Engineered Cas12i nuclease and its effector protein and use

Abstract

The present application provides an engineered Cas12i nuclease; it comprises one, two, three or four of the following mutations based on a reference Cas12i nuclease: (1) one or more amino acids interacting with PAM in the reference Cas12i nuclease are replaced with positively charged amino acids; and / or (2) one or more amino acids involved in opening the DNA double helix in the reference Cas12i nuclease are replaced with amino acids with aromatic rings; and / or (3) one or more amino acids located in the RuvC domain of the reference Cas12i nuclease and interacting with a single-stranded DNA substrate are replaced with positively charged amino acids; and / or (4) one or more amino acids interacting with the DNA-RNA double helix in the reference Cas12i nuclease are replaced with positively charged amino acids; in particular, the reference Cas12i nuclease is a natural Cas12i nuclease, such as a natural Cas12i2 nuclease, whose amino acid sequence is defined as SEQ ID NO.1.

Description

[0001] This case is a divisional application of the invention patent application with application date of May 27, 2021, application number 2021105812903, and invention name “Engineered Cas12i nuclease, its effector protein and use”. Technical Field

[0002] The present application belongs to the field of biotechnology. More specifically, the present application relates to Cas12i nucleases, effector proteins and uses thereof with improved catalytic activity (e.g., gene editing activity). Background Art

[0003] Genome editing is an important and useful technology in genome research. There are multiple systems available for genome editing, including the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, the transcription activator-like effector nuclease (TALEN) system, and the zinc finger nuclease (ZFN) system.

[0004] The CRISPR-Cas system is an efficient and cost-effective genome editing technology that can be widely used in a range of eukaryotic organisms from yeast, plants to zebrafish and humans (see reviews: Van der Oost 2013, Science 339:768-770, and Charpentier and Doudna, 2013, Nature 495:50-51). The CRISPR-Cas system provides adaptive immunity in archaea and bacteria by combining Cas12i effector protein and CRISPR RNA (crRNA). To date, based on the outstanding functional and evolutionary modularity of the system, two types (type 1 and type 2) of CRISPR-Cas systems have been characterized, including six types (types I–VI). Among the type 2 CRISPR-Cas systems, the type II Cas9 system and the type V-A / B / E / J Cas12a / Cas12b / Cas12e / Cas12j system have been used for genome editing and provide broad prospects for biomedical research.

[0005] However, current CRISPR-Cas systems have multiple limitations, including limited gene editing efficiency. Therefore, improved methods and systems are needed for efficient genome editing across multiple loci. SUMMARY OF THE INVENTION

[0006] This application provides the following technical solutions:

[0007] 1. An engineered Cas12i nuclease; comprising one, two, three or four mutations based on a reference Cas12i nuclease, the mutations being selected from:

[0008] (1) replacing one or more amino acids that interact with PAM in the reference Cas12i nuclease with positively charged amino acids; and / or

[0009] (2) replacing one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease with amino acids with aromatic rings; and / or

[0010] (3) replacing one or more amino acids in the reference Cas12i nuclease located in the RuvC domain and interacting with the single-stranded DNA substrate with positively charged amino acids; and / or

[0011] (4) One or more amino acids that interact with the DNA-RNA double helix in the reference Cas12i nuclease are replaced with positively charged amino acids.

[0012] In some embodiments, the reference Cas12i nuclease is a natural wild-type Cas12i enzyme. In some embodiments, the reference Cas12i nuclease is Cas12i1, Cas12i2, or a homologous nuclease thereof. In some embodiments, the reference enzyme is a natural Cas12i2 nuclease, and its amino acid sequence is defined as SEQ ID NO.1. In some embodiments, the reference enzyme is a natural Cas12i1 enzyme, and its amino acid sequence is defined as SEQ ID NO.13. In some embodiments, the reference Cas12i nuclease is an engineered Cas12i nuclease.

[0013] 2. The engineered Cas12i nuclease of item 1, wherein the one or more amino acids interacting with PAM are amino acids within 9 angstroms of PAM in a three-dimensional structure. In some embodiments, the one or more amino acids interacting with PAM are one or more amino acids at the following positions: 176, 178, 226, 227, 229, 237, 238, 264, 447 and / or 563. In some embodiments, the one or more amino acids interacting with PAM are one or more amino acids: E176, E178, Y226, A227, N229, E237, K238, K264, T447 and / or E563. In some embodiments, the one or more amino acids interacting with PAM are one or more amino acids: E176, K238, T447 and / or E563. Wherein, the amino acid position numbering is as defined in SEQ ID NO.1.

[0014] 3. The engineered Cas12i nuclease of item 2, wherein the positively charged amino acid is R or K. In some embodiments, the positively charged amino acid is R.

[0015] 4. The engineered Cas12i nuclease of item 3, wherein the replacement of one or more amino acids interacting with PAM in the reference Cas12i nuclease with positively charged amino acids refers to one or more of the following replacements: E176R, K238R, T447R and / or E563R. In some embodiments, the Cas12i nuclease comprises any one or combination of the following mutations: 1) E563R; (2) E176R, T447R, E176R and E563R; (3) K238R and E563R; (4) E176R, K238R and T447R; (5) E176R, K238R and E563R; (6) E176R, T447R and E563R; and / or (7) E176R, K238R, T447R and E563R. Wherein, the amino acid position number is as defined in SEQ ID NO.1.

[0016] 5. An engineered Cas12i nuclease as described in any one of items 1 to 4, wherein the one or more amino acids involved in opening the DNA double strand are amino acids that interact with the last base pair at the 3' end of the target strand in the PAM. In some embodiments, the one or more amino acids involved in opening the DNA double strand are one or more amino acids at the following positions: 163 and / or 164. ; In some embodiments, the one or more amino acids involved in opening the DNA double strand are the following one or more amino acids: Q163 and / or N164. In some embodiments, the one or more amino acids involved in opening the DNA double strand are N164. Wherein, the amino acid position number is as defined in SEQ ID NO.1.

[0017] 6. The engineered Cas12i nuclease of item 5, wherein the one or more amino acids involved in opening the double-stranded DNA are replaced with an amino acid with an aromatic ring, and the amino acid with an aromatic ring is F, Y or W. In some embodiments, the amino acid is F or Y.

[0018] 7. The engineered Cas12i nuclease of item 6, wherein the replacement of one or more amino acids involved in opening the double-stranded DNA in the reference Cas12i enzyme with amino acids with aromatic rings refers to: Q163F, Q163Y, Q163W, N164F and / or N164Y. In some embodiments, the Cas12i nuclease comprises a N164Y or N164F mutation, such as N164Y.

[0019] 8. The engineered Cas12i nuclease of any one of items 1 to 7, wherein the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are amino acids within 9 angstroms of the single-stranded DNA substrate in the three-dimensional structure. In some embodiments, the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are one or more amino acids at the following positions: 323, 362, 425, 925, 926, 390, 391, 392, 751, 755, 840, 848, 851, 856, 885, 897, 929, 932, 327, 355, 359, 360, 361, 414, 421, 650, 652, 705, 708, 709, 752, 928, 388, 393, 417, 418, 424, 653, 696 and / or 1022. In some embodiments, the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are one or more of the following amino acids: E323, D362, Q425, N925, I926, N390, N391, F392, L751, E755, N840, N848, S851, A856, Q885, M897, G929, Y932, L327, V355, G359, G360, K361, Q414, K421, S650, E652, K705, K708, E709, S752, T928, L388, K393, L417, A418, Q424, G653, I696 and / or A1022. In some embodiments, the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are one or more of the following amino acids: E323, D362, Q425, N925, I926, N391, Q424 and / or G929, wherein the amino acid position numbering is as defined in SEQ ID NO.1.

[0020] 9. An engineered Cas12i nuclease as described in item 8, wherein one or more amino acids involved in the cleavage of double-stranded DNA in the reference Cas12i nuclease are replaced with a positively charged amino acid, and the positively charged amino acid is R or K. In some embodiments, the amino acid is R.

[0021] 10. The engineered Cas12i nuclease of item 9, wherein replacing one or more amino acids of the reference Cas12i nuclease located in the RuvC domain and interacting with the single-stranded DNA substrate with positively charged amino acids refers to one or more of the following replacements: E323R, D362R, N391R, Q424R, Q425R, N925R, I926R and / or G929R. In some embodiments, the Cas12i nuclease comprises any one or combination of the following mutations: 1) E323R; (2) D362R; (3) Q425R; (4) N925R; (5) I926R; (6) E323R and D362R; (7) E323R and Q425R; (8) E323R and I926R; (9) Q425R and I926R; (10) D 362R and I926R; (11) N925R and I926R; (12) E323R, D362R and Q425R; (13) E323R, D362R and I926R; (14) E323R, Q425R and I926R; (15) D362R, N925R and I926R; and / or (16) E323R, D362R, Q425R and I926R. Wherein, the amino acid position numbers are as defined in SEQ ID NO.1.

[0022] 11. The engineered Cas12i nuclease of any one of items 1 to 10, wherein the one or more amino acids interacting with the DNA-RNA double helix are amino acids within 9 angstroms of the DNA-RNA double helix in the three-dimensional structure. In some embodiments, the one or more amino acids interacting with the DNA-RNA double helix are one or more amino acids at the following positions: 116, 117, 156, 159, 161, 301, 305, 306, 308, 312, 313, 427, 433, 438, 441, 442, 852, 855, 861, 865, 160, 316, 319, 320, 247, 343, 348, 349, 679, 683, 691, 782, 783, 797, 800, 853, 957, 958, 293, 294 and / or 297. In some embodiments, the one or more amino acids that interact with the DNA-RNA double helix are one or more of the following amino acids: G116, E117, A156, T159, S161, T301, 1305, K306, T308, N312, F313, D427, K433, V438, N441, Q442, M852, L855, N861, Q865, E160, Q316, E319, Q320, E247, E343, E348, E349, N679, E683, E691, D782, E783, E797, E800, D853, S957, D958, G293, E294 and / or N297. In some embodiments, the one or more amino acids that interact with the DNA-RNA double helix are one or more of the following amino acids: G116, E117, T159, S161, E319, E343 and / or D958, wherein the amino acid position numbers are as defined in SEQ ID NO.1.

[0023] 12. The engineered Cas12i nuclease of claim 11, wherein the positively charged amino acid is R or K. In some embodiments, the positively charged amino acid is R.

[0024] 13. The engineered Cas12i nuclease of item 12, wherein the replacement of one or more amino acids interacting with the DNA-RNA double helix in the reference Cas12i nuclease with positively charged amino acids refers to one or more of the following replacements: G116R, E117R, T159R, S161R, E319R, E343R and / or D958R. In some embodiments, the Cas12i nuclease comprises D958R. Wherein, the amino acid position numbering is as defined in SEQ ID NO.1.

[0025] 14. An engineered Cas12i nuclease as described in any one of items 1 to 13, comprising one or more flexible region mutations that increase the flexibility of the flexible region in the reference Cas12i nuclease, the flexible region being selected from the group corresponding to the following region: amino acid residues 439-443 or amino acid residues 925-929, wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the flexible region mutation is located at one or more of the following positions: 439 and / or 926. In some embodiments, the flexible region mutation is one or more of the following amino acids: L439 and / or I926.

[0026] 15. An engineered Cas12i nuclease as described in item 14, wherein the one or more flexible region mutations are: replacing the amino acid with G, and / or inserting one or two Gs thereafter. In some embodiments, the one or more flexible region mutations comprise I926G. In some embodiments, the one or more flexible region mutations comprise 439G or 439GG. Wherein the amino acid position numbering is as defined in SEQ ID NO.1.

[0027] 16. An engineered Cas12i nuclease (such as Cas12i2 nuclease), whose amino acid position numbering is as defined in SEQ ID NO.1; the engineered Cas12i nuclease comprises any one or more of the following groups (such as 2, 3, 4, 5, 6 or more groups) of mutations: (1) E563R; (2) E176R and T447R; (3) E176R and E563R; (4) K238R and E563R; (5) E176R, K238R and T447R; (6) E176R, T447R and E563R ; (7) E176R, K238R and E563R; (8) E176R, K238R, T447R and E563R; (9) N164Y; (10) N164F; (11) E323R; (12) D362R; (13) Q425R; (14) N925R; (15) I926R; (16) D958R; (17) E323R and D362R; (18) E3 23R and Q425R; (19) E323R and I926R; (20) Q425R and I926R; (21) D362R and I926R; (22) N925R and I926R; (23) E323R, D362R and Q425R; (24) E323R, D362R and I926R; (25) E323R, Q425R and I926R; (26) D362R, N925R and I926R; (27) E323R, D362R, Q425R and I926R; (28) D362R and I926G; (29) N925R and I926G; (30) D362R, N925R and I926G; (31) I926R and 439G; (32) I926R and 439GG; and / or (33) E323R, D362R and I926G. In some embodiments, the engineered Cas12i nuclease comprises any one or more of the following groups (e.g., 2, 3, 4, or 5 groups) of mutations: (1) E176R, K238R, T447R, and E563R; (2) N164Y; (3) I926R; (4) E323R and D362R; (4) I926G; (5) I926R and 439G; (6) I926R and 439GG; and / or (7) D958R.In some embodiments, the engineered Cas12i nuclease comprises any of the following mutations: ((1) E176R, K238R, T447R, E563R and N164Y; (2) E176R, K238R, T447R, E563R and I926R; (3) N164Y, E323R and D362R; (4) E176R, K238R, T447R, E563R, E323R and D362R; (5) N164Y and I926R; (6) E176R, K238R, T447R, E563R, N164Y and I926R; (7) E176R, K238R, T447R, E563R, N164Y, E323R and D362R; (8) E176R, K238R, T447R, E563R, N164Y, I926R, E323R and D362R; (9) E176R, K238R, T447R, E563R, N164Y, E323R, D362R and I926G; (10) E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G and 439GG; (11) E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G and 439G; (12) E176R, K238R, T447 R, E563R, N164Y and D958R; (13) E176R, K238R, T447R, E563R, I926R and D958R; (14) E176R, K238R, T447R, E563R, E323R, D362R and D958R; (15) N164Y, I926R and D9 58R; (16) N164Y, E323R, D362R and D958R; (17) E176R, K238R, T447R, E563R, N164Y, I926R and D958R; (18) E176R, K238R, T447R, E563R, N164Y, E323R, D362R and D 958R; (19) E176R, K238R, T447R, E563R, N164Y, I926R, E323R, D362R and D958R; (20) E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G and D958R; (21 )E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G, 439GG and D958R; or (22) E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G, 439G and D958R.In some embodiments, the mutation is based on the natural wild-type Cas12i2 nuclease, whose amino acid sequence is SEQ ID NO.1.

[0028] 17. An engineered Cas12i nuclease comprising an amino acid sequence as shown in any one of SEQ ID NO. 2 to 12. In some embodiments, the engineered Cas12i nuclease comprises an amino acid sequence having at least 85% (e.g., at least 87%, 89%, 91%, 93%, 95%, 97%, or 99%) sequence identity to the amino acid sequence shown in any one of SEQ ID NO. 2 to 12.

[0029] 18. An engineered Cas12i effector protein comprising an engineered Cas12i nuclease or a functional derivative thereof as described in any one of items 1 to 17. In some embodiments, the engineered Cas12i nuclease or a functional derivative thereof has enzymatic activity. In some embodiments, the effector protein comprises an enzymatically inactive mutant of the engineered Cas12i nuclease.

[0030] 19. An engineered Cas12i effector protein as described in claim 18, wherein the effector protein is capable of inducing double-strand breaks or single-strand breaks in DNA molecules.

[0031] 20. An engineered Cas12i effector protein as described in claim 18, wherein the functional derivative of the engineered Cas12i nuclease is an enzyme-inactive mutant, such as an enzyme-inactive mutant containing D599A, E833A, S883A, H884A, D886A, R900A and / or D1019A.

