P806A, V936F, S962G, S962A, S962V, S962L, S962I, S962P, S962F, S962Y, S962W, S962T, S962C, S962M, S962N, S962Q, S96 2D, S962E, S962K, S962R, S962H, M957L, I976L, M1035R, C1090D, F304I, T307F, P342T, V377D, D384K, R385I, R386K, V428K, A451E, V452K, V463K, V500R, T814D, V842S, Y854K, H873Y, S874K, F884D, Q888K, D959N, C965N, A966E and I996V.

5. The Cas12 protein according to any one of claims 1 to 4, characterized in that The mutation is selected from any two of the following: C10L, L59K, M746Y, V936F, S962X, M957L, I976L, C1090D, T307F, A451E, and A966E; wherein X is G, A, V, L, I, P, F, Y, W, T, C, M, N, Q, D, E, K, R, or H; and / or, The mutations are selected from at least three, at least four, or at least five of the following: C10L, G49K, L59K, M456I, Y549K, M701L, H733L, M746Y, V936F, S962X, M957L, I976L, C1090D, T307F, R385I, A451E, V452K, V500R, and A966E; Preferably, the mutation is selected from any one of the following: S962K / C1090D、M957L / C1090D、I976L / C1090D、S962K / I976L、M957L / I976L、C10L / C1090D、C10L / S962K、C10L / M957L、C10L / V936F、C10L / S962K / C1090D、C10L / M957L / C1090D、C10L / V936F / C1090D、C10L / I976L / C1090D、C10L / S962K / I976L、C10L / Y549K / C1090D、C10L / M957L / I976L、C10L / H733L / C1090D、C10L / V936F / I976L、Y549K / S962K / I976L、G49K / S962K / I976L、S962K / I976L / C1090D、G49K / Y549K / C1090D、M456I / S962K / I976L、C10L / M957L / S962K / C1090D、C10L / V936F / M957L / C1090D、C10L / S962K / I976L / C1090D、C10L / M965L / I976L / C1090D、C10L / Y549K / M957L / C1090D、C10L / V936F / I976L / C1090D、C10L / M957L / S962K / I976L、C10L / Y549K / I976L / C1090D、C10L / H733L / I976L / C1090D、C10L / M456I / I976L / C1090D、C10L / S962G / I976L / C1090D、 C10L / S962A / I976L / C1090D、C10L / S962V / I976L / C1090D、C10L / S962L / I976L / C1090D、C10L / S962I / I976L / C1090D、C10L / S962P / I976L / C1090D、C10L / S962F / I976L / C1090D、C10L / S962Y / I976L / C1090D、C10L / S962W / I976L / C1090D、C10L / S962T / I976L / C1090D、C10L / S962C / I976L / C1090D、C10L / S962M / I976L / C1090D、C10L / S962N / I976L / C1090D、C10L / S962Q / I976L / C1090D、C10L / S962D / I976L / C1090D、C10L / S962E / I976L / C1090D、C10L / S962R / I976L / C1090D、C10L / S962H / I976L / C1090D、Y549K / S962K / I976L / C1090D、G49K / Y549K / S962K / I976L、Y549K / M957L / S962K / I976L、M456I / Y549K / S962K / I976L、L59K / Y549K / S962K / I976L、C10L / Y549K / S962K / I976L / C1090D、G49K / Y549K / S962K / I976L / C1090D、C10L / Y549K / M957L / S962K / I976L、G49K / Y549K / M957L / S962K / I976L、M456I / Y549K / S962K / I976L / C1090D、C10L / G49K / Y549K / S962K / I976L / C1090D、C10L / G49K / Y549K / M957L / S962K / I976L、C10L / M456I / Y549K / S962K / I976L / C1090D、G49K / M456I / Y549K / S962K / I976L / C1090D、C10L / M456I / Y549K / M957L / S962K / I976L、C10L / G49K / L59K / M456I / Y549K / V936F / M957L / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / M957L / S962K / I976L / C1090D、<h2 style=";text-align:left;direction:ltr">C10L / G49K / L59K / M456I / Y549K / H733L / M957L / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / V936F / S962K / I976L / C1090D、C10L / G49K / L59 K / M456I / Y549K / V936F / M957L / S962K / I976L、C10L / G49K / M456I / Y549K / V9 36F / M957L / S962K / I976L / C1090D、G49K / L59K / M456I / Y549K / V936F / M957L / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / H733L / V936F / S962K / I976L / C1090D 、C10L / G49K / L59K / M456I / Y549K / M957L / S962K / I976L、T307F / M746Y、S962 K / A966E、L59K / A451E、A451E / S962K、T307F / M701L / M746Y、T307F / M746Y / S 962K, T307F / R385I / M746Y, T307F / V500R / M701L / M746Y, T307F / M701L / M746Y / A966E, T307F / M701L / M746Y / S962K, T307F / R385I / M746Y / A966E, T307 F / R385I / M701L / M746Y, T307F / R385I / V500R / M746Y, T307F / R385I / M746Y / S962K, T307F / M746Y / S962K / A966E, T307F / V500R / M701L / M746Y / A966E, T3 07F / R385I / V500R / M701L / M746Y、T307F / R385I / M701L / M746Y / A966E、T307F / R385I / V500R / M746Y / A966E、T307F / V500R / M746Y / S962K / A966E、T307F / R385I / M701L / M746Y / S962K, T307F / R385I / V500R / M746Y / S962K, A451E / S962K / A966E, L59K / S962K / A966E, R385I / S962K / A966E, C10L / S962K / A966E<h2 style=";text-align:left;direction:ltr">S962K / A966E / C1090D L59K / A451E / S962K / A966E, R385I / A451E / S962K / A966E, C10L / A451E / S962K / A966E, L59K / R385I / S962K / A966E, C10L / L59K / S962K / A966E, L59K / R385I / A451E / S962K / A966E, C10L / L5 9K / A451E / S962K / A966E, C10L / R385I / A451E / S962K / A966E, L59K / A451E / S962K / A966E / C1 090D, C10L / L59K / R385I / S962K / A966E, C10L / L59K / R385I / A451E / S962K / A966E / C1090D, C1 0L / L59K / R385I / A451E / S962K / A966E, C10L / L59K / R385I / A451E / V936F / S962K / A966E, L59 K / R385I / A451E / S962K / A966E / C1090D, C10L / L59K / A451E / S962K / A966E / C1090D, C10L / L59 K / R385I / A451E / M701L / S962K / A966E, C10L / L59K / R385I / A451E / V452K / S962K / A966E, C10L / L59K / R385I / A451E / V500R / S962K / A966E and C10L / L59K / R385I / A451E / M957L / S962K / A966E; More preferably, the mutation is selected from any one of the following: <h2 style=";text-align:left;direction:ltr">S962K / C1090D、I976L / C1090D、S962K / I976L、C10L / S962K、C10L / S962K / C1090D、C10L / S962K / I976L、C10L / M957L / I976L、Y549K / S962K / I976L、G 49K / S962K / I976L、S962K / I976L / C1090D、G49K / Y549K / C1090D、M456I / S 962K / I976L、C10L / M957L / S962K / C1090D、C10L / S962K / I976L / C1090D、C 10L / M965L / I976L / C1090D、C10L / Y549K / M957L / C1090D、C10L / V936F / I9 76L / C1090D、C10L / M957L / S962K / I976L、C10L / Y549K / I976L / C1090D、C1 0L / H733L / I976L / C1090D、C10L / M456I / I976L / C1090D、Y549K / S962K / I9 76L / C1090D、G49K / Y549K / S962K / I976L、Y549K / M957L / S962K / I976L、M4 56I / Y549K / S962K / I976L, L59K / Y549K / S962K / I976L, C10L / Y549K / S962K / I976L / C1090D, G49K / Y549K / S962K / I976L / C1090D, C10L / Y549K / M957 L / S962K / I976L、G49K / Y549K / M957L / S962K / I976L、M456I / Y549K / S962K / I976L / C1090D、C10L / G49K / Y549K / S962K / I976L / C1090D、C10L / G49K / Y 549K / M957L / S962K / I976L、C10L / M456I / Y549K / S962K / I976L / C1090D、G 49K / M456I / Y549K / S962K / I976L / C1090D、C10L / M456I / Y549K / M957L / S9 62K / I976L, C10L / G49K / L59K / M456I / Y549K / V936F / M957L / S962K / I976L / C1090D, C10L / G49K / L59K / M456I / Y549K / M957L / S962K / I976L / C1090D<h2 style=";text-align:left;direction:ltr">C10L / G49K / L59K / M456I / Y549K / H733L / M957L / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / V936F / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / V936F / M957L / S962K / I 976L, C10L / G49K / M456I / Y549K / V936F / M957L / S962K / I976L / C1090D, G49K / L59K / M456I / Y549K / V936F / M957L / S962K / I976L / C1090D, C10L / G49K / L59K / M456I / Y549K / S962K / I976L / C1090D <h2 style=";text-align:left;direction:ltr">C10L / G49K / L59K / M456I / Y549K / H733L / V936F / S962K / I976L / C1090D、C10L / G49K / L59K / M456I / Y549K / M957L / S962K / I976L、T307F / M746Y、S962K / A96 6E, L59K / A451E, A451E / S962K, T307F / M701L / M746Y, T307F / M746Y / S962K, T307F / R385I / M746Y, T307F / V500R / M701L / M746Y, T307F / M701L / M746Y / A9 66E, T307F / M701L / M746Y / S962K, T307F / R385I / M746Y / A966E, T307F / R385I / M701L / M746Y, T307F / R385I / V500R / M746Y, T307F / R385I / M746Y / S962K, T307F / M746Y / S962K / A966E, T307F / V500R / M701L / M746Y / A966E, T307F / R385I / V500R / M701L / M746Y, T307F / R385I / M701L / M746Y / A966E, T307F / R385I / V500R / M746Y / A966E、T307F / V500R / M746Y / S962K / A966E、T307F / R385I / M701L / M746Y / S962K、T307F / R385I / V500R / M746Y / S962K、A451E / S962K / A96 6E、L59K / S962K / A966E、R385I / S962K / A966E、C10L / S962K / A966E、S962K / A 966E / C1090D、L59K / A451E / S962K / A966E、R385I / A451E / S962K / A966E、C10L / A451E / S962K / A966E、L59K / R385I / S962K / A966E、C10L / L59K / S962K / A966E、L59K / R385I / A451E / S962K / A966E、C10L / L59K / A451E / S962K / A966E、C10 L / R385I / A451E / S962K / A966E、L59K / A451E / S962K / A966E / C1090D、C10L / L59K / R385I / S962K / A966E、C10L / L59K / R385I / A451E / S962K / A966E / C1090D、C10L / L59K / R385I / A451E / S962K / A966E, C10L / L59K / R385I / A451E / V936F / S962K / A966E, L5 9K / R385I / A451E / S962K / A966E / C1090D, C10L / L59K / A451E / S962K / A966E / C1090D, C10L / L5 9K / R385I / A451E / M701L / S962K / A966E, C10L / L59K / R385I / A451E / V452K / S962K / A966E, C10 L / L59K / R385I / A451E / V500R / S962K / A966E and C10L / L59K / R385I / A451E / M957L / S962K / A966E. , 6. The Cas12 protein according to any one of claims 2 to 5, characterized in that The mutations include C10L, S962K and / or C1090D; for example, S962K; And / or, the homologous sequence is selected from Cas12a proteins derived from different species or Cas12 proteins derived from different subtypes; Preferably, the homologous sequence of the Cas12 protein is selected from the amino acid sequence of AsCas12a, BbCas12a, BoCas12a, FnCas12a, HkCas12a, Lb4Cas12a, Lb5Cas12a, OsCas12a or TsCas12a; and / or, the homologous sequence of the Cas12 protein is selected from Cas12a protein, Cas12b protein, Cas12c protein, Cas12d protein, Cas12e protein, Cas12f protein, Cas12g protein, Cas12i protein, Cas12j protein, Cas12k protein, Cas12l protein and TnpB protein.

