Cas12 protein and application thereof

Abstract

The invention discloses a Cas12 protein and an application thereof. The Cas12 protein has an amino acid sequence which has at least 90% sequence identity with a sequence as shown in SEQ ID NO: 1 or a homologous sequence of the sequence as shown in SEQ ID NO: 1, and the amino acid sequence of the Cas12 protein is not SEQ ID NO: 1. The Cas12 protein has excellent thermal stability and trans-cleavage activity, storage and transportation of the Cas12 protein in practical application are facilitated, application scenes of the Cas12 protein and a CRISPR (clustered regularly interspaced short palindromic repeats) system based on the Cas12 protein are increased, and more choices are provided for users; in addition, on the basis of the existing protein engineering theory, the Cas12 protein mutant with improved thermal stability can also be used as a template / starting point sequence of subsequent protein engineering.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a Cas12 protein and an application thereof. Background Art

[0002] CRISPR and CRISPR-associated genes (Cas) are the immune systems of bacteria to resist exogenous viral infections. Cas nuclease can bind to substrate nucleic acid under the guidance of guide RNA, thereby cutting nucleic acid. The modular characteristics of CRISPR make this technology widely used in genome engineering. In addition to gene editing related applications (such as base editing, guide editing, epigenetic editing, transcriptional regulation editing, etc.), CRISPR technology can also be applied to nucleic acid detection. CRISPR nucleic acid detection relies on the trans-cleavage activity of Cas nuclease, that is, it can non-specifically cut single-stranded nucleic acid after binding to substrate nucleic acid. Summary of the invention

[0003] The problem to be solved by the present invention is the defect that the Cas12 protein in the prior art has few choices and strict use conditions, 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 the Cas12 protein and the CRISPR system based thereon, and provides users with more choices.

[0004] The present invention solves the above technical problems through the following technical solutions.

[0005] A first aspect of the present invention provides a Cas12 protein, the Cas12 protein having an amino acid sequence having at least 90% sequence identity with 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;

[0006] The Cas12 protein has:

[0007] (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,

[0008] (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;

[0009] or,

[0010] 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.

[0011] In some embodiments of the present invention, the amino acid sequence having at least 90% sequence identity with 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.

[0012] 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 domains of the Cas12 protein.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] In some examples of the present invention, the mutation is S962 / C10L / I976L / C1090D; wherein S962 mutates 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.

[0017] 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, wherein X is G, A, V, L, I, P, F, Y, W, T, C, M, N, Q, D, E, K, R, or H.

[0018] 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.

[0019] In some other preferred embodiments of the present invention, the mutation is selected from any two of the following:

[0020] 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.

[0021] 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.

[0022] In some specific embodiments of the present invention, the mutation is selected from any one of the following:

[0023] 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 / 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 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 / M7 01L / 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 / 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 / A966E, 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 / A966EC10L / L59K / R385I / A451E / V936F / S962K / A966E, L59K / R385I / A451E / S962K / A966E / C1090D , C10L / L59K / A451E / 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.

[0024] In some examples of the present invention, the mutation is selected from any one of the following:

[0025] <h2 style=";text-align:left;direction:ltr">S962K / C1090D、I976L / C1090D、S962K / I976L、C10L / S962K、C10L / S962K / C1 090D、C10L / S962K / I976L、C10L / M957L / I976L、Y549K / S962K / I976L、G49K / S 962K / I976L, S962K / I976L / C1090D, G49K / Y549K / C1090D, M456I / S962K / I976L, C10L / M957L / S962K / C1090D, C10L / S962K / I976L / C1090D, C10L / M965L / I976L / C1090D、C10L / Y549K / M957L / C1090D、C10L / V936F / I976L / C1090D、C 10L / 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 / 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<h2 style=";text-align:left;direction:ltr">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 / 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 / M 701L / 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 / 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 / A966E, C10L / L59K / S962K / A966E, L59K / R385I / A451E / S962K / A966E, C10L / L59K / A451E / S962K / A966E C10L / R385I / A451E / S962K / A966E, L59K / A451E / S962K / A966E / C1090DC10L / L59K / R385I / S962K / A966E, C10L / L59K / R385I / A451E / S962K / A966E / C 1090D, C10L / L59K / R385I / A451E / S962K / A966E, C10L / L59K / R385I / A451E / V 936F / S962K / A966E, L59K / R385I / A451E / S962K / A966E / C1090D, C10L / L59K / A451E / 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.