[0032] 21. The engineered Cas12i effector protein of items 18 to 20, further comprising a functional domain fused to the engineered Cas12i nuclease.

[0033] 22. An engineered Cas12i effector protein as described in claim 21, wherein the functional domain is selected from the group consisting of a translation initiation domain, a transcription repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain (e.g., a CBE or ABE domain), a reverse transcriptase domain, a reporter molecule domain (e.g., a fluorescent domain), and a nuclease domain.

[0034] 23. An engineered Cas12i effector protein as described in items 18 to 22, wherein the engineered Cas effector protein comprises: a first polypeptide containing the N-terminal portion of the engineered Cas12i nuclease or a functional derivative thereof and a second polypeptide containing the C-terminal portion of the engineered Cas12i nuclease or a functional derivative thereof, wherein the first polypeptide and the second polypeptide are capable of associating with each other in the presence of a guide RNA comprising a guide sequence to form a clustered regularly interspaced short palindromic repeats (CRISPR) complex that specifically binds to a target nucleic acid, wherein the target nucleic acid comprises a target sequence complementary to the guide sequence. In some embodiments, the first polypeptide comprises the N-terminal portion of amino acid residues 1 to X of the engineered Cas12i nuclease described in any one of items 1-17, and the second polypeptide comprises the amino acid residues X+1 of the engineered Cas12i nuclease described in any one of items 1-17 to the C-terminus of the Cas12i nuclease, wherein the first polypeptide and the second polypeptide are capable of associating with each other in the presence of a guide RNA comprising a guide sequence to form a clustered regularly spaced short palindromic repeat (CRISPR) complex specifically bound to a target nucleic acid, the target nucleic acid comprising a target sequence complementary to the guide sequence. In some embodiments, the first polypeptide and the second polypeptide each comprise a dimerization domain. In some embodiments, the first dimerization domain and the second dimerization domain associate with each other in the presence of an inducer. In some embodiments, the first polypeptide and the second polypeptide do not comprise a dimerization domain.

[0035] 24. An engineered CRISPR-Cas12i system comprising:

[0036] (a) the engineered Cas12i effector protein of any one of items 18-23; and

[0037] (b) a guide RNA comprising a guide sequence complementary to the target sequence, or one or more nucleic acids encoding the guide RNA,

[0038] Wherein the engineered Cas12i effector protein and the guide RNA are capable of forming a CRISPR complex, and the CRISPR complex specifically binds to a target nucleic acid comprising the target sequence and induces modification of the target nucleic acid. In some embodiments, the guide RNA is a crRNA comprising the guide sequence. In some embodiments, the system includes a precursor guide RNA array encoding multiple crRNAs. In some embodiments, wherein the engineered Cas effector protein is a primary editor, and the guide RNA is a pegRNA.

[0039] 25. An engineered CRISPR-Cas12i system as described in item 24, comprising one or more vectors encoding the engineered Cas12i effector protein. In some embodiments, the one or more vectors are selected from the group consisting of retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors. In some embodiments, the one or more vectors are adeno-associated virus (AAV) vectors. In some embodiments, the AAV vector also encodes the guide RNA (e.g., crRNA, or a precursor guide RNA array).

[0040] 26. A method for detecting a target nucleic acid in a sample, comprising:

[0041] (a) contacting the sample with the engineered CRISPR-Cas12i system of item 24 and a tagged detection nucleic acid that is single-stranded and does not hybridize to the guide sequence of the guide RNA; and

[0042] (b) measuring a detectable signal generated by cleavage of the tagged detection nucleic acid by the engineered Cas12i effector protein, thereby detecting the target nucleic acid.

[0043] 27. A method for modifying a target nucleic acid comprising a target sequence, comprising contacting the target nucleic acid with the engineered CRISPR-Cas12i system described in item 24. In some embodiments, the method is performed in vitro. In some embodiments, the target nucleic acid is present in a cell. In some embodiments, the cell is a bacterial cell, a yeast cell, a mammalian cell, a plant cell, or an animal cell. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo.

[0044] In some embodiments according to any one of the methods for modifying a target nucleic acid, the target nucleic acid is cut or the target sequence in the target nucleic acid is changed by the engineered CRISPR-Cas12i system. In some embodiments, the expression of the target nucleic acid is changed by the engineered CRISPR-Cas12i system. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target sequence is associated with a disease or condition.

[0045] In some embodiments according to any of the above methods of modifying a target nucleic acid, the engineered CRISPR-Cas12i system comprises a precursor guide RNA array encoding a plurality of crRNAs, wherein each crRNA comprises a different guide sequence.

[0046] 28. Use of the engineered CRISPR-Cas12i system of item 24 in the preparation of a medicament for treating a disease or condition associated with a target nucleic acid in a cell of an individual. In some embodiments, the disease or condition is selected from the group consisting of cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection.

[0047] 29. A method for treating a disease or condition associated with a target nucleic acid in a cell of an individual; the method comprising modifying a target nucleic acid in a cell of the individual using any of the methods of using the engineered CRISPR-Cas12i system of item 27, thereby treating the disease or condition. In some embodiments, the disease or condition is selected from the group consisting of cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection.

[0048] 30. An engineered cell comprising a modified target nucleic acid, wherein the target nucleic acid has been modified using the method as described in item 27.

[0049] 31. An engineered non-human animal comprising one or more engineered cells as described in claim 27.

[0050] It should be understood that certain features of the disclosure described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the disclosure described in the context of a single embodiment for brevity may also be provided individually or in any suitable subcombination. All combinations of embodiments involving specific method steps, reagents or conditions, or composition components are expressly contemplated by the disclosure and are disclosed herein as if each and every combination were individually and expressly disclosed.

[0051] Beneficial effects achieved by the technical solution of this application

[0052] The Cas12i nuclease and its effector protein engineered in this application have higher activity, such as the catalytic efficiency of cutting nucleic acid substrates and the gene editing efficiency in cells. The engineered Cas12i nuclease in this application has a gene editing efficiency in mammalian cells (such as human cells) that is more excellent than existing conventional Cas gene editing tools; for example, some exemplary Cas12i2 nuclease mutants in this application tested gene editing efficiency at multiple sites (such as 62 sites) in human cells, and found that the gene editing efficiency of 57 sites exceeded about 60%, and the average gene editing efficiency was close to 70%. In some embodiments, the Cas12i nuclease and its effector protein engineered in this application also have one or more of the following advantages: small protein (1,054aa), simple crRNA components, simple PAM sequences, and the protein itself can process precursor crRNA. These advantages make the efficient engineered Cas12i nuclease and its effector protein of this application very suitable for gene editing or gene regulation in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1a :Replace the amino acid that interacts with PAM in the reference Cas12i nuclease with a positively charged amino acid to improve gene editing efficiency. As shown in the figure, the four mutants E176R, K238R, T447R, and E563R can significantly improve the gene editing efficiency in human 293T cells.

[0054] Figure 1b :Will Figure 1a The amino acid mutations (E176R, K238R, T447R, E563R) that can significantly improve the gene editing efficiency were combined, and it was found that the combined mutants could show higher gene editing efficiency in human 293T cells.

[0055] Figure 2 :Replace the amino acids involved in opening the DNA double strand in the reference Cas12i nuclease with amino acids with aromatic rings to improve the efficiency of gene editing. As shown in the figure, the mutants Q163F, Q163Y, Q163W, N164F, and N164Y can significantly improve the gene editing efficiency in human 293T cells.

[0056] Figure 3a , 3b, 3c: The amino acids in the RuvC domain of the reference Cas12i nuclease that interact with the single-stranded DNA substrate are replaced with positively charged amino acids to improve the efficiency of gene editing. As shown in the figure, mutants such as E323R, L327R, V355R, G359R, G360R, D362R, N391R, Q424R, Q425R, N925R, I926R and G929R can significantly improve the gene editing efficiency in human 293T cells.

[0057] Figure 3d :Will Figure 3a , 3b The point mutations that improve efficiency were combined and it was found that the combined mutants could show higher gene editing efficiency in human 293T cells.

[0058] Figure 3e :Will Figure 3a , 3b The point mutations that improve efficiency and the mutations modified according to the principle of molecular flexibility (439GG, I926G) were combined, and it was found that the combined mutants could show higher gene editing efficiency in human 293T cells.

[0059] Figure 4 : The amino acids that interact with the DNA-RNA double helix in the reference Cas12i enzyme reference Cas12i nuclease are replaced with positively charged amino acids to improve the gene editing efficiency. As shown in the figure, the mutants G116R, E117R, T159R, S161R, E319R, E343R, and D958R can significantly improve the gene editing efficiency in human 293T cells. Among them, D958R is the best.

[0060] Figure 5 :Will Figure 1a to Figure 3e The high-efficiency mutants obtained by the three transformation strategies and the mutations transformed according to the principle of molecular flexibility (439GG, I926G) were combined, and it was found that the combined mutants can show higher gene editing efficiency in human 293T cells. The combination can greatly improve the gene editing efficiency. The mutant with the best gene editing effect was selected and named CASXX for subsequent experiments.

[0061] Figure 6a : Summary of gene editing efficiency of CasXX at 62 human genome sites. PAM=NTTN. Here, CasXX represents an engineered enzyme (based on the reference Cas12i2 of amino acid sequence SEQ ID NO.1) with the E176R+K238R+T447R+E563R+N164Y+E323R+D362R mutation combination.

[0062] Figure 6b: Comparison of gene editing efficiency between CasXX and AsCas12a, BhCas12b v4.

[0063] Figure 6c : Comparison of gene editing efficiency between CasXX and SpCas9, SaCas9, and SaCas9-KKH.

[0064] Figure 6d : Statistics of the gene editing efficiency of CasXX in the mouse Hepa1-6 cell line show that CasXX exhibits strong gene editing capabilities at 65 sites, with an average gene editing efficiency of over 60%.

[0065] Figure 7 : Homology comparison of the amino acid sequences of Cas12i2 (SEQ ID NO.1) and Cas12i1 (SEQ ID NO.13). The shaded amino acids represent the same amino acids of the two Cas12i proteins, and the amino acids marked with white boxes represent the amino acids with similar properties of the two Cas12i proteins. DETAILED DESCRIPTION

[0066] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present invention, but the description is based on the general principles of the specification and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be defined by the attached claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by ordinary technicians in the field to which the present disclosure belongs.

[0067] I. Terminology

[0068] As used herein, "effector protein" refers to a protein having an activity such as site-specific binding activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, single-stranded RNA cleavage activity, or transcriptional regulatory activity.

[0069] As used herein, "guide RNA" and "gRNA" are used interchangeably herein and refer to RNA that can form a complex with a Cas12i effector protein and a target nucleic acid (e.g., double-stranded DNA). Precursor guide RNA arrays that can be processed into multiple crRNAs are also contemplated herein. "crRNA" or "CRISPR RNA" comprises a guide sequence that is sufficiently complementary to a target sequence of a target nucleic acid (e.g., double-stranded DNA) that directs the CRISPR complex to bind to the sequence-specific binding of the target nucleic acid.

[0070] The terms "nucleic acid", "polynucleotide" and "nucleotide sequence" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof and analogs thereof. "Oligonucleotide" and "oligonucleotide" are used interchangeably to refer to short polynucleotides having no more than about 50 nucleotides.

[0071] As used herein, "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid by traditional Watson-Crick base pairing. The complementarity percentage represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., 5 / 10, 6 / 7, 8 / 9, 10, respectively complementary to about 50%, 60%, 70%, 80%, 90% and 100%). "Complete complementarity" refers to the formation of hydrogen bonds between all continuous residues of a nucleic acid sequence and the same number of continuous residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, and the degree of complementarity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100, or refers to two nucleic acids hybridized under stringent conditions.

[0072] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity with a target sequence hybridizes primarily with the target sequence, but does not substantially hybridize with a non-target sequence. Stringent conditions are generally sequence-dependent and vary depending on many factors. Generally, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes with its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology - Hybridization With Nucleic Acid Probes, Part I, Chapter II "Overview of principles of hybridization and the strategy of nucleic acid probe assay," Elsevier, N, Y.

[0073] "Hybridization" refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. A sequence that is capable of hybridizing to a given sequence is referred to as the "complement" of the given sequence.

[0074] "Percentage (%) sequence identity" for nucleic acid sequences is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in a particular nucleic acid sequence, after aligning the sequences by allowing gaps (if necessary) to achieve the maximum percentage of sequence identity. "Percentage (%) sequence identity" for peptide, polypeptide, or protein sequences is the percentage of amino acid residues in a candidate sequence that are identically replaced with the amino acid residues in a particular peptide or amino acid sequence, after aligning the sequences by allowing gaps (if necessary) to achieve the maximum percentage of sequence homology. For the purpose of determining the percentage of amino acid sequence identity, alignment can be achieved in various ways within the skill of the art, for example, using a BLAST, BLAST-2, ALIGN, or MEGALIGN sequence. TM Publicly available computer software such as (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0075] The terms "polypeptide" and "peptide" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and may be interrupted by non-amino acids. A protein may have one or more polypeptides. The term also encompasses amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as conjugation with a labeling component).

[0076] As used herein, "variant" is interpreted as a polynucleotide or polypeptide that is different from a reference polynucleotide or polypeptide, but retains the necessary characteristics. A typical variant of a polynucleotide is different from the nucleic acid sequence of another reference polynucleotide. Changes in the variant nucleic acid sequence may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide is different from another reference polypeptide in amino acid sequence. Usually, the difference is limited, so that the sequences of the reference polypeptide and the variant are generally very similar, and are identical in many regions. The amino acid sequences of the variant and the reference polypeptide may differ by any combination of one or more substitutions, additions, and deletions. The amino acid residues replaced or inserted may or may not be amino acid residues encoded by the genetic code. The variant of a polynucleotide or polypeptide may be naturally occurring (such as an allele variant), or may be an unknown naturally occurring variant. Non-naturally occurring variants of polynucleotides and polypeptides may be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0077] As used herein, the term "wild type" has a meaning generally understood by those skilled in the art, and refers to an organism, strain, gene or characteristic in a typical form that distinguishes it from a mutant or variant when it exists in nature. It can be isolated from resources in nature and has not been intentionally modified.

[0078] As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and refer to human involvement. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it is naturally associated or naturally occurring.

[0079] As used herein, the term "orthologue" has the meaning commonly understood by those of ordinary skill in the art. As a further guide, the "orthologue" of a protein referred to herein refers to a protein belonging to a different species that performs the same or similar function as its orthologue.

[0080] As used herein, the term "identity" is used to indicate sequence matching between two polypeptides or between two nucleic acids. When one position in the two comparison sequences is occupied by the same base or amino acid monomer subunit (for example, one position in each of the two DNA molecules is occupied by adenine, or one position in each of the two polypeptides is occupied by lysine), then each molecule is identical at that position. The "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of the two sequences match, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 matches in a total of 6 positions). Typically, this comparison is performed when two sequences are aligned to produce maximum identity. This alignment can be achieved, for example, by the method in Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The PAM 120 weight residue table can also be used, using the algorithm of E.Meyers and W.Miller (Comput.Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0). A gap length penalty of 12 and a gap penalty of 4 are used to determine the percent identity between two amino acid sequences. In addition, the Needleman and Wunsch (JMoI Biol.48:444-453 (1970)) algorithm integrated into the GAP program of the GCG software package (available from www.gcg.com) can be used, using the Blossum 62 matrix or the PAM250 matrix, with a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences.

[0081] "Cell" as used herein should be understood to refer not only to a particular individual cell, but also to the progeny or potential progeny of that cell. Because certain modifications may occur in progeny due to mutation or environmental influences, such progeny may not in fact be the same as the parent cell, but are still included within the scope of the term herein.