7. A fusion protein or conjugate, characterized in that The fusion protein or conjugate comprises the Cas12 protein according to any one of claims 1 to 6 fused or conjugated to a protein active functional domain or a non-protein active functional domain; Preferably, the fusion or conjugation comprises fusion or conjugation directly or through a linker; and / or, the protein active functional domain is selected from a polypeptide and a protein domain; and / or, the non-protein active functional domain is selected from a nucleic acid molecule, a small molecule cytotoxic agent, a small molecule immunomodulator and a small molecule pathway regulator; More preferably, the polypeptide is selected from an affinity tag, a reporter tag and a fusion tag; And / or, the protein domain is selected from a cytosine deaminase domain, an adenosine deaminase domain, a translation regulatory domain, a transcription regulatory domain, a nuclease domain, an epigenetic regulatory domain, a guide editing domain, a recombinase domain, a reverse transcriptase domain, a transposase domain and a transcription factor domain.

8. A combination of Cas12 proteins, characterized in that The combination comprises one or more Cas12 proteins as described in any one of claims 1 to 6, or a fusion protein or conjugate as described in claim 7; Optionally, the combination further comprises a Cas12 protein mutant, wherein the Cas12 protein mutant is not the Cas12 protein as described in any one of claims 1 to 6; preferably, the Cas12 protein mutant has a sequence as shown in SEQ ID K632R / F863V / W890A / Q1108L / S1132T / S1214P, F863V / F884L / W890A / D952N / C965Y / V1011A / Q1108L / A1113V / S1132T / S1214P, K632R / F863V / Q1108L / S1132T / S1214P, F863V / F884L / D952N / C965Y / V1011A / Q1108L / A1113V / S1132T / S1214P, amino acid sequences of Q1108L / A1113V / S1132T / S1214P, D156R / D235R / E292R / D350R, D156R / G532R / K538V / Y542R / K595R, D156R / R883K / R887K, G532R / K595R, D156R / G532R / K538V / Y542R / K595R, D832A / D925A / D1180A, R1138A, G146R / R182V / E795Q, G532R / K595R, G532R / K538V / Y542R, or G532R / K595R mutations.

9. A CRISPR-Cas12 system, characterized in that It comprises the Cas12 protein according to any one of claims 1 to 6, or the fusion protein or conjugate according to claim 7, or the combination according to claim 8, and a guide polynucleotide; Wherein, the guide polynucleotide forms a CRISPR complex with the Cas12 protein or fusion protein or conjugate, the guide polynucleotide is partially complementary to the target nucleic acid, and guides the CRISPR complex to hybridize with the target nucleic acid.

10. A vector system or a recombinant cell, characterized in that: The vector system or recombinant cell comprises one or more vectors encoding a nucleic acid of the Cas12 protein according to any one of claims 1 to 6, or a fusion protein or conjugate according to claim 7, or a combination according to claim 8, and a nucleic acid encoding a guide polynucleotide; Wherein, the guide polynucleotide and the Cas12 protein or fusion protein or conjugate or combination form a CRISPR complex, the guide polynucleotide is partially complementary to the target nucleic acid, and guides the CRISPR complex to hybridize with the target nucleic acid; Preferably, the vector is selected from plasmids, viruses, bacteriophages, extracellular vesicles, liposomes and lipid nanoparticles; and / or, the starting cell of the recombinant cell is a eukaryotic cell or a prokaryotic cell; More preferably, the eukaryotic cell is selected from plant cells, fungal cells, animal cells or human cells, such as CHO cells, insect cells or HEK293 cells; the prokaryotic cell is selected from bacterial cells, such as Escherichia coli cells.

11. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Cas12 protein according to any one of claims 1 to 6, the fusion protein or conjugate according to claim 7, the combination according to claim 8, and / or a guide polynucleotide; Wherein, the guide polynucleotide and the Cas12 protein or fusion protein or conjugate or combination form a CRISPR complex, and the guide polynucleotide is partially complementary to the target nucleic acid, guiding the CRISPR complex to hybridize with the target nucleic acid.

12. A composition, characterized in that The composition comprises the Cas12 protein according to any one of claims 1 to 6, the fusion protein or conjugate according to claim 7, the combination according to claim 8, the CRISPR-Cas12 system according to claim 9, the vector system or recombinant cell according to claim 10, and / or the nucleic acid molecule according to claim 11; Preferably, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier and / or excipient.

13. A kit, characterized in that: The kit comprises the Cas12 protein according to any one of claims 1 to 6, the fusion protein or conjugate according to claim 7, the combination according to claim 8, the CRISPR-Cas12 system according to claim 9, the vector system or recombinant cell according to claim 10, the nucleic acid molecule according to claim 11, and / or the composition according to claim 12.

14. A use of a Cas12 protein as claimed in any one of claims 1 to 6, a fusion protein or conjugate as claimed in claim 7, a combination as claimed in claim 8, a CRISPR-Cas12 system as claimed in claim 9, a vector system or a recombinant cell as claimed in claim 10, a nucleic acid molecule as claimed in claim 11, a composition as claimed in claim 12, or a kit as claimed in claim 13 in the preparation of a drug for in vivo or in vitro gene editing; Preferably, the drug is selected from diagnostic, preventive and / or therapeutic drugs or agents.

15. A method for in vivo or in vitro gene editing, characterized in that: The method comprises contacting the target cell with the Cas12 protein of any one of claims 1 to 6, the fusion protein or conjugate of claim 7, the combination of claim 8, the CRISPR-Cas12 system of claim 9, the vector system or recombinant cell of claim 10, the nucleic acid molecule of claim 11, the composition of claim 12, or the kit of claim 13.

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