[0026] In some embodiments of the present invention, the mutation comprises C10L, S962K and / or C1090D; for example, S962K.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] In some embodiments of the present invention, the fusion or conjugation includes fusion or conjugation directly or through a linker.

[0032] In some embodiments of the present invention, the protein active functional domain is selected from the group consisting of a polypeptide and a protein domain.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] The third aspect of the present invention provides a combination of Cas12 proteins, the combination comprising one or more Cas12 proteins as described in the first aspect, or the fusion protein or conjugate as described in the second aspect;

[0037] Optionally, the combination further includes a Cas12 protein mutant, and the Cas12 protein mutant is not the Cas12 protein described in the first aspect.

[0038] 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 / S1132T / S1214P , F863V / D952N / C965Y / V1011A / 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 mutant amino acid sequences.

[0039] 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;

[0040] 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.

[0041] The fifth aspect of the present invention provides a vector system or a 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;

[0042] 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.

[0043] 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.

[0044] In some embodiments of the present invention, the starting cell of the recombinant cell is a eukaryotic cell or a prokaryotic cell.

[0045] In the present invention, the eukaryotic cell is selected from plant cells, fungal cells, animal cells or human cells, such as CHO cells, insect cells or HEK293 cells.

[0046] In the present invention, the prokaryotic cells are selected from bacterial cells, such as Escherichia coli cells.

[0047] 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;

[0048] 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.

[0049] 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.

[0050] The eighth aspect of the present invention provides a pharmaceutical composition, which comprises 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.

[0051] The ninth aspect of the present invention provides a kit, which comprises 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.

[0052] 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 use in the preparation of a drug for in vivo or in vitro gene editing.

[0053] In some embodiments of the present invention, the drug is selected from diagnostic, preventive and / or therapeutic drugs or agents.

[0054] The eleventh aspect of the present invention provides a method for in vivo or in vitro gene editing, the method comprising administering to a subject in need 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.

[0055] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0056] 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 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.

[0057] In the present invention, the numbers represented by the sites refer to the positions of the amino acid residues corresponding to the amino acid sequence of the wild-type Cas12 protein SEQ ID NO: 1.

[0058] 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.

[0059] Sequence identity

[0060] 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 each molecule is 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 of the 10 positions of the two sequences match, then the two sequences have 60% sequence identity. Typically, two sequences are compared when they are aligned to produce maximum sequence identity. Such an alignment 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, and those of ordinary skill in the art can reasonably choose to use. 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 local area of ​​the sequences being compared.

[0061] CRISPR-Cas12 system

[0062] As used herein, the term "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated (Cas) (CRISPR-Cas) System" or "CRISPR System" is used interchangeably and has a meaning generally understood by those skilled in the art, which generally includes a transcription product or other element related to the expression of a CRISPR-associated ("Cas") gene, or a transcription product or other element capable of directing the activity of the Cas gene. Such a transcription product or other element may include a sequence encoding a Cas effector protein and a guide polynucleotide.

[0063] 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.

[0064] 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, wherein the guide sequence is engineered to hybridize with a 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.

[0065] Cas12 mutants

[0066] In some embodiments, the Cas12 protein provided herein comprises one or more mutations, such as a single amino acid insertion, a single amino acid deletion, a single amino acid substitution, or a combination thereof, compared to the wild-type Cas12 protein (SEQ ID NO: 1). In some examples, the Cas12 protein provided herein comprises a single amino acid substitution. In other examples, the Cas12 protein provided herein comprises multiple amino acid substitutions. In other examples, the Cas12 protein provided herein comprises multiple amino acid substitutions, including S962X in the multiple amino acid substitutions. In other examples, the Cas12 protein provided herein comprises multiple amino acid substitutions, including S962X / C10L / I976L / C1090D, wherein the meaning of "X" is as described above.

[0067] 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 Cas12 proteins that do not substantially affect the binding process of Cas12 proteins to target nucleic acid molecules complementary to the guide sequence in the guide polynucleotide.

[0068] Protein domain

[0069] In some embodiments, the Cas12 protein is covalently linked or fused to a homologous or heterologous protein domain.

[0070] 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.

[0071] Vector system

[0072] 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.

[0073] 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.

[0074] In some embodiments, the polynucleotide sequence encoding Cas12 protein and / or the polynucleotide sequence encoding guide polynucleotide are operably connected to regulatory elements.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 constitutively express nucleotide sequences in many types of host cells, and regulatory elements (e.g., tissue-specific regulatory sequences) that make nucleotide sequences 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.