[0082] As used herein, the terms "transduction" and "transfection" include methods known in the art to introduce DNA into cells using infectious agents (such as viruses) or other means to express a protein or molecule of interest. In addition to viruses or virus-like agents, there are chemical-based transfection methods, such as transfection methods using calcium phosphate, dendrimers, liposomes or cationic polymers (such as DEAE-dextran or polyethyleneimine); non-chemical methods, such as electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, plasmid delivery or transposon; particle-based methods, such as using a gene gun, magnetofection or magnet-assisted transfection, particle bombardment; and hybrid methods (such as nuclear transfection).

[0083] As used herein, the term "transfected," "transformed," or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid.

[0084] The term "in vivo" refers to inside the organism from which the cell was obtained. "Ex vivo" or "in vitro" refers to outside the organism from which the cell was obtained.

[0085] As used herein, "treatment" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, reducing the recurrence rate of the disease, delaying or slowing the progression of the disease, improving the disease state, providing (partial or complete) relief of the disease, reducing the dosage of one or more other drugs required for treating the disease, delaying the progression of the disease, improving the quality of life, and / or prolonging survival. "Treatment" also includes reducing the pathological consequences of a disease, condition, or disease. The method of the present invention contemplates any one or more of these therapeutic aspects.

[0086] As used herein, the term "effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition or disease (such as improving, alleviating, alleviating and / or delaying one or more symptoms thereof). As understood in the art, an "effective amount" can be administered in one or more doses, i.e., a single dose or multiple doses may be required to achieve a desired treatment endpoint.

[0087] "Subject," "individual," or "patient" are used interchangeably herein for purposes of treatment and refer to any animal classified as a mammal, including humans, livestock and farm animals, and zoo, farm or pet animals such as dogs, horses, cats, cows, etc. In some embodiments, the individual is a human individual.

[0088] It is to be understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments. Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0089] As used herein, reference to a "not" value or parameter generally means and describes an "except" value or parameter. For example, the method is not used to treat cancer type X, meaning that the method is used to treat cancers other than type X.

[0090] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0091] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. This statement is therefore intended to serve as antecedent basis for the use of exclusive terminology such as "only", "only" and the like in connection with the recitation of claim elements, or the use of a limitation of "no".

[0092] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0093] II. Cas12i nuclease and effector protein

[0094] Engineered Cas12i nuclease

[0095] In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises one, two, three or four of the following mutations based on a reference Cas12i nuclease:

[0096] (1) replacing the amino acid that interacts with PAM in the reference Cas12i nuclease with a positively charged amino acid; and / or

[0097] (2) replacing the amino acids involved in opening the DNA double strand in the reference Cas12i nuclease with amino acids with aromatic rings; and / or

[0098] (3) replacing the amino acid in the reference Cas12i nuclease located in the RuvC domain and interacting with the single-stranded DNA substrate with a positively charged amino acid; and / or

[0099] (4) replacing the amino acid that interacts with the DNA-RNA double helix in the reference Cas12i nuclease with a positively charged amino acid; the reference Cas12i nuclease is a natural Cas12i nuclease or an engineered Cas12i nuclease, such as a natural Cas12i2 nuclease (e.g., its amino acid sequence is defined as SEQ ID NO.1).

[0100] The application provides a method for engineering enzymes by introducing amino acid mutations, which are based on any one or more combinations of the above three transformation principles, which result in an increase in enzyme activity in vitro and in vivo. The engineered Cas12i nuclease contains one or more specific mutations as described in the following 1)-4) sections. In some embodiments, any one or more mutations described in this application can be combined with existing Cas12i mutations (e.g., mutations described in the following 5) sections) to provide an engineered Cas12i nuclease with higher activity.

[0101] In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises a mutation in which one or more amino acids interacting with PAM in a reference Cas12i nuclease are replaced with positively charged amino acids. In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises replacing one or more amino acids involved in opening a double-stranded DNA in a reference Cas12i nuclease with amino acids with aromatic rings. In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises replacing one or more amino acids located in the RuvC domain of a reference Cas12i nuclease and interacting with a single-stranded DNA substrate with positively charged amino acids. In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises replacing one or more amino acids interacting with a DNA-RNA double helix in a reference Cas12i nuclease with positively charged amino acids.

[0102] In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) a mutation in which one or more amino acids interacting with PAM in a reference Cas12i nuclease are replaced with positively charged amino acids; and 2) one or more amino acids involved in opening a double-stranded DNA in a reference Cas12i nuclease are replaced with amino acids with aromatic rings. In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) a mutation in which one or more amino acids interacting with PAM in a reference Cas12i nuclease are replaced with positively charged amino acids; and 2) one or more amino acids in a reference Cas12i nuclease located in the RuvC domain and interacting with a single-stranded DNA substrate are replaced with positively charged amino acids. In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) replacing one or more amino acids involved in opening a double-stranded DNA in a reference Cas12i nuclease with amino acids with aromatic rings; and 2) replacing one or more amino acids in a reference Cas12i nuclease located in the RuvC domain and interacting with a single-stranded DNA substrate with positively charged amino acids.

[0103] In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) replacing one or more amino acids interacting with PAM in a reference Cas12i nuclease with a mutation of a positively charged amino acid; and 2) replacing one or more amino acids interacting with a DNA-RNA double helix in a reference Cas12i nuclease with a positively charged amino acid. In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) replacing one or more amino acids involved in opening a DNA double helix in a reference Cas12i nuclease with an amino acid with an aromatic ring; and 2) replacing one or more amino acids interacting with a DNA-RNA double helix in a reference Cas12i nuclease with a positively charged amino acid. In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) one or more amino acids in the reference Cas12i nuclease that are located in the RuvC domain and interact with a single-stranded DNA substrate are replaced with positively charged amino acids; and 2) one or more amino acids in the reference Cas12i nuclease that interact with a DNA-RNA double helix are replaced with positively charged amino acids.

[0104] In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) a mutation in which one or more amino acids interacting with PAM in a reference Cas12i nuclease are replaced with positively charged amino acids; 2) one or more amino acids involved in opening a double-stranded DNA in a reference Cas12i nuclease are replaced with amino acids with aromatic rings; and 3) one or more amino acids located in the RuvC domain and interacting with a single-stranded DNA substrate in a reference Cas12i nuclease are replaced with positively charged amino acids. In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) a mutation in which one or more amino acids interacting with PAM in a reference Cas12i nuclease are replaced with positively charged amino acids; 2) one or more amino acids involved in opening a double-stranded DNA in a reference Cas12i nuclease are replaced with amino acids with aromatic rings; and 3) one or more amino acids interacting with a DNA-RNA double helix in a reference Cas12i nuclease are replaced with positively charged amino acids. In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) a mutation in which one or more amino acids interacting with PAM in a reference Cas12i nuclease are replaced with positively charged amino acids; 2) one or more amino acids in a reference Cas12i nuclease that are located in the RuvC domain and interact with a single-stranded DNA substrate are replaced with positively charged amino acids; and 3) one or more amino acids in a reference Cas12i nuclease that interact with a DNA-RNA double helix are replaced with positively charged amino acids. In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) replacing one or more amino acids in the reference Cas12i nuclease that are located in the RuvC domain and interact with a single-stranded DNA substrate with positively charged amino acids; 2) replacing one or more amino acids in the reference Cas12i nuclease that are located in the RuvC domain and interact with a single-stranded DNA substrate with positively charged amino acids; and 3) replacing one or more amino acids in the reference Cas12i nuclease that interact with a DNA-RNA double helix with positively charged amino acids.

[0105] In some embodiments, an engineered Cas12i nuclease is provided; the engineered Cas12i nuclease comprises: 1) a mutation in which one or more amino acids interacting with PAM in a reference Cas12i nuclease are replaced with positively charged amino acids; 2) one or more amino acids involved in opening a double-stranded DNA in a reference Cas12i nuclease are replaced with amino acids with aromatic rings; 3) one or more amino acids in a reference Cas12i nuclease located in the RuvC domain and interacting with a single-stranded DNA substrate are replaced with positively charged amino acids; and 4) one or more amino acids interacting with a DNA-RNA double helix in a reference Cas12i nuclease are replaced with positively charged amino acids.

[0106] 1) Replace the amino acids that interact with PAM in the reference Cas12i nuclease with positively charged amino acids

[0107] In some embodiments, the engineered Cas12i nuclease comprises one or more mutations based on a reference Cas12i nuclease (e.g., Cas12i2), wherein the mutation is to replace an amino acid that interacts with a PAM in the reference Cas12i nuclease with a positively charged amino acid. In some embodiments, the engineered Cas12i nuclease comprises replacement of one, two, three, four, five, or six of the amino acid residues.

[0108] In some embodiments, the amino acid that interacts with PAM is an amino acid that is within 9 angstroms of PAM in the three-dimensional structure, for example, it can be: an amino acid that is within 9 angstroms of PAM in the three-dimensional structure, an amino acid that is within 8 angstroms of PAM in the three-dimensional structure, an amino acid that is within 7 angstroms of PAM in the three-dimensional structure, an amino acid that is within 6 angstroms of PAM in the three-dimensional structure, an amino acid that is within 5 angstroms of PAM in the three-dimensional structure, an amino acid that is within 4 angstroms of PAM in the three-dimensional structure, or an amino acid that is within 3 angstroms of PAM in the three-dimensional structure.

[0109] In some embodiments, the one or more mutations based on the reference Cas12i nuclease are one or more amino acids at the following positions: 176, 178, 226, 227, 229, 237, 238, 264, 447 and 563. In some embodiments, the one or more mutations based on the reference Cas12i nuclease are at one or more amino acids: E176, E178, Y226, A227, N229, E237, K238, K264, T447, E563. In some embodiments, the one or more mutations based on the reference Cas12i nuclease are at one or more amino acids: E176, K238, T447, E563. In some embodiments, the mutation based on the reference Cas12i nuclease is at amino acid residue 563, such as E563. In some embodiments, the amino acid position numbering is as defined in SEQ ID NO.1.

[0110] In the context of this specification, E176 means: in the cited amino acid sequence, the 176th amino acid E (glutamic acid); here, common amino acids and their three-letter and one-letter abbreviations are listed as follows:

[0111] Alanine AlaA; Arginine Arg R;

[0112] Aspartic acid Asp D; Cysteine ​​Cys C;

[0113] Glutamine Gln Q; Glutamate Glu E;

[0114] Histidine His H; Isoleucine Ile I;

[0115] Glycine Gly G; Asparagine AsnN;

[0116] Leucine Leu L; Lysine Lys K;

[0117] Methionine Met M; Phenylalanine Phe F;

[0118] Proline Pro P; Serine Ser S;

[0119] Threonine ThrT; Tryptophan Trp W;

[0120] Tyrosine TyrY; Valine ValV.

[0121] As used herein, "the amino acid is at position X, wherein the amino acid position number is as defined in SEQ ID NO.1" means that the amino acid residue is located at a position of the reference enzyme Cas12i, which is equivalent to position X of SEQ ID NO:1, and the amino acid sequence of the reference enzyme Cas12i and the amino acid sequence of SEQ ID NO:1 are aligned with each other based on sequence homology. For example, Figure 7 The homology comparison of the amino acid sequences of CAS12i2 (SEQ ID NO.1) and CAS12i1 (SEQ ID NO.13) is shown. Those skilled in the art can use software commonly used in the art, such as Clustal Omega, to compare and align the amino acid sequence of any reference Cas12i nuclease with SEQ ID NO.1 for sequence identity, and then obtain the amino acid sites in the reference Cas12i nuclease corresponding to the amino acid sites defined based on SEQ ID NO.1 described in the present application.

[0122] In some embodiments, the mutation based on the reference Cas12i nuclease is to replace the corresponding amino acid residue in the reference Cas12i nuclease with R or K. In some embodiments, the mutation based on the reference Cas12i nuclease is to replace the corresponding amino acid residue in the reference Cas12i nuclease with R.

[0123] In some embodiments, the engineered Cas12i nuclease comprises one or more of the following amino acid residues: R176, R238, R447 and R563, wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises one or more of the following mutations based on the reference Cas12i nuclease: E176R, K238R, T447R and E563R; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises an E563R mutation; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, for the purpose of improving gene editing efficiency, an engineered enzyme having at least 85% sequence identity with the above-mentioned engineered Cas12i nuclease can also be used; in some embodiments, an engineered enzyme having at least 87%, 89%, 91%, 93%, 95%, 97%, 99% sequence identity therewith can be used.

[0124] In the context of the present specification, E176R means that in the cited amino acid sequence, the 176th amino acid E (glutamic acid) is replaced by R (arginine).

[0125] In some embodiments, the engineered Cas12i nuclease comprises any one or combination of mutations at the following amino acid residue positions: 176, 238, 264, 447, 563, 176 and 238, 176 and 447, 176 and 563, 238 and 447, 238 and 563, 447 and 563, 176 and 238 and 447, 176 and 238 and 563, 176 and 447 and 563, 238 and 447 and 563, 176 and 238 and 447 and 563; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the mutation is to replace the corresponding amino acid residue in the reference Cas12i nuclease with R or K, such as R. In some embodiments, the engineered Cas12i nuclease comprises any one of the following amino acid residues or combinations: R176, R238, R264, R447, R563, R176+R238, R176+R447, R176+R563, R238+R447, R238+R563, R447+R563, R176+R238+R447, R176+R238+R563, R176+R447+R563, R238+R447+R563, R176+R238+R447+R563; wherein the amino acid position numbering is as defined in SEQ ID NO.1.

[0126] In some embodiments, the engineered Cas12i nuclease comprises any one of the following mutations / mutation combinations: E176R, K238R, E264R, T447R, E563R, E176R+K238R, E176R+T447R, E176R+E563R, K238R+T447R, K238R+E563R, T447R+E563R, E176R+K238R+T447R, E176R+K238R+E563R, E176R+T447R+E563R, K238R+T447R+E563R, E176R+K238R+T447R+E563R; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises any of the following mutations / mutation combinations: E563R, E176R+T447R, E176R+E563R, K238R+E563R, E176R+K238R+T447R, E176R+K238R+E563R, E176R+T447R+E563R, E176R+K238R+T447R+E563R; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises the E176R+K238R+T447R+E563R mutation combination; wherein the amino acid position numbering is as defined in SEQ ID NO.1. For the purpose of improving gene editing efficiency, engineered enzymes having at least 85% sequence identity with the above-mentioned engineered Cas12i can also be used; in some embodiments, engineered enzymes having at least 87%, 89%, 91%, 93%, 95%, 97%, 99% sequence identity therewith can be used.

[0127] 2) Reference Cas12i Nucleic Acids The amino acids involved in opening the double-stranded DNA in the enzyme are replaced with amino acids with aromatic rings

[0128] In some embodiments, the engineered Cas12i nuclease comprises one or more mutations based on a reference Cas12i nuclease (e.g., Cas12i2), wherein the mutation is to replace the amino acid involved in opening the double-stranded DNA in the reference Cas12i nuclease with an amino acid with an aromatic ring. In some embodiments, the engineered Cas12i nuclease comprises one, two, three, four, five, or six replacements of the amino acid residues.

[0129] Among them, the one or more amino acids involved in opening the DNA double strand are amino acids that interact with the last base pair at the 3' end of the PAM relative to the target strand. For example, the PAM sequence recognized by Cas12i2 is a 5'-NTTN-3' base pair, in which the base pair formed by the N base at the 3' end of the PAM sequence and the target strand is the "last base pair at the 3' end of the PAM relative to the target strand" described in the text, and the sequence of the target site is followed by the base pair.

[0130] In some embodiments, the one or more amino acids are located at the following positions: 163 and / or 164. In some embodiments, the one or more amino acids are the following one or more amino acids: Q163, N164. In some embodiments, the amino acid is N164; wherein the amino acid position numbering is as defined in SEQ ID NO.1.

[0131] In some embodiments, the amino acid involved in opening the double-stranded DNA is replaced by F, Y or W. In some embodiments, the amino acid involved in opening the double-stranded DNA is replaced by F. In some embodiments, the amino acid involved in opening the double-stranded DNA is replaced by Y.

[0132] In some embodiments, the engineered Cas12i nuclease comprises any one or more of the following amino acid residues: F163, Y163, W163, F164, or Y164; wherein the amino acid position numbering is as defined in SEQ ID NO.1.

[0133] In some embodiments, the engineered Cas12i nuclease comprises any of the following mutations: Q163F, Q163Y, Q163W, N164F or N164Y. In some embodiments, the amino acid position number is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises N164Y or N164F mutations. In some embodiments, the engineered Cas12i nuclease comprises N164Y mutations. In some embodiments, for the purpose of improving gene editing efficiency, an engineered enzyme having at least 85% sequence identity with the above-mentioned engineered enzyme can also be used; in some embodiments, an enzyme having at least 87%, 89%, 91%, 93%, 95%, 97%, 99% sequence identity can be used.

[0134] 3) Replace the amino acid located in the RuvC domain of the reference Cas12i nuclease and interacting with the single-stranded DNA substrate Replace with positively charged amino acids

[0135] In some embodiments, the engineered Cas12i nuclease comprises one or more mutations based on a reference Cas12i nuclease (e.g., Cas12i2), wherein the mutation is to replace an amino acid located in the RuvC domain of the reference Cas12i enzyme and interacting with a single-stranded DNA substrate with a positively charged amino acid. In some embodiments, the engineered Cas12i enzyme comprises one, two, three, four, five, or six replacements of the amino acid residues.

[0136] Wherein, the one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are amino acids within 9 angstroms of the single-stranded DNA substrate in the three-dimensional structure, for example, can be: amino acids within 8 angstroms of the single-stranded DNA substrate in the three-dimensional structure, amino acids within 7 angstroms of the single-stranded DNA substrate in the three-dimensional structure, amino acids within 6 angstroms of the single-stranded DNA substrate in the three-dimensional structure, amino acids within 5 angstroms of the single-stranded DNA substrate in the three-dimensional structure, amino acids within 4 angstroms of the single-stranded DNA substrate in the three-dimensional structure, and amino acids within 3 angstroms of the single-stranded DNA substrate in the three-dimensional structure. The RuvC domain is the enzymatic domain responsible for cutting single-stranded DNA or double-stranded DNA in the Cas12i protein. In the primary sequence of the protein, the RuvC domain of Cas12i is divided into three parts: RuvC-1, RuvC-2 and RuvC-3. These three parts are adjacent to each other in the three-dimensional structure and together form a catalytic pocket with enzymatic activity. The three-dimensional crystal structure of Cas12i2, its domain composition, and the description of interaction with DNA substrates are shown in Huang X.etal., Nature Communications, 11, Article number: 5241 (2020). The three-dimensional crystal structure of Cas12i1, its domain composition, and the description of interaction with DNA substrates are shown in Zhang H.et al. Nature Structural & Molecular Biology 27, 1069-1076 (2020). The three-dimensional structural model of the interaction between reference Cas12i and substrate can be obtained by homology structure comparison and modeling (homologymodeling) by the known Cas12i three-dimensional crystal structure. A modeling method is described in Example 3 to obtain an amino acid located in the RuvC domain in Cas12i2 and within 9 angstroms of a single-stranded DNA substrate.

[0137] In some embodiments, the one or more amino acids are one or more amino acids at the following positions: 323, 362, 425, 925, 926, 390, 391, 392, 751, 755, 840, 848, 851, 856, 885, 897, 929, 932, 327, 355, 359, 360, 361, 414, 421, 650, 652, 705, 708, 709, 752, 928, 388, 393, 417, 418, 424, 653, 696, 1022. In some embodiments, the one or more amino acids are one or more of the following: E323, D362, Q425, N925, I926, N390, N391, F392, L751, E755, N840, N848, S851, A856, Q885, M897, G929, Y932, L327, V355, G359, G360, K361, Q414, K421, S650, E652, K705, K708, E709, S752, T928, L388, K393, L417, A418, Q424, G653, I696, A1022. In some embodiments, the one or more amino acids are one or more of the following amino acids: E323, D362, Q425, N925, I926, N391, Q424, G929, L388, L417. In some embodiments, the amino acid position numbering is as defined in SEQ ID NO.1.

[0138] In some embodiments, the engineered Cas12i nuclease comprises a mutation in which one or more amino acids involved in the cleavage of double-stranded DNA in a reference Cas12i nuclease are replaced with R or K. In some embodiments, the engineered Cas12i nuclease comprises a mutation in which one or more amino acids involved in the cleavage of double-stranded DNA in a reference Cas12i nuclease are replaced with R.

[0139] In some embodiments, the engineered Cas12i nuclease comprises a mutation or combination of mutations at any of the following amino acid residue positions: 323, 362, 425, 925, 926, and 929; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises a mutation or combination of mutations at any of the following amino acid residue positions: 323, 362, 425, 925, 926, 323 and 362, 323 and 425, 323 and 926, 362 and 425, 362 and 926, 425 and 926, 925 and 926, 323 and 362 and 425, 323 and 362 and 926, 323 and 425 and 926, 362 and 925 and 926, 362 and 425 and 926; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the mutation is a mutation that replaces the amino acid residue at the position with R or K (such as R). In some embodiments, the engineered Cas12i nuclease comprises any one of the following amino acids or amino acid combinations: R323, R362, R425, R925, R926, R323+R362, R323+R425, R323+R926, R362+R425, R362+R926, R425+R926, R925+R926, R323+R362+R425, R323+R362+R926, R323+R425+R926, R362+R925+R926, R362+R425+R926; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises any one or combination of the following mutations: E323R, D362R, Q424R, Q425R, N925R, I926R, and G929R; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises any one of the following mutations or combinations of mutations: E323R, D362R, Q425R, N925R, I926R, E323R+D362R, E323R+Q425R, E323R+I926R, Q425R+I926R, D362R+I926R, N925R+I926R, E323R+D362R+Q425R, E323R+D362R+I926R, E323R+Q425R+I926R, D362R+N925R+I926R, E323R+D362R+Q425R+I926R; wherein the amino acid position numbering is as defined in SEQ ID NO.1.In some embodiments, the engineered Cas12i nuclease comprises an I926R mutation; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises an E323R+D362R mutation; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, for the purpose of improving gene editing efficiency, an engineered enzyme having at least 85% sequence identity with the above-mentioned engineered enzyme may also be used. In some embodiments, enzymes having at least 87%, 89%, 91%, 93%, 95%, 97%, 99% sequence identity therewith may be used.

[0140] 4) Replace one or more amino acids that interact with the DNA-RNA double helix in the reference Cas12i nuclease with Positively charged amino acids

[0141] In some embodiments, the engineered Cas12i nuclease comprises one or more mutations based on a reference Cas12i nuclease (e.g., Cas12i2), wherein the mutation is to replace one or more amino acids interacting with the DNA-RNA double helix in the reference Cas12i nuclease with positively charged amino acids. In some embodiments, the engineered Cas12i enzyme comprises replacement of one, two, three, four, five, or six of the amino acid residues.

[0142] Among them, the one or more amino acids that interact with the DNA-RNA double helix are amino acids within 9 angstroms of the DNA-RNA double helix in the three-dimensional structure, for example, they can be: amino acids within 8 angstroms of the DNA-RNA double helix in the three-dimensional structure, amino acids within 7 angstroms of the DNA-RNA double helix in the three-dimensional structure, amino acids within 6 angstroms of the DNA-RNA double helix in the three-dimensional structure, amino acids within 5 angstroms of the DNA-RNA double helix in the three-dimensional structure, amino acids within 4 angstroms of the DNA-RNA double helix in the three-dimensional structure, or amino acids within 3 angstroms of the DNA-RNA double helix in the three-dimensional structure. The working principle of some Cas nucleases is as follows: Cas forms a complex with a guide RNA (such as crRNA), in which the crRNA and the target DNA are paired with each other to form a DNA-RNA double helix, and interact with the Cas nuclease to open the double-stranded target DNA and form an R-loop, so that the Cas enzyme active site can complete the cutting of dsDNA. The three-dimensional crystal structure of Cas12i2, its domain composition, and a description of its interaction with the DNA-RNA double helix are described in Huang X. et al., Nature Communications, 11, Article number: 5241 (2020).

[0143] In some embodiments, the one or more amino acids are one or more amino acids at the following positions: 116, 117, 156, 159, 161, 301, 305, 306, 308, 312, 313, 427, 433, 438, 441, 442, 852, 855, 861, 865, 160, 316, 319, 320, 247, 343, 348, 349, 679, 683, 691, 782, 783, 797, 800, 853, 957, 958, 293, 294, or 297. In some embodiments, the one or more amino acids are one or more of the following: G116, E117, A156, T159, S161, T301, 1305, K306, T308, N312, F313, D427, K433, V438, N441, Q442, M852, L855, N861, Q865, E160, Q316, E319, Q320, E247, E343, E348, E349, N679, E683, E691, D782, E783, E797, E800, D853, S957, D958, G293, E294, or N297. In some embodiments, the one or more amino acids are one or more of the following amino acids: G116, E117, T159, S161, E319, E343, or D958. In some embodiments, the one or more amino acids are D958. In some embodiments, the one or more amino acids are In some embodiments, the amino acid position numbering is as defined in SEQ ID NO.1.

[0144] In some embodiments, the engineered Cas12i nuclease comprises a mutation in which one or more amino acids interacting with the DNA-RNA double helix in the reference Cas12i nuclease are replaced with R or K. In some embodiments, the engineered Cas12i nuclease comprises a mutation in which one or more amino acids involved in the DNA-RNA double helix interaction in the reference Cas12i nuclease are replaced with R.

[0145] In some embodiments, the engineered Cas12i nuclease comprises a mutation or combination of mutations at any of the following amino acid residue positions: 116, 117, 159, 161, 319, 343, or 958; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the mutation is a mutation that replaces the amino acid residue at the position with R or K (such as R). In some embodiments, the engineered Cas12i nuclease comprises any of the following amino acids or combinations of amino acids: R116, R117, R159, R161, R319, R343, or R958; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises any of the following mutations or combinations of mutations: E323R, D362R, Q424R, Q425R, N925R, I926R, and G929R; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises any of the following mutations or combinations of mutations: G116R, E117R, T159R, S161R, E319R, E343R, or D958R; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises an I926R mutation; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the engineered Cas12i nuclease comprises a D958R mutation; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, for the purpose of improving gene editing efficiency, an engineered enzyme having at least 85% sequence identity with the above-mentioned engineered enzyme may also be used. In some embodiments, enzymes having at least 87%, 89%, 91%, 93%, 95%, 97%, 99% sequence identity therewith may be used.

[0146] 5) Other mutations

[0147] Any one or more mutations described in sections 1) to 4) can be combined with any one or more known mutations that increase Cas12i activity, such as target binding, double-stranded cleavage activity, nickase activity, and / or gene editing activity. Exemplary mutations can be found, for example, in the following documents PCT / CN2020 / 0134249 and CN 112195164A, which are incorporated herein by reference as a whole.

[0148] In some embodiments, the engineered Cas12i nuclease further comprises one or more flexible region mutations, which increase the flexibility of the flexible region in the reference Cas12i nuclease. The flexible region in the reference Cas12i nuclease can be determined using any method known in the art. In some embodiments, multiple flexible regions are determined based only on the amino acid sequence of the reference enzyme. In some embodiments, multiple flexible regions are determined based on the structural information of the reference enzyme, including, for example, secondary structure, crystal structure, NMR structure, etc.

[0149] The method for engineering a flexible region of a Cas12i nuclease described herein includes: (a) obtaining a plurality of engineered Cas12i nucleases, each engineered Cas12i nuclease comprising one or more mutations, the mutations increasing the flexibility of the flexible region in a plurality of flexible regions of a reference Cas12i nuclease; and (b) selecting one or more engineered Cas12i nucleases from the plurality of engineered Cas12i nucleases, wherein the one or more engineered Cas12i nucleases have increased activity compared to the reference Cas12i nuclease. In some embodiments, the method further includes determining a plurality of flexible regions in the reference Cas12i nuclease. In some embodiments, activity is measured in eukaryotic cells such as mammalian cells (eg, human cells).

[0150] In some embodiments, multiple flexible regions are determined using a program selected from the group consisting of PredyFlexy, FoldUnfold, PROFbval, Flexserv, FlexPred, DynaMine, and Disomine. In some embodiments, the multiple flexible regions are located at random coils. In some embodiments, the multiple flexible regions are in the domains of the reference Cas12i nucleases that interact with DNA and / or RNA. In some embodiments, the length of the flexible region is at least about 5 (e.g., 5) amino acids.

[0151] In some embodiments, the one or more mutations include inserting one or more (e.g., 2) glycine (G) residues in the flexible region. In some embodiments, the one or more G residues are inserted at the N-terminus of the flexible amino acid residues in the flexible region, wherein the flexible amino acid residues are selected from the group consisting of G, serine (S), asparagine (N), aspartic acid (D), histidine (H), methionine (M), threonine (T), glutamic acid (E), glutamine (Q), lysine (K), arginine (R), alanine (A), and proline (P). In some embodiments, the flexible amino acid residues are selected according to the following priority: G>S>N>D>H>M>T>E>Q>K>R>A>P. In some embodiments, the one or more mutations include replacing one or more non-G residues with one or more G residues.

[0152] In some embodiments, the one or more mutations comprise replacing a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of leucine (L), isoleucine (I), valine (V), cysteine ​​(C), tyrosine (Y), phenylalanine (F), and tryptophan (W).

[0153] In some embodiments, the activity is site-specific nuclease activity. In some embodiments, the activity is gene editing activity in eukaryotic cells (e.g., human cells). In some embodiments, the gene editing efficiency is measured using the following method: T7 endonuclease 1 (T7E1) assay, sequencing of target DNA, insertion and deletion tracking by decomposition (TIDE) assay, or insertion and deletion detection (IDAA) assay by amplicon analysis.

[0154] In some embodiments, the engineered Cas12i nuclease comprises one or more mutations that increase the flexibility of a flexible region in a reference Cas12i nuclease (such as a Cas12i2 nuclease), the flexible region being selected from a group corresponding to the following regions: amino acid residues 228-232, amino acid residues 439-443, amino acid residues 478-482, amino acid residues 500-504, amino acid residues 775-779, and amino acid residues 925-929, wherein the amino acid residues are numbered based on SEQ ID NO: 1. In some embodiments, the flexible region corresponds to amino acid residues 439-443 or amino acid residues 925-929, wherein the amino acid residues are numbered based on SEQ ID NO: 1. In some embodiments, the reference Cas12i enzyme is Cas12i2. In some embodiments, the one or more mutations include inserting one or more (e.g., 2) G residues in the flexible region. In some embodiments, the one or more G residues are inserted at the N-terminus of the flexible amino acid residues in the flexible region, wherein the flexible amino acid residues are selected from the group consisting of G, S, N, D, H, M, T, E, Q, K, R, A, and P. In some embodiments, the flexible amino acid residues are selected according to the following priority: G>S>N>D>H>M>T>E>Q>K>R>A>P. In some embodiments, the one or more mutations comprise replacing a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of L, I, V, C, Y, F, and W.

[0155] In some embodiments, the flexible region mutations are located at one or more of the following positions: 439, 926. In some embodiments, they are one or more of the following amino acids: L439, I926.