[0075] cell

[0076] 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, archaea, 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, Xenopus laevis 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).

[0077] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0078] The reagents and raw materials used in the present invention are commercially available.

[0079] The positive and progressive effects of the present invention are:

[0080] 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

[0081] Figure 1a Comparison of the stability (characterized by Tm) and activity of some mutants with wild-type (WT) (native sequence) Cas12a.

[0082] Figure 1b Comparison of relative trans-cleavage activities of some mutants and wild-type (WT) (native sequence) Cas12a at different temperatures.

[0083] Figure 1c : Comparison of the relative trans-cleavage activities of some mutants and wild-type (native sequence) Cas12a at different reaction times.

[0084] Figure 2 Schematic diagram of Cas12a protein sequence alignment from different species.

[0085] Figure 3Schematic diagram of the saturation mutagenesis study targeting site 962.

[0086] Figure 4a Schematic diagram for evaluating the tolerance of MutA and MutB to nucleic acid mismatches.

[0087] Figure 4b Schematic diagram of the evaluation of mutant tolerance to different buffers.

[0088] Figure 5 Schematic diagram of the evaluation of the trans-cleavage activity of mutants on different substrates.

[0089] Figure 6a Schematic diagram of the proportion of soluble protein expressed in Escherichia coli by mutation combinations a, b, and c.

[0090] Figure 6b Schematic diagram showing that mutation combinations a, b, and c can restore the stability (Tm) of the inactive mutant Comb17.

[0091] Figure 6c Schematic diagram showing that mutation combinations a, b, and c can restore the trans-cleavage activity of the inactivated mutant Comb17; NC is the blank control. DETAILED DESCRIPTION

[0092] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0093] Example 1: Determination of thermal stability (melting temperature) and activity of mutants ( Figure 1a-1c )

[0094] 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 mutations at some sites to screen for Cas12a protein mutants with good thermal stability and trans-cleavage activity.

[0095] SEQ ID NO: 1:

[0096]

[0097] After obtaining the simulated Cas12a protein mutant, the mutant was evaluated for stability and activity by experiment. 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, and uses a fluorescently labeled single-stranded DNA probe to detect the activated trans-cleavage activity of the Cas12a protein after it recognizes the substrate to cut 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.).

[0098] The results are as follows Figure 1a-1c As shown. Figure 1a As shown, 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 asterisk of the mutant with the first wild-type asterisk, many mutants have higher trans-cleavage activity than the wild-type.

[0099] like Figure 1b As shown, 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.

[0100] like Figure 1c As shown, the activities of different mutants relative to the wild type at different reaction temperatures vary, and many mutants have higher trans-cleavage activities than the wild type at 20 or 30 minutes of reaction.

[0101] Example 2: Comparison of Cas12a protein sequences

[0102] The inventors used ESPript 3.0 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi) to compare the amino acid sequence of LbCas12a protein according to the website instructions. The results are as follows Figure 2 shown.

[0103] Through comparative studies, it was found that some mutations, such as I976L, are actually conserved sequences in Cas12a of different species. The inventors have proved through a large number of experiments that this mutation will improve stability or activity. Therefore, those skilled in the art can reasonably expect that mutations of this site in different species may produce similar results.

[0104] Example 3: Saturation mutation study on site 962 proves that it is one of the important mutation sites

[0105] like Figure 3 As shown, in the experiment of saturation mutation (replaced with other 19 kinds of amino acids) for 962 sites of SEQ ID NO: 1 (Ser amino acid in wild type), it was found that, except Trp (W), these mutations either in the wild type background or in the C10L / I976L / C1090D background improved the stability of the protein. 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 Cas12 protein.

[0106] The experimental method of saturation mutagenesis can be performed by those skilled in the art based on existing textbooks or technical manuals. For example, overlap extension PCR can be used to generate 19 mutants using primers for other 19 amino acid mutations. The primers can be obtained by designing and preparing primers according to conventional technical means in the art, or obtained through services provided by suppliers.

[0107] Example 4: Evaluation of the gain-of-function of MutA and MutB

[0108] In this example, the gain effect of two combined mutants, MutA and MutB, was evaluated.

[0109] MutA mutant: G49K / Y549K / S962K / I976L

[0110] MutB mutant: C10L / G49K / L59K / M456I / Y549K / S962K / I976L / C1090D.