[0156] In some embodiments, the engineered Cas12i nuclease comprises amino acid residues G926 and / or G439, ​​and the amino acid residues are numbered based on SEQ ID NO: 1. In some embodiments, the engineered Cas12i nuclease comprises one or more of the following flexible region mutations: I926G; and / or 439G or 439GG. In some embodiments, the engineered Cas12i nuclease comprises an I926G mutation. In some embodiments, the engineered Cas12i nuclease comprises a 439G mutation. In some embodiments, the engineered Cas12i nuclease comprises a 439GG mutation.

[0157] In the context of this specification, 439G means that in the cited amino acid sequence, one glycine (G) is inserted after amino acid No. 439. And 439GG means that in the cited amino acid sequence, two glycines (GG) are inserted after amino acid No. 439.

[0158] In some embodiments, the engineered Cas12i nuclease comprises a mutation or combination of mutations at any of the following amino acid residue positions: 926, 439, 925 and 926, 326 and 925 and 926, 926 and 439, 323 and 362 and 926; wherein the amino acid position numbering is as defined in SEQ ID NO.1. In some embodiments, the mutation at amino acid position 323, 362, 925 or 926 is a mutation that replaces the amino acid residue at the position with R or K (such as R). In some embodiments, the mutation at amino acid position 439 or 926 is a mutation that replaces the amino acid residue at the position with G or inserts G or GG after the amino acid residue.

[0159] In some embodiments, the engineered Cas12i nuclease comprises any one of the following amino acid residues or combinations of amino acid residues: G926, 439GG, R925+G926, R326+R925+G926, R926+439GG, or R323+R362+G926; wherein the amino acid position numbering is as defined in SEQ ID NO.1.

[0160] In some embodiments, the engineered Cas12i nuclease comprises any one of the following mutations or combinations of mutations: I926G, 439GG, I926R+439GG, N925R+I926G, D362R+N925R+I926G, E323R+D362R+I926G; wherein the amino acid position numbering is as defined in SEQ ID NO.1.

[0161] In some embodiments, to achieve the purpose of improving gene editing efficiency, an engineered enzyme having at least 85% sequence identity with the above-mentioned engineered enzyme may also be used. In some embodiments, an enzyme having at least 87%, 89%, 91%, 93%, 95%, 97%, 99% sequence identity therewith may be used.

[0162] 6) Combined mutation

[0163] Engineered enzymes obtained by combining the mutations described in Sections 1)-5) of this specification and multiple amino acid substitutions / insertions in Tables 1 to 5 are all within the scope of protection of this application.

[0164] In some embodiments, the engineered Cas12i nuclease comprises a mutation or combination of mutations at any of the following amino acid residue positions: 176, 238, 447, 563, 164, 926, 323, 362, 439, 958, 176 and 238 and 447 and 563, 323 and 362, 176 and 238 and 447 and 563 and 164, 176 and 238 and 447 and 563 and 926, 176 and 238 and 447 and 563 63 and 323 and 362, 164 and 926, 164 and 323 and 362, 176 and 238 and 447 and 563 and 164 and 926, 176 and 238 and 447 and 563 and 164 and 323 and 362, 176 and 238 and 447 and 563 and 164 and 926 and 323 and 362, 176 and 238 and 447 and 563 and 164 and 926 and 323 and 362, 176 and 238 and 447 and 563 and 164 and 323 and 362 and 926 and 439; wherein the amino acid position numbers are as defined in SEQ ID NO. 1. In some embodiments, the mutation at amino acid position 176, 238, 447, 563, 926, 323, 362 or 958 is a mutation that replaces the amino acid residue at the position with R or K (such as R). In some embodiments, the mutation at amino acid position 164 is a mutation that replaces the amino acid residue at the position with Y or F (such as Y). In some embodiments, the mutation at amino acid position 439 or 926 is a mutation that replaces the amino acid residue at the position with G or inserts G or GG after the amino acid residue.

[0165] In some embodiments, the engineered Cas12i nuclease comprises any one of the following amino acid residues or combinations of amino acid residues: R176, R238, R447, R563, Y164, R926, G926, R958, R323, R362, 439G, 439GG, R176+R238+R447+R563, R323+R362, R176+R238+R447+R563+Y164, R176+R238+R447+R563+R926, R176+R238+R447+R563+R323+R362, Y164+R926, Y 164+R323+R362, R176+R238+R447+R563+Y164+R926, R176+R238+R447+R563+Y164+R323+R362, R176+R238+R447+R563+Y164+R926+R323 +R362、R176+R238+R447+R563+Y164+R323+R362+G926、R176+R238+R447+R563+Y164+R323+R362+G926+439G、R176+R238+R447+R563+Y1 64+R323+R362+G926+439GG; R176+R238+R447+R563+Y164+R958; R176+R238+R447+R563+R926+R958; R176+R238+R447+R563+R323+R362 +R958; Y164Y+R926+R958; 238+R447+R563+Y164+R323+R362+R958; R176+R238+R447+R563+Y164+R926+R323+R362+R958; R176+R238+R447+R563+Y164+R323+R362+G926+R958; R176+R238+R447+R563+Y164+R323+R362+G926+439GG+R958; or R176+R238+R447+R563+Y164+R323+R362+G926+439G+R958;

[0166] The amino acid position numbers are as defined in SEQ ID NO.1.

[0167] In some embodiments,The engineered Cas12i nuclease comprises any one of the following mutations or combinations of mutations: E176R+K238R+T447R+E563R, N164Y, I926R, E323R+D362R, E176R+K238R+T447R+E563R+N164Y, E176R+K238R+T447R+E563R+I926R, N164Y+E323R+D362R, E176R+K238R+T447R+E563R+E323R+D362R, N164Y+I926R, E176R+K238R+T447R+E563R+N164Y+I926R, E323R+D362R R+T447R+E563R+N164Y+E323R+D362, E176R+K238R+T447R+E563R+N164Y+I 926R+E323R+D362R, E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I92 6G, E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+439GG, E176R +K238R+T447R+E563R+N164Y+E323R+D362R+I926G+439G, E176R+K238R+T44 7R+E563R+N164Y+D958R, E176R+K238R+T447R+E563R+I926R+D958R, E176R +K238R+T447R+E563R+E323R+D362R+D958R、N164Y+I926R+D958R、N164Y+E 323R+D362R+D958R, E176R+K238R+T447R+E563R+N164Y+D958R, E176R+K23 8R+T447R+E563R+N164Y+I926R+D958R, E176R+K238R+T447R+E563R+N164Y+ E323R+D362R+D958R, E176R+K238R+T447R+E563R+N164Y+I926R+E323R+D3 62R+D958R, E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+D958 R, E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+439GG+D958R, or E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+439G+D958R;The amino acid position numbers are as defined in SEQ ID NO. 1.

[0168] In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, and D362R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, and I926R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, E323R, and D362R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, and I926G mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G, and 439GG mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G, and 439G mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, and D958R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, I926R, and D958R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, and D958R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, I926R, E323R, D362R, and D958R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G, and D958R mutations. In some embodiments, the engineered Cas12i nuclease comprises E176R, K238R, T447R, E563R, N164Y, E323R, D362R, I926G, 439GG, and D958R mutations.

[0169] In some embodiments, in order to achieve the purpose of improving gene editing efficiency, engineered enzymes having at least 85% sequence identity with the above-mentioned engineered enzymes can also be used; in some embodiments, enzymes having at least 87%, 89%, 91%, 93%, 95%, 97%, or 99% sequence identity therewith can be used.

[0170] In some embodiments, an engineered Cas12i nuclease is provided, comprising any one of the amino acid sequences shown in SEQ ID NOs. 1 to 12, or an amino acid sequence having at least 85% (e.g., at least 87%, 89%, 91%, 93%, 95%, 97%, or 99%) sequence identity to any one of the amino acid sequences shown in SEQ ID NOs. 1 to 12.

[0171] 6) Reference Cas12i nuclease

[0172] In some embodiments, the reference Cas12i nuclease is Cas12i1, Cas12i2, or a direct homolog thereof. In some embodiments, the reference Cas12i nuclease is a natural Cas12i1. In some embodiments, the reference Cas12i nuclease is a natural Cas12i2. In some embodiments, the reference Cas12i nuclease is an engineered Cas12i nuclease.

[0173] Type VI CRISPR-Cas12i has been identified as an RNA-guided DNA endonuclease system. Unlike CRISPR-Cas systems such as Cas12b or Cas9, the CRISPR system based on Cas12i does not require a tracrRNA sequence. In some embodiments, the RNA guide sequence includes crRNA. Typically, the crRNA described herein includes a direct repeat sequence and a spacer sequence. In certain embodiments, the crRNA includes a direct repeat sequence connected to a guide sequence or a spacer sequence, substantially consisting of or consisting of it. In some embodiments, the crRNA includes a direct repeat sequence, a spacer sequence, and a direct repeat sequence (DR-spacer-DR), which is a typical feature of the precursor crRNA (pre-crRNA) configuration in other CRISPR systems. In some embodiments, the crRNA includes a truncated direct repeat sequence and a spacer sequence, which is a typical feature of a processed or mature crRNA. In some embodiments, the CRISPR-Cas12i effector protein forms a complex with an RNA guide sequence, and the spacer sequence guides the complex to sequence-specific binding with a target nucleic acid, and the target nucleic acid is complementary to the spacer sequence.

[0174] In some embodiments, the engineered Cas12i of the present application is an endonuclease that binds to a specific site of a target sequence and cuts under the guidance of a guide RNA, and has DNA and RNA endonuclease activity. In some embodiments, the Cas12i can carry out autonomous crRNA biogenesis by processing a precursor crRNA array. Autonomous precursor crRNA processing can promote the delivery of Cas12i, thereby realizing double incision applications, because two separate genomic sites can be targeted by a single crRNA transcript. Then, the Cas12i protein processes the CRISPR array into two homologous crRNAs, thereby forming a paired incision complex. The multiplexing of type VI (Cas12i) effector proteins is accomplished using the precursor crRNA processing capabilities of the effector proteins, wherein multiple targets with different sequences can be programmed on a single RNA guide sequence. In this way, multiple genes or DNA targets can be manipulated simultaneously for therapeutic applications. In some embodiments, the guide RNA comprises a pre-crRNA expressed by a CRISPR array consisting of a target sequence interlaced with unprocessed DR sequences, repeated by intrinsic pre-crRNA processing of the effector protein to enable simultaneous targeting of one, two, or more sites.

[0175] In some embodiments, the Type VI CRISPR-Cas12i effector protein is capable of recognizing a protospacer adjacent motif (PAM), and the target nucleic acid comprises or consists of the PAM, and the PAM comprises or consists of the nucleic acid sequence 5'-TTN-3', 5'-TTH-3', 5'-TTY-3', or 5'-TTC-3'.

[0176] Cas12i nucleases from a variety of organisms can be used as the reference Cas12i nucleases to provide engineered Cas12i nucleases and effector proteins of the present application. Exemplary Cas12i nucleases have been described in, for example, WO2019 / 201331A1 and US2020 / 0063126A1, which are incorporated herein by reference as a whole. In some embodiments, the reference Cas12i nuclease has enzymatic activity. In some embodiments, the reference Cas12i is a nuclease, i.e., two chains of a target double-helix nucleic acid (e.g., double-helix DNA) are cut. In some embodiments, the reference Cas12i is a nickase, i.e., a single chain of a target double-helix nucleic acid (e.g., double-helix DNA) is cut. In some embodiments, the reference Cas12i nuclease is enzyme-inactivated. In some embodiments, the reference Cas12i enzyme is Cas12i1, Cas12i2, or Cas12i-Phi. Orthologs having a certain sequence identity (e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 98% or more) with Cas12i or its functional derivatives can be used as the basis for designing the engineered Cas12i nuclease or effector protein of the present application.

[0177] In some embodiments, the engineered Cas12i nuclease is based on a functional variant of a naturally occurring Cas12i nuclease. In some embodiments, the functional variant has one or more mutations, such as amino acid substitutions, insertions and deletions. For example, compared to a wild-type naturally occurring Cas12i nuclease, the functional variant may include any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions. In some embodiments, the one or more substitutions are conservative substitutions. In some embodiments, the functional variant has all domains of a naturally occurring Cas12i nuclease. In some embodiments, the functional variant does not have one or more domains of a naturally occurring Cas12i nuclease.

[0178] Also provided is an engineered Cas12i effector protein based on any one of the engineered Cas12i2 nucleases described herein. In some embodiments, the engineered Cas12i effector protein has enzymatic activity. In some embodiments, the engineered Cas12i effector protein is a nuclease that cuts two chains of a target double-helix nucleic acid (e.g., double-helix DNA). In some embodiments, the engineered Cas12i effector protein is a nickase, i.e., a single strand that cuts a target double-helix nucleic acid (e.g., double-helix DNA). In some embodiments, the engineered Cas12i effector protein comprises an enzyme-inactive mutant of the engineered Cas12i nuclease. Mutations of one or more amino acid residues in the active site of the Cas12i nuclease can result in Cas12i that loses enzymatic activity. In some embodiments, the engineered Cas12i enzyme provided herein can be modified to have reduced nuclease activity, for example, the nuclease is inactivated by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% compared to the wild-type Cas12i enzyme. The nuclease activity can be reduced by several methods, for example, by introducing mutations into the PAM interaction domain of the nuclease or Cas12i enzyme. In some embodiments, the catalytic residues of the nuclease activity are identified, and these amino acid residues can be replaced by different amino acid residues (e.g., glycine or alanine) to reduce the nuclease activity. Examples of such mutations of Cas12i1 include D647A, E894A or D948A. Examples of such mutations of Cas12i2 include D599A, E833A, S883A, H884A, D886A, R900A and / or D1019A.

[0179] 7) Activity of engineered Cas12i nuclease

[0180] The engineered Cas12i nuclease has increased activity compared to the reference Cas12i nuclease. In some embodiments, the activity is target DNA binding activity. In some embodiments, the activity is site-specific nuclease activity. In some embodiments, the activity is double-stranded DNA cleavage activity. In some embodiments, the activity is single-stranded DNA cleavage activity, including, for example, site-specific DNA cleavage activity or non-specific DNA cleavage activity. In some embodiments, the activity is single-stranded RNA cleavage activity, such as site-specific RNA cleavage activity or non-specific RNA cleavage activity. In some embodiments, the activity is measured in vitro. In some embodiments, the activity is measured in cells such as bacterial cells, plant cells, or eukaryotic cells. In some embodiments, the activity is measured in mammalian cells such as rodent cells or human cells. In some embodiments, the activity is measured in human cells such as 293T cells. In some embodiments, the activity is measured in mouse cells, such as Hepa1-6 cells. In some embodiments, the engineered Cas12i nuclease has a site-specific nuclease activity that is increased by at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times or more compared to a reference Cas12i nuclease. The site-specific nuclease activity of the engineered Cas12i nuclease can be measured using methods known in the art, including, for example, gel shift assays, in vitro cleavage assays based on agarose gel electrophoresis as described in the Examples provided herein.

[0181] In some embodiments, the activity is gene editing activity in cells. In some embodiments, the cell is a bacterial cell, a plant cell or a eukaryotic cell. In some embodiments, the cell is a mammalian cell such as a rodent cell or a human cell. In some embodiments, the cell is a 293T cell. In some embodiments, the activity is measured in mouse cells, such as Hepa1-6 cells. In some embodiments, the activity is an insertion and deletion activity at a target genomic site in a cell, such as site-specific cutting of a target nucleic acid by the engineered Cas12i nuclease and DNA repair by a non-homologous end joining (NHEJ) mechanism. In some embodiments, the activity is the insertion of an exogenous nucleic acid sequence at a target genomic site in a cell, such as site-specific cutting of a target nucleic acid by the engineered Cas12i nuclease and DNA repair by a homologous recombination (HR) mechanism. In some embodiments, the engineered Cas12i nuclease increases gene editing (e.g., insertion and deletion formation) activity by at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times or more at a genomic site in a cell (e.g., a human cell such as a 293T cell or a mouse Hepa1-6 cell) compared to a reference Cas12i nuclease. In some embodiments, the engineered Cas12i nuclease increases at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times or more of any one of the gene editing (e.g., insertion and deletion formation) activity compared to the reference Cas12i2 nuclease in a plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) genomic sites of cells (e.g., human cells such as 293T cells or mouse Hepa1-6 cells). In some embodiments, the engineered Cas12i nuclease can edit a greater number of genomic sites compared to the reference Cas12i nuclease. In some embodiments, the common PAM sequence of the engineered Cas12i nuclease is the same as that of the reference Cas12i nuclease.

[0182] The gene editing efficiency of the engineered Cas12i nuclease in the cell can be determined using methods known in the art, including, for example, T7 endonuclease 1 (T7E1) assay, sequencing of target DNA (including, for example, Sanger sequence, and second-generation sequencing), assayed by decomposition tracking insertion and deletion (TIDE) or assayed by insertion and deletion detection (IDAA) by amplicon analysis. See, for example, SentmanatMF et al., "A survey of validation strategies for CRISPR-Cas9editing," ScientificReports, 2018, 8, article number 888, which is incorporated herein by reference as a whole. In some embodiments, for example, as described in the Examples herein, targeted second-generation sequencing (NGS) is used to measure the gene editing efficiency of the engineered Cas12i nuclease in the cell. Exemplary genomic sites for determining the gene editing efficiency of the engineered Cas12i nuclease include, but are not limited to, CCR5, AAVS, CD34, RNF2, and EMX1. In some embodiments, the gene editing efficiency of the engineered Cas12i nuclease is the average gene editing efficiency of the engineered Cas12i nuclease at at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or more sites (into human cell genome sites). In some embodiments, the gene editing efficiency (eg, insertion and deletion rate) of the engineered Cas12i nuclease reaches at least 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or higher.

[0183] Engineered Cas12i effector proteins

[0184] The present application provides engineered Cas12i (such as Cas12i2) effector proteins having improved activities, such as target binding, double-stranded cleavage activity, nickase activity and / or gene editing activity. In some embodiments, an engineered Cas12i effector protein (e.g., Cas12i nuclease, Cas12i nickase, Cas12i fusion effector protein or split Cas12i effector protein) is provided, comprising any one of the engineered Cas12i nucleases described herein or their functional derivatives.

[0185] Variants

[0186] The present application provides engineered Cas12i effector proteins, which include functional variants of engineered Cas12i nucleases described herein. In some embodiments, when compared with the amino acid sequence of the corresponding engineered Cas12i nuclease, the amino acid sequence of the functional variant has a difference of at least one amino acid residue (e.g., with deletion, insertion, substitution and / or fusion). In some embodiments, the functional variant has one or more mutations, such as amino acid substitutions, insertions and / or deletions. For example, compared with engineered Cas12i nucleases, functional variants may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in any one replacement. In some embodiments, the one or more replacements are conservative replacements. In some embodiments, the functional variant has all domains of engineered Cas12i nucleases. In some embodiments, the functional variant does not have one or more domains of engineered Cas12i nucleases.

[0187] For any of the Cas12i variant proteins described herein (e.g., nickase Cas12i protein, inactivated or catalytically inactivated Cas12i (dCas12i), fusion Cas12i), the Cas12i variant may include a Cas12i protein sequence having the same parameters as described above (e.g., domains present, percent identity, etc.).

[0188] Catalytic activity

[0189] In some embodiments, the functional variant has different catalytic activity compared to the non-mutated form of the Cas12i nuclease engineered by it. In some embodiments, the mutation (e.g., amino acid substitution, insertion and / or deletion) is in the catalytic domain (e.g., RuvC domain) of the Cas12i effector protein. In some embodiments, the variant comprises mutations in multiple catalytic domains. The Cas12i effector protein that cuts one chain of a double-stranded target nucleic acid without cutting the other chain is referred to as a "nickase" (e.g., "Cas12i nickase") herein. In this article, the Cas12i protein that is substantially free of nuclease activity is referred to as an inactivated Cas12i protein ("dCas12i") (statement: in the case of fusion Cas12i effector protein, a heterologous polypeptide (fusion partner) can provide nuclease activity, which will be described in detail below). In some embodiments, relative to its non-mutated form, when the DNA cleavage activity of the mutant enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less, it is considered that the Cas12i effector protein substantially lacks all DNA cleavage activity.

[0190] Split Cas12i effector protein

[0191] The present application also provides a split Cas12i effector protein based on any of the engineered Cas12i effector proteins described herein. Split Cas12i effector proteins may be advantageous for delivery. In some embodiments, the engineered Cas12i effector protein is split into two parts of the enzyme, which can be reconstituted together to provide a substantially functional Cas12i effector protein. Cas effector proteins can be provided using known methods, for example, split forms of Cas12 and Cas9 proteins have been described in, for example, WO2016 / 112242, WO2016 / 205749, and PCT / CN 2020 / 111057, which are incorporated herein by reference in their entirety.

[0192] In some embodiments, a split Cas12i effector protein is provided, comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the N-terminal portion of any one of the engineered Cas12i nucleases described herein or a functional derivative thereof, the second polypeptide comprising the C-terminal portion of the engineered Cas12i nuclease or a functional derivative thereof, wherein the first polypeptide and the second polypeptide are capable of associating with each other in the presence of a guide RNA comprising a guide sequence to form a CRISPR complex that specifically binds to a target nucleic acid, the target nucleic acid comprising a target sequence complementary to the guide sequence. In some embodiments, the first polypeptide and the second polypeptide each comprise a dimerization domain. In some embodiments, the first dimerization domain and the second dimerization domain associate with each other in the presence of an inducer (e.g., rapamycin). In some embodiments, the first polypeptide and the second polypeptide do not comprise a dimerization domain. In some embodiments, the split Cas12i effector protein is self-inducing.

[0193] The cleavage can be performed in a manner that does not affect the catalytic domain. The Cas12i effector protein can function as a nuclease (including a nickase) or can be an inactivated enzyme, which is essentially an RNA-guided DNA-binding protein with little or no catalytic activity (e.g., due to a mutation in its catalytic domain).

[0194] In some embodiments, the nuclease leaf and α-helical leaf of the Cas12i protein are expressed as separate polypeptides. Although the leaves do not interact with each other by themselves, the RNA guide sequence recruits them into a complex that summarizes the activity of the full-length Cas12i enzyme and catalyzes site-specific DNA cleavage. In some embodiments, the modified RNA guide sequence can be used to eliminate the activity of the segmentation enzyme by preventing dimerization, thereby allowing the development of an inducible dimerization system. The segmentation enzyme is described in, for example, Wright, Addison V., et al. "Rational design of a split-Cas9 enzyme complex," Proc. Nat'l. Acad. Sci., 112.10 (2015): 2984-2989, which is incorporated herein by reference in its entirety.

[0195] The split Cas12i effector protein portion described herein can be designed to be divided (i.e., split) into two halves by dividing a reference engineered Cas12i effector protein (e.g., full-length engineered Cas12i) at a split position, which is the point where the N-terminal portion of the reference Cas12i effector protein is separated from the C-terminal portion. In some embodiments, the N-terminal portion comprises amino acid residues 1 to X, and the C-terminal portion comprises amino acid residues X+1 to the C-terminus of the reference Cas12i effector protein. In this example, the numbering is continuous, but this is not necessary, because it is also considered that amino acids (or nucleotides encoding them) can be trimmed from any of the split ends and / or mutations (e.g., insertions, deletions, and substitutions) in the internal region of the polypeptide chain, provided that the reconstructed Cas12i effector protein retains sufficient DNA binding activity (if necessary), DNA nickase or cleavage activity, for example, at least 40%, 50%, 60%, 70%, 80%, 90% or 95% activity compared to the reference Cas12i effector protein.

[0196] The split point can be designed by computer (in silico) and cloned into the construct. In the process, mutations can be introduced into the split Cas12i effector protein, and non-functional domains can be removed. In some embodiments, the two parts or fragments of the split Cas12i effector protein (i.e., N-terminal and C-terminal fragments) can form a complete Cas12i effector protein, which contains, for example, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the complete Cas12i effector protein sequence.

[0197] The split Cas12i effector proteins may each comprise one or more dimerization domains. In some embodiments, the first polypeptide comprises a first dimerization domain fused to a first split Cas12i effector protein portion, and the second polypeptide comprises a second dimerization domain fused to a second split Cas12i effector protein portion. The dimerization domain may be fused to the split Cas12i effector protein portion by a peptide linker (e.g., a flexible peptide linker such as a GS linker) or a chemical bond. In some embodiments, the dimerization domain is fused to the N-terminus of the split Cas12i effector protein portion. In some embodiments, the dimerization domain is fused to the C-terminus of the split Cas12i effector protein portion.

[0198] In some embodiments the split Cas12i effector protein does not comprise any dimerization domain.

[0199] In some embodiments, the dimerization domain promotes the association of two split Cas12i effector protein parts. In some embodiments, the split Cas12i effector protein parts are induced by an inducer to associate or dimerize into a functional Cas12i effector protein. In some embodiments, the split Cas12i effector protein comprises an inducible dimerization domain. In some embodiments, the dimerization domain is not an inducible dimerization domain, that is, the dimerization domain dimerizes in the absence of an inducer.

[0200] The inducer can be an induction energy source or an induction molecule other than a guide RNA (eg, sgRNA). The inducer reconstructs two segmented Cas12i effector protein portions into functional Cas12i effector proteins by dimerization of the induced dimerization domain. In some embodiments, the inducer brings two segmented Cas12i effector protein portions together by the induced association of the inducible dimerization domain. In some embodiments, in the absence of an inducer, the two segmented Cas12i effector protein portions do not associate with each other to reconstruct a functional Cas12i effector protein. In some embodiments, in the absence of an inducer, two separate Cas12i effector protein portions can associate with each other in the presence of a guide RNA (eg, crRNA) to reconstruct a functional Cas12i effector protein.

[0201] The inducing agent of the present application can be heat, ultrasound, electromagnetic energy or a compound. In some embodiments, the inducing agent is an antibiotic, a small molecule, a hormone, a hormone derivative, a steroid or a steroid derivative. In some embodiments, the inducing agent is abscisic acid (ABA), doxycycline (DOX), isopropyl benzoic acid (cumate), rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. In some embodiments, the segmented Cas12i effector system is a system controlled by an inducing agent selected from the group consisting of an antibiotic-based induction system, an electromagnetic energy-based induction system, a small molecule-based induction system, a nuclear receptor-based induction system, and a hormone-based induction system. In some embodiments, the split Cas12i effector system is an inducer-controlled system selected from the group consisting of a tetracycline (Tet) / DOX induction system, a light induction system, an ABA induction system, a cumate repressor / operator system, a 4OHT / estrogen induction system, an ecdysone-based induction system, and a FKBP12 / FRAP (FKBP12-rapamycin complex) induction system. Such inducers are also discussed herein and PCT / US2013 / 051418, which are incorporated herein by reference in their entirety. The FRB / FKBP / rapamycin system has been described in Paulmurugan and Gambhir, Cancer Res, August 15, 200565; 7413, and Crabtree et al., Chemistry & Biology 13, 99-107, Jan 2006, which are incorporated herein by reference in their entirety.

[0202] In some embodiments, the paired split Cas12i effector proteins are separate and inactive until dimerization of the dimerization domains (e.g., FRB and FKBP) is induced, which results in the reassembly of functional Cas12i effector protein nucleases. In some embodiments, the first split Cas12i effector protein comprising the first half of the inducible dimer (e.g., FRB) is delivered separately and / or is in a separate position from the second split Cas12i effector protein comprising the second half of the inducible dimer (e.g., FKBP).

[0203] Other exemplary FKBP-based inducible systems that can be used in the inducer-controlled split-Cas12i effector system described herein include, but are not limited to: FKBP dimerized with calcineurin (CNA) in the presence of FK506; FKBP dimerized with CyP-Fas in the presence of FKCsA; FKBP dimerized with FRB in the presence of rapamycin; GyrB dimerized with GryB in the presence of coumermycin; GAI dimerized with GID1 in the presence of gibberellins; or Snap-tag dimerized with HaloTag in the presence of HaXS.

[0204] Alternatives within the FKBP family itself are also contemplated. For example, in the presence of FK1012, the FKBPs homodimerize (ie, one FKBP dimerizes with another FKBP).

[0205] In some embodiments, the dimerization domain is FKBP and the inducer is FK1012. In some embodiments, the dimerization domain is GryB and the inducer is coumermycin. In some embodiments, the dimerization domain is ABA and the inducer is gibberellin.

[0206] In some embodiments, the split Cas12i effector protein portion can be automatically induced (i.e., automatically activated or self-induced) in the absence of an inducer to associate / dimerize into a functional Cas12i effector protein. Without being bound by any theory or hypothesis, the automatic induction of the split Cas12i effector protein portion can be mediated by binding to a guide RNA such as crRNA. In some embodiments, the first polypeptide and the second polypeptide do not comprise a dimerization domain. In some embodiments, the first polypeptide and the second polypeptide comprise a dimerization domain.

[0207] In some embodiments, the reconstructed Cas12i effector protein of the split Cas12i effector system described herein (including inducer-controlled and auto-induced systems) has an editing efficiency of at least 70% (such as at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more efficiency or 100% efficiency) relative to the editing efficiency of the reference Cas12i effector protein.

[0208] In some embodiments, the reconstructed Cas12i effector protein of the inducer-controlled split Cas12i effector system described herein has an editing efficiency of no more than 50% (such as no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less efficiency, or 0% efficiency) relative to the editing efficiency of the reference Cas12i effector protein in the absence of an inducer (i.e., due to auto-induction).

[0209] Fusion Cas12i effector protein

[0210] The present application also provides an engineered Cas12i effector protein comprising additional protein domains and / or components, such as a linker, a nuclear localization / export sequence, a functional domain and / or a reporter protein.

[0211] In some embodiments, the engineered Cas12i effector protein is a protein complex comprising one or more heterologous protein domains (e.g., about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains) and the nucleic acid targeting domain of the engineered Cas12i nuclease or a functional derivative thereof. In some embodiments, the engineered Cas12i effector protein is a fusion protein comprising one or more heterologous protein domains (e.g., about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains) fused to the engineered Cas12i nuclease.

[0212] In some embodiments, the engineered Cas12i effector protein of the present application may include (for example, through a fusion protein, such as through one or more peptide linkers, such as a GS peptide linker, etc.) one or more functional domains or associate (for example, through the co-expression of multiple proteins) with it. In some embodiments, the one or more functional domains are enzyme domains. These functional domains can have a variety of activities, such as DNA and / or RNA methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity and switch activity (for example, light-induced). In some embodiments, the one or more functional domains are transcriptional activation domains (ie, transactivation domains) or repressor domains. In some embodiments, the one or more functional domains are histone modification domains. In some embodiments, the one or more functional domains are transposase domains, HR (homologous recombination) mechanism domains, recombinase domains and / or integrase domains. In some embodiments, the functional domain is Krüppel-associated box (KRAB), VP64, VP16, Fok1, P65, HSF1, MyoD1, biotin-APEX, APOBEC1, AID, PmCDA1, Tad1, and M-MLV reverse transcriptase. In some embodiments, the functional domain is selected from the group consisting of a translation initiation domain, a transcription repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain (e.g., a CBE or ABE domain), a reverse transcriptase domain, a reporter domain (e.g., a fluorescent domain), and a nuclease domain.