[0111] The evaluation experimental plan is as follows:

[0112] The activity evaluation method described in Example 1 was used to observe the fluorescence change within 40 minutes.

[0113] The results are as follows Figure 4a and Figure 4b shown. Figure 4a The tolerance of MutA and MutB to nucleic acid mismatches was evaluated, and the results showed that the MutA and MutB mutants basically maintained the tolerance of the wild type to nucleic acid mismatches, that is, no adverse effects were introduced. Figure 4b The following buffers were used:

[0114] 1×NEBuffer 2.1: 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 100μg / mL BSA, pH7.9, 25℃;

[0115] 1×NEBuffer 3.1: 100mM NaCl, 50mM Tris-HCl, 10mM MgCl2, 100μg / mL BSA, pH7.9, 25℃;

[0116] CutSmart Buffer: 50mM potassium acetate, 50mM Tris-acetate, 10mM magnesium acetate, 100μg / mL BSA, pH 7.9, 25℃.

[0117] 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 a variety of buffer systems was better than that of the wild type, indicating that mutations in MutA and MutB can give Cas12 protein better chemical tolerance.

[0118] Example 5: Evaluation of trans-cleavage activity of mutants on different substrates

[0119] The trans-cleavage activity of the mutant on different substrates (target 1, target 2 and target 3) was detected using the experimental conditions of trans-cleavage activity in Example 4. The results are as follows Figure 5As shown, the experiment shows that the mutants described in the present invention have better trans-cleavage activity than the wild type (WT) when cleaving different substrates (such as target 1, target 2 and target 3), indicating that the effects of these mutations are widely 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.

[0120] Example 6: Mutations in the present invention can "restore" the stability and activity of the inactivated Cas12 mutant (Comb17)

[0121] 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:

[0122] Mutation combination a: G49K / S962K / I976L

[0123] Mutation combination b: G49K / Y549K / S962K / I976L

[0124] Mutation combination c: G49K / M456I / Y549K / S962K / I976L / C1090D

[0125] Com17 is:

[0126] G146R / D156R / R182V / D235R / E292R / D350R / E795Q / F863V / R883K / R887K / D952N / C965Y / V1011A / Q1108L / A1113V / S1132T / S1214P

[0127] The results are as follows Figure 6a-6c shown. Figure 6a It showed that the mutation combinations a, b, and c could restore the proportion of soluble protein expressed by the inactive mutant Comb17 in E. coli; Figure 6b This indicates that the mutation combinations a, b, and c can restore the stability of the inactivated mutant Comb17 (T m ); Figure 6c It shows that mutation combinations a, b, and c can restore the trans-cleavage activity of the inactivated mutant Comb17.

[0128] Example 7

[0129] We cloned MutA, MutB and other mutants into mammalian cell expression vectors, transfected them into HEK293, HeLa and other cells using transfection reagents, and then evaluated the effects of these amino acid mutations on the gene editing effect of Cas12a cells.

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、<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、M 456I / 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 / Y54 9K / 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, C10L / G49K / L59K / M456I / Y549K / H733L / M957L / S962K / I976L / C1090D、C10L / G49 K / L59K / M456I / Y549K / V936F / S962K / I976L / C1090D、C10L / G49K / L59K / M4 56I / Y549K / V936F / M957L / S962K / I976L、C10L / G49K / M456I / Y549K / V936F / M957L / S962K / I976L / C1090D、G49K / L59K / M456I / Y549K / V936F / M957L / S 962K / 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 / A966E / C1090D, C10L / L59K / A451E / S962K / A966E / C1090D, C10L / L59K / R385I / A451E / M70 1L / 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 / 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 / A966E, C10L / A451E / S 962K / A966E, L59K / R385I / S962K / A966E, C10L / L59K / S962K / A966E, L59K / R385I / A451E / S962K / A966E、C10L / L59K / A451E / S962K / A966E、C10L / R385I / A451E / S962K / A966E、L59K / A451E / S96 2K / 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 / A96 6E, L59K / R385I / A451E / S962K / A966E / C1090D, C10L / L59K / A451E / 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.

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.

13. A pharmaceutical composition, characterized in that The pharmaceutical 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, the nucleic acid molecule according to claim 11, and / or the composition according to claim 12, and a pharmaceutically acceptable carrier and / or excipient.

14. 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, the composition according to claim 12, and / or the pharmaceutical composition according to claim 13.

15. 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, a pharmaceutical composition as claimed in claim 13, or a kit as claimed in claim 14 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.

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