[0213] In some embodiments, the positioning of one or more functional domains in the engineered Cas12i effector protein allows the correct spatial orientation of the functional domain to affect the target with the assigned functional effect. For example, if the functional domain is a transcriptional activator (e.g., VP16, VP64, or p65), the transcriptional activator is placed in a spatial orientation that enables it to affect the transcription of the target. Similarly, the transcriptional repressor is positioned to affect the transcription of the target, and the nuclease (e.g., Fok1) is positioned to cut or partially cut the target. In some embodiments, the functional domain is located at the N-terminus of the engineered Cas12i effector protein. In some embodiments, the functional domain is located at the C-terminus of the engineered Cas12i effector protein. In some embodiments, the engineered Cas12i effector protein comprises a first functional domain at the N-terminus and a second functional domain at the C-terminus. In some embodiments, the engineered Cas12i effector protein comprises a catalytically inactive mutant of any one of the engineered Cas12i nucleases described herein fused with one or more functional domains.

[0214] In some embodiments, the engineered Cas12i effector protein is a transcriptional activator. In some embodiments, the engineered Cas12i effector protein comprises an enzymatically inactive variant of any one of the engineered Cas12i nucleases described herein fused to a transactivation domain. In some embodiments, the transactivation domain is selected from the group consisting of VP64, p65, HSF1, VP16, MyoD1, HSF1, RTA, SET7 / 9, and combinations thereof. In some embodiments, the transactivation domain comprises VP64, p65, and HSF1. In some embodiments, the engineered Cas12i effector protein comprises two split Cas12i effector polypeptides, each fused to a transactivation domain.

[0215] In some embodiments, the engineered Cas12i effector protein is a transcriptional repressor. In some embodiments, the engineered Cas12i effector protein comprises an enzyme-inactive variant of any of the engineered Cas12i nucleases described herein fused to a transcriptional repression domain. In some embodiments, the transcriptional repressor domain is selected from the group consisting of Krüppel-associated box (KRAB), EnR, NuE, NcoR, SID, SID4X, and combinations thereof. In some embodiments, the engineered Cas12i effector protein comprises two split Cas12i effector polypeptides, each fused to a transcriptional repression domain.

[0216] In some embodiments, the engineered Cas12i effector protein is a base editor, such as a cytosine editor or an adenosine editor. In some embodiments, the engineered Cas12i effector protein comprises an enzyme-inactive variant of any of the engineered Cas12i nucleases described herein fused to a nuclear base editing domain, such as a cytosine base editor (CBE) domain or an adenosine base editor (ABE) domain. In some embodiments, the nuclear base editing domain is a DNA editing domain. In some embodiments, the nuclear base editing domain has deaminase activity. In some embodiments, the nuclear base editing domain is a cytosine deaminase domain. In some embodiments, the nuclear base editing domain is an adenosine deaminase domain. Exemplary base editors based on Cas nucleases are described in, for example, WO2018 / 165629A1 and WO2019 / 226953A1, which are incorporated herein by reference in their entirety. Exemplary CBE domains include, but are not limited to, activation-induced cytidine deaminase or AID (e.g., hAID), apolipoprotein B mRNA editing complex or APOBEC (e.g., rat APOBEC1, hAPOBEC3 A / B / C / D / E / F / G), and PmCDA1. Exemplary ABE domains include, but are not limited to, TadA, ABE8, and variants thereof (see, e.g., Gaudelli et al., 2017, Nature 551:464-471; and Richter et al., 2020, Nature Biotechnology 38:883-891). In some embodiments, the functional domain is an APOBEC1 domain, e.g., a rat APOBEC1 domain. In some embodiments, the functional domain is a TadA domain, e.g., an Escherichia coli (E. coli) TadA domain. In some embodiments, the engineered Cas12i effector protein further comprises one or more nuclear localization sequences.

[0217] In some embodiments, the engineered Cas12i effector protein is a primary editor. The primary editor based on Cas9 is described in, for example, A.Anzalone et al., Nature, 2019, 576 (7785): 149-157, which is incorporated herein by reference as a whole. In some embodiments, the engineered Cas12i effector protein comprises a nickase variant of any one of the engineered Cas12i nucleases described herein fused to a reverse transcriptase domain. In some embodiments, the functional domain is a reverse transcriptase domain. In some embodiments, the reverse transcriptase domain is an M-MLV reverse transcriptase or a variant thereof, such as an M-MLV reverse transcriptase with one or more mutations of D200N, T306K, W313F, T330P, and L603W. In some embodiments, an engineered CRISPR / Cas12i system comprising the primary editor is provided. In some embodiments, the engineered CRISPR / Cas12i system further comprises a second Cas12i nickase, for example, based on the same engineered Cas12i nuclease as the primary editor. In some embodiments, the engineered CRISPR / Cas12i system comprises a primary editor guide RNA (pegRNA) comprising a primer binding site and a reverse transcriptase (RT) template sequence.

[0218] In some embodiments, the present application provides a split Cas12i effector system having one or more (e.g., 1, 2, 3, 4, 5, 6 or more) functional domains associated (i.e., bound or fused) with one or both of the split Cas12i effector protein portions. The functional domain can be provided as part of the first and / or second split Cas12i effector protein as a fusion within the construct. The functional domain is typically fused to other parts of the split Cas12i effector protein (e.g., a split Cas12i effector protein portion) via a peptide linker (such as a GS linker). These functional domains can be used to re-replace the function of the split Cas12i effector system based on the catalytically inactive Cas12i effector protein.

[0219] In some embodiments, the engineered Cas12i effector protein comprises one or more nuclear localization sequences (NLS) and / or one or more nuclear export sequences (NES). Exemplary NLS sequences include, for example, PKKKRKVPG and ASPKKKRKV. NLS and / or NES can be operably linked to the N-terminus and / or C-terminus of the engineered Cas12i effector protein or a polypeptide chain in the engineered Cas12i effector protein.

[0220] In some embodiments, the engineered Cas12i effector protein may encode additional components, such as a reporter protein. In some embodiments, the engineered Cas12i effector protein comprises a fluorescent protein, such as GFP. Such a system can allow for imaging of genomic sites (e.g., see “Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR / Cas System” Chen B et al. Cell 2013). In some embodiments, the engineered Cas12i effector protein is an inducible segmentation Cas12i effector system that can be used for imaging genomic sites.

[0221] In another specific embodiment, an engineered Cas12i effector protein is provided, wherein the effector protein is capable of inducing double-strand breaks or single-strand breaks in DNA molecules.

[0222] In another specific embodiment, an engineered Cas12i effector protein is provided, wherein the functional derivative of the engineered Cas12i nuclease is an enzyme-inactivated mutant, such as a Cas12i2 nuclease-inactivated mutant containing D599A, E833A, S883A, H884A, D886A, R900A and / or D1019A, and a Cas12i1 nuclease-inactivated mutant containing D647A, E894A and / or D948A. Known enzyme-inactivated mutants of Cas12i2 nucleases, such as any enzyme-inactivated mutants of Cas12i2 nucleases described in US10808245B2 and Huang X.et al., Nature Communications, 11, Article number: 5241 (2020), can be combined with the mutants in the present application to provide functional derivatives of engineered Cas12i nucleases and their corresponding effector proteins.

[0223] In another specific embodiment, an engineered Cas12i effector protein is provided, which further comprises a functional domain fused to the engineered Cas12i nuclease.

[0224] In yet another specific embodiment, an engineered Cas12i effector protein is provided, wherein the functional domain is selected from the group consisting of a translation initiation domain, a transcription repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain, a reverse transcriptase domain, a reporter domain, and a nuclease domain.

[0225] Engineered CRISPR-Cas12i system

[0226] In some embodiments, an engineered CRISPR-Cas12i system is provided, comprising: (a) any one of the engineered Cas12i effector proteins described herein (such as engineered Cas12i nucleases); and (b) a guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding the guide RNA,

[0227] The engineered Cas12i effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to a target nucleic acid comprising the target sequence and induces modification of the target nucleic acid.

[0228] In some embodiments, the engineered CRISPR-Cas12i system includes: (a) any one of the engineered Cas12i effector proteins described herein (e.g., engineered Cas12i nucleases, nickases, split-type Cas12i, transcriptional repressors, transcriptional activators, base editors, or primary editors); and (b) a guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding the guide RNA, wherein the engineered Cas12i effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to a target nucleic acid comprising the target sequence and induces modification of the target nucleic acid. In some embodiments, the engineered CRISPR-Cas12i system comprises one or more nucleic acids encoding the engineered Cas12i effector protein and / or the guide RNA. In some embodiments, the engineered CRISPR-Cas12i system comprises a precursor guide RNA array, which can be processed into multiple crRNAs, for example, by the engineered Cas12i effector protein. In some embodiments, the engineered CRISPR-Cas12i system comprises one or more vectors encoding the engineered Cas12i effector protein and / or the guide RNA. In some embodiments, the engineered CRISPR-Cas12i system comprises a ribonucleoprotein (RNP) complex comprising the engineered Cas12i effector protein bound to the guide RNA.

[0229] The engineered CRISPR-Cas12i system of the present application may include any suitable guide RNA.Guide RNA (gRNA) may include a guide sequence that can hybridize with a target sequence in a target nucleic acid (such as a target genomic site in a cell). In some embodiments, the gRNA includes a CRISPR RNA (crRNA) sequence containing the guide sequence.

[0230] Typically, crRNA as described herein includes direct repeats and spacer sequences. In certain embodiments, the crRNA includes a direct repeat connected to a guide sequence or a spacer sequence, is essentially composed of or is composed of it. In some embodiments, the crRNA includes direct repeats, spacer sequences and direct repeats (DR-spacer sequences-DR), which are typical features of the precursor crRNA (pre-crRNA) configuration. In some embodiments, the crRNA includes truncated direct repeats and spacer sequences, which are typical features of processed or mature crRNA. In some embodiments, the CRISPR-Cas12i effector protein forms a complex with the RNA guide sequence, and the spacer sequence guides the complex to sequence-specific binding with the target nucleic acid, and the target nucleic acid is complementary to the spacer sequence.

[0231] In some embodiments, the guide RNA is a crRNA comprising a guide sequence. In some embodiments, the engineered CRISPR-Cas12i system comprises a precursor guide RNA array encoding multiple crRNAs. In some embodiments, the Cas12i effector protein cuts the precursor guide RNA array to produce multiple crRNAs. In some embodiments, the engineered CRISPR-Cas12i system comprises a precursor guide RNA array encoding multiple crRNAs, wherein each crRNA comprises a different guide sequence.

[0232] The guide sequence can have a suitable length. In some embodiments, the guide sequence is between about 18 to about 35 nucleotides, including, for example, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides. The guide sequence can have at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% complementarity with the target sequence of the target nucleic acid.

[0233] Constructs and vectors

[0234] Also provided herein are constructs, vectors, and expression systems encoding any of the engineered Cas12i effector proteins described herein (eg, engineered Cas12i nucleases). In some embodiments, the construct, vector, or expression system further comprises one or more gRNA or crRNA arrays.

[0235] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphipathic compounds, plasmids and viruses. Typically, a suitable vector comprises an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selective markers. The term "vector" should also be interpreted to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc.

[0236] In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors and derivatives thereof. In some embodiments, the vector is a phage vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals.

[0237] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used.

[0238] In certain embodiments, the vector is an adeno-associated virus (AAV) vector, such as AAV2, AAV8, or AAV9, which can be expressed as a vector comprising at least 1×10 5 A single administration of particles (also referred to as particle units, pu) of adenovirus or adeno-associated virus is administered. In some embodiments, the administration amount is at least about 1×10 6 particles, at least about 1×10 7 particles, at least about 1×10 8 particles, or at least about 1×10 9 The delivery method and dosage are described in, for example, WO2016205764 and U.S. Patent No. 8,454,972, which are incorporated herein by reference in their entirety.

[0239] In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. For example, in some embodiments, a modified AAV vector can be used for delivery. The modified AAV vector can be based on one or more of several capsid types, including AAV1, AV2, AAV5, AAV6, AAV8, AAV8.2, AAV9, AAVrh10, modified AAV vectors (e.g., modified AAV2, modified AAV3, modified AAV6) and pseudotyped AAV (e.g., AAV2 / 8, AAV2 / 5 and AAV2 / 6). Exemplary AAV vectors and techniques that can be used to produce rAAV particles are known in the art (see, e.g., Aponte-Ubillus et al. (2018) Appl. Microbiol. Biotechnol. 102(3): 1045-54; Zhong et al. (2012) J. Genet. Syndr. Gene Ther. S1: 008; West et al. (1987) Virology 160: 38-47 (1987); Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-60; U.S. Pat. Nos. 4,797,368 and 5,173,414; International Publication Nos. WO2015 / 054653 and WO93 / 24641, each of which is incorporated herein by reference).

[0240] Any known AAV vector for delivering Cas9 and other Cas proteins can be used to deliver the engineered Cas12i system of the present application.

[0241] Methods for introducing vectors into mammalian cells are known in the art. Vectors can be transferred into host cells by physical, chemical or biological methods.

[0242] Physical methods for introducing vectors into host cells include: calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector is introduced into the cell by electroporation.

[0243] Biological methods for introducing heterologous nucleic acids into host cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, such as human cells.

[0244] Chemical methods for introducing vectors into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery carrier is a liposome (e.g., an artificial membrane vesicle). In some embodiments, the engineered CRISPR-Cas12i system is delivered in nanoparticles in the form of RNPs.

[0245] In some embodiments, the vector or expression system encoding the CRISPR-Cas12i system or its components comprises one or more selectable or detectable markers that provide a means for isolating or effectively selecting cells that contain and / or have been modified by the CRISPR-Cas12i system (e.g., at early stages and on a large scale).

[0246] Reporter gene can be used for identifying the cell of potential transfection and evaluating the function of regulating sequence.Usually, reporter gene is a gene that does not exist or is not expressed in a receptor organism or tissue, and the expression of its encoded polypeptide is proved by some properties (such as enzymatic activity) that are easy to detect.The expression of reporter gene is measured at the appropriate time after DNA is introduced into the receptor cell.Suitable reporter gene can include the gene of coding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein gene (such as Ui-Tei et al.FEBS Letters 479:79-82 (2000)).

[0247] Other methods for confirming the presence of heterologous nucleic acid in host cells include, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, for example, detecting the presence or absence of specific peptides by immunological methods (such as ELISA and Western blotting).

[0248] In some embodiments, the nucleic acid sequence encoding the engineered Cas12i effector protein and / or the guide RNA is operably connected to a promoter. In some embodiments, the promoter is an endogenous promoter relative to the cell engineered using the engineered CRISPR-Cas12i system. For example, the nucleic acid encoding the engineered Cas12i effector protein can be knocked into the genome of the engineered mammalian cell using any method known in the art and located downstream of the endogenous promoter. In some embodiments, the endogenous promoter is a promoter of an abundant protein (such as β-actin). In some embodiments, the endogenous promoter is an inducible promoter, for example, it can be induced by an endogenous activation signal of an engineered mammalian cell. In some embodiments, wherein the engineered mammalian cell is a T cell, the promoter is a T cell activation-dependent promoter (such as an IL-2 promoter, a NFAT promoter, or a NFκB promoter).

[0249] In some embodiments, the promoter is a heterologous promoter relative to the cell engineered using the engineered CRISPR-Cas12i system. A variety of promoters have been explored to express genes in mammalian cells, and any promoter known in the art can be used in the present application. Promoters can be roughly divided into constitutive promoters or regulated promoters, such as inducible promoters.

[0250] In some embodiments, the nucleic acid sequence encoding the engineered Cas12i effector protein and / or the guide RNA is operably connected to a constitutive promoter. A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, cytomegalovirus (CMV) promoter, human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken β-actin promoter coupled to CMV early enhancer (CAG). In some embodiments, the promoter is a CAG promoter, which comprises a cytomegalovirus (CMV) early enhancer element, a promoter, the first exon and the first intron of the chicken β-actin gene, and a splicing acceptor of a rabbit β-globin gene.

[0251] In some embodiments, the nucleic acid sequence encoding the engineered CRISPR-Cas12i effector protein and / or the guide RNA is operably connected to an inducible promoter. Inducible promoters belong to regulated promoter types. The inducible promoter can be induced by one or more conditions such as physical conditions, microenvironment or physiological state of host cells, inducers (ie, inducers) or combinations thereof. In some embodiments, the induction conditions are selected from the group consisting of inducers, irradiation (such as ionizing radiation, light), temperature (such as heat), redox state, tumor environment and activation state of cells to be engineered by the engineered CRISPR-Cas12i system. In some embodiments, the promoter can be induced by small molecule inducers such as compounds. In some embodiments, the small molecule is selected from the group consisting of doxycycline, tetracycline, alcohol, metal or steroid. Chemically induced promoters have been most widely studied. Such promoters include promoters whose transcriptional activity is regulated by the presence or absence of small molecule chemicals such as doxycycline, tetracycline, alcohol, steroid, metal and other compounds. The doxycycline induction system with reverse tetracycline controlled transactivator (rtTA) and tetracycline response element promoter (TRE) is the most mature system at present. WO9429442 describes strict control of gene expression in eukaryotic cells by tetracycline responsive promoter. WO9601313 discloses tetracycline-regulated transcriptional regulators. In addition, Tet technologies such as Tet-on systems have been described on websites such as TetSystems.com. In the present application, any known chemically regulated promoter can be used to drive the expression of the engineered CRISPR-Cas12i protein and / or the guide RNA.

[0252] In some embodiments, the nucleic acid sequence encoding the engineered Cas12i effector protein is codon optimized.

[0253] In some embodiments, an expression construct is provided, comprising a codon-optimized sequence encoding the engineered Cas12i effector protein ligated to a BPK2104-ccdB vector. In some embodiments, the expression construct encodes a tag (e.g., a 10xHis tag) operably linked to the C-terminus of the engineered Cas12i effector protein.

[0254] In some embodiments, each engineered segmented Cas12i construct encodes a fluorescent protein such as GFP or RFP. The reporter protein can be used to assess the co-localization and / or dimerization of the engineered Cas12i protein, for example using a microscope. The nucleic acid sequence encoding the engineered Cas12i effector protein can be fused to the nucleic acid sequence encoding the additional component using a sequence encoding a self-cleaving peptide such as a T2A, P2A, E2A or F2A peptide.

[0255] In some embodiments, an expression construct for a mammalian cell (eg, a human cell) is provided, the construct comprising a nucleic acid sequence encoding the engineered Cas12i effector protein. In some embodiments, the expression construct comprises a codon-optimized sequence encoding the engineered Cas12i effector protein inserted into a pCAG-2A-eGFP vector, thereby operably linking the Cas12i protein to eGFP. In some embodiments, a second vector is provided for expressing a guide RNA (eg, crRNA or a precursor crRNA array) in a mammalian cell (eg, a human cell). In some embodiments, the sequence encoding the guide RNA is expressed in the pUC19-U6-i2-crRNA vector backbone.

[0256] III. How to use

[0257] The present application provides methods for detecting a target nucleic acid or modifying a nucleic acid in vitro, ex vivo or in vivo using any of the engineered Cas12i effector proteins or CRISPR-Cas12i systems described herein, and methods for treating or diagnosing using the engineered Cas12i effector proteins or CRISPR-Cas12i systems. Also provided are uses of the engineered Cas12i effector proteins or CRISPR-Cas12i systems described herein for detecting or modifying nucleic acids in cells, and for treating or diagnosing a disease or condition in a subject; and uses of compositions comprising any of the engineered Cas12i effector proteins or one or more components of the engineered CRISPR-Cas12i system in the preparation of drugs for detecting or modifying nucleic acids in cells and for treating or diagnosing a disease or condition in a subject.

[0258] Method for detecting target nucleic acid in a sample

[0259] The application also provides a method for detecting a target nucleic acid using any of the engineered Cas12i effector proteins or CRISPR-Cas12i systems with improved activity. The use of Cas12i effector proteins as detection agents can utilize the following findings: once activated by detecting a target DNA, a V-type CRISPR / Cas protein (e.g., Cas12i) can promiscuously cut non-targeted single-stranded DNA (ssDNA or RNA, i.e., a single-stranded nucleic acid to which the guide sequence of a guide RNA does not hybridize). Therefore, when there is a target DNA (double-stranded or single-stranded) in a sample (e.g., in some cases exceeding a threshold amount), the result is the cutting of a single-stranded nucleic acid in the sample, which can be detected using any convenient detection method (e.g., using a labeled single-stranded detection nucleic acid such as DNA or RNA). Cas12i can cut ssDNA and ssRNA. For example, methods using Cas proteins as detection agents are described in US10253365 and WO2020 / 056924, which are incorporated herein by reference in their entirety.

[0260] In some embodiments, a method for detecting a target DNA (e.g., double-stranded or single-stranded) in a sample is provided, comprising: (a) contacting the sample with: (i) any one of the engineered Cas12i effector proteins described herein; (ii) a guide RNA comprising a guide sequence that hybridizes with the target DNA; and (iii) a detection nucleic acid that is single-stranded (i.e., "single-stranded detection nucleic acid") and does not hybridize with the guide sequence of the guide RNA; and (b) measuring a detectable signal generated by cleavage of the single-stranded detection nucleic acid by the engineered Cas12i effector protein. In some cases, the single-stranded detection nucleic acid includes a fluorescent dye pair (e.g., a fluorescent dye pair is a fluorescence resonance energy transfer (FRET) pair, a quencher / fluorescence pair). In some cases, the target DNA is viral DNA (e.g., papillomavirus, hepatovirus, herpesvirus, adenovirus, poxvirus, parvovirus, etc.). In some embodiments, the single-stranded detection nucleic acid is DNA. In some embodiments, the single-stranded detection nucleic acid is RNA.

[0261] The method for detecting a target DNA (single-stranded or double-stranded) in a sample disclosed herein can detect the target DNA with high sensitivity. In some cases, the method disclosed herein can be used to detect a target DNA present in a sample containing a plurality of DNAs (including the target DNA and a plurality of non-target DNAs), wherein the target DNA is present at a concentration of 10 7 One or more copies of non-target DNA are present (e.g., 6 One or more copies of non-target DNA per 10 5 One or more copies of non-target DNA per 10 4One or more copies of non-target DNA per 10 3 One or more copies of non-target DNA per 10 2 In some embodiments, the engineered Cas12i effector protein described herein can detect target DNA with higher sensitivity compared to the reference Cas12i nuclease. In some embodiments, the engineered Cas12i effector protein can detect target DNA with 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more sensitivity compared to the reference Cas12i nuclease.

[0262] Modification method

[0263] In some embodiments, the application provides a method for modifying a target nucleic acid comprising a target sequence, the method comprising contacting the target nucleic acid with any one of the engineered CRISPR-Cas12i systems described herein. In some embodiments, the method is performed in vitro. In some embodiments, the target nucleic acid is present in a cell. In some embodiments, the cell is a bacterial cell, a yeast cell, a mammalian cell, a plant cell, or an animal cell. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo.

[0264] In some embodiments, the target nucleic acid is cut or the target sequence in the target nucleic acid is changed by the engineered CRISPR-Cas12i system. In some embodiments, the expression of the target nucleic acid is changed by the engineered CRISPR-Cas12i system. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target sequence is associated with a disease or condition. In some embodiments, the engineered CRISPR-Cas12i system comprises a precursor guide RNA array encoding multiple crRNAs, wherein each crRNA comprises a different guide sequence.

[0265] In some embodiments, the application provides a method for treating a disease or condition associated with a target nucleic acid in an individual cell, comprising modifying the target nucleic acid in the individual cell using any of the methods described herein, thereby treating the disease or condition. In some embodiments, the disease or condition is selected from the group consisting of cancer, cardiovascular disease, hereditary disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection.

[0266] The engineered CRISPR-Cas12i system described herein can modify the target nucleic acid in the cell in a variety of ways, depending on the type of engineered Cas12i effector protein in the CRISPR-Cas12i system. In some embodiments, the method induces site-specific cutting in the target nucleic acid. In some embodiments, the method cuts genomic DNA in cells such as bacterial cells, plant cells, or animal cells (e.g., mammalian cells). In some embodiments, the method kills the cell by cutting the genomic DNA in the cell. In some embodiments, the method cuts viral nucleic acids in the cell.

[0267] In some embodiments, the method changes (such as increasing or decreasing) the expression level of the target nucleic acid in the cell. In some embodiments, the method is based on, for example, an inactive Cas12i protein fused to a transactivation domain, and an engineered Cas12i effector protein is used to increase the expression level of the target nucleic acid in the cell. In some embodiments, the method is based on, for example, an inactive Cas12i protein fused to a transcription repression domain, and an engineered Cas12i effector protein is used to reduce the expression level of the target nucleic acid in the cell. In some embodiments, the method is based on, for example, an inactive Cas12i protein fused to an epigenetic modification domain, and an engineered Cas12i effector protein is used to introduce epigenetic modifications into the target nucleic acid in the cell. The engineered Cas12i system described herein can be used to introduce other modifications into the target nucleic acid, depending on the functional domains contained in the engineered Cas12i effector protein.

[0268] In some embodiments, the method changes the target sequence in the target nucleic acid described in the cell. In some embodiments, the method introduces mutations into the target nucleic acid in the cell. In some embodiments, the method uses one or more endogenous DNA repair pathways, such as non-homologous end joining (NHEJ) or homology-directed recombination (HDR), to repair double-strand breaks induced in the target DNA in the cell as a result of sequence-specific cutting by the CRISPR complex. Exemplary mutations include, but are not limited to, insertions, deletions, substitutions, and frameshifts. In some embodiments, the method inserts donor DNA at the target site. In some embodiments, the insertion of donor DNA results in the introduction of a selective marker or reporter protein into the cell. In some embodiments, the insertion of donor DNA results in the knock-in of a gene. In some embodiments, the insertion of donor DNA results in a knockout mutation. In some embodiments, the insertion of donor DNA results in a replacement mutation such as a single nucleotide replacement. In some embodiments, the method induces phenotypic changes in cells.

[0269] In some embodiments, the engineered CRISPR-Cas12i system is used as part of a genetic circuit, or for inserting a genetic circuit into the genomic DNA of a cell. The engineered segmented Cas12i effector protein controlled by the inducible described herein is particularly useful as a component of a genetic circuit. Genetic circuits can be used for gene therapy. Methods and techniques for designing and using genetic circuits are known in the art. Reference may be further made to, for example, Brophy, Jennifer AN, and Christopher A. Voigt. "Principles of genetic circuit design." Nature methods 11.5 (2014): 508.

[0270] The engineered CRISPR-Cas12i system described herein can be used to modify a variety of target nucleic acids. In some embodiments, the target nucleic acid is in a cell. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid is extrachromosomal DNA. In some embodiments, the target nucleic acid is exogenous to a cell. In some embodiments, the target nucleic acid is a viral nucleic acid such as viral DNA. In some embodiments, the target nucleic acid is a plasmid in a cell. In some embodiments, the target nucleic acid is a plasmid transferred horizontally. In some embodiments, the target nucleic acid is RNA.

[0271] In some embodiments, the target nucleic acid is an isolated nucleic acid such as an isolated DNA. In some embodiments, the target nucleic acid is present in a cell-free environment. In some embodiments, the target nucleic acid is an isolated vector such as a plasmid. In some embodiments, the target nucleic acid is an isolated linear DNA fragment.

[0272] The methods described herein are applicable to any suitable cell type. In some embodiments, the cell is a bacterium, a yeast cell, a fungal cell, an algae cell, a plant cell or an animal cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cell is a cell of natural origin, such as a cell isolated by a tissue biopsy. In some embodiments, the cell is a cell isolated from a cell line cultured in vitro. In some embodiments, the cell is from a primary cell line. In some embodiments, the cell is from an immortalized cell line. In some embodiments, the cell is a genetically engineered cell.

[0273] In some embodiments, the cell is an animal cell of an organism selected from the group consisting of cow, sheep, goat, horse, pig, deer, chicken, duck, goose, rabbit, and fish.

[0274] In some embodiments, the cell is a plant cell of an organism selected from the group consisting of corn, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, pearl millet, corn, sorghum, rapeseed, hemp, vegetable crops, forage crops, industrial crops, tree crops, and biomass crops.

[0275] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a human embryonic kidney 293T (HEK293T or 293T) cell or a HeLa cell. In some embodiments, the cell is a human embryonic kidney (HEK293T) cell. In some embodiments, the cell is a mouse Hepa1-6 cell. In some embodiments, the mammalian cell is selected from the group consisting of an immune cell, a hepatocyte, a tumor cell, a stem cell, a zygote, a muscle cell, and a skin cell.

[0276] In some embodiments, the cell is an immune cell selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) T cells, iNK-T cells, NK-T-like cells, γδT cells, tumor-infiltrating T cells, and dendritic cells (DC) activated T cells. In some embodiments, the method produces modified immune cells, such as CAR-T cells or TCR-T cells.

[0277] In some embodiments, the cell is an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a gamete progenitor cell, a gamete, a zygote, or a cell in an embryo.

[0278] The methods described herein can be used to modify target cells in vivo, in vitro or in vitro, and can be performed in a manner that changes the cells so that once modified, the descendants or cell lines of the modified cells retain the altered phenotype. The modified cells and descendants can be part of a multicellular organism, such as a plant or animal with in vitro or in vivo applications (such as genome editing and gene therapy).

[0279] In some embodiments, the method is performed in vitro. In some embodiments, after the engineered CRISPR-Cas12i system is introduced into cells, the modified cells (e.g., mammalian cells) are propagated in vitro. In some embodiments, the modified cells are cultured to propagate at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the modified cells are cultured for no more than about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the modified cells are further evaluated or screened to select cells with one or more desired phenotypes or characteristics.

[0280] In some embodiments, the target sequence is a sequence associated with a disease or condition. Exemplary diseases or conditions include, but are not limited to, cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection. In some embodiments, the disease or condition is a genetic disease. In some embodiments, the disease or condition is a monogenic disease or condition. In some embodiments, the disease or condition is a polygenic disease or condition.

[0281] In some embodiments, the target sequence has a mutation compared to the wild-type sequence. In some embodiments, the target sequence has a single nucleotide polymorphism (SNP) associated with a disease or condition.

[0282] In some embodiments, the donor DNA inserted into the target nucleic acid encodes a biological product selected from the group consisting of reporter proteins, antigen-specific receptors, therapeutic proteins, antibiotic resistance proteins, RNAi molecules, cytokines, kinases, antigens, antigen-specific receptors, cytokine receptors, and suicide polypeptides. In some embodiments, the donor DNA encodes therapeutic proteins. In some embodiments, the donor DNA encodes therapeutic proteins that can be used for gene therapy. In some embodiments, the donor DNA encodes therapeutic antibodies. In some embodiments, the donor DNA encodes engineered receptors, such as chimeric antigen receptors (CAR) or engineered TCRs. In some embodiments, the donor DNA encodes therapeutic RNAs, such as small RNAs (e.g., siRNAs, shRNAs, or miRNAs) or long non-coding RNAs (lincRNAs).

[0283] The methods described herein can be used to perform multiple gene editing or regulation at two or more (e.g., 2, 3, 4, 5, 6, 8, 10 or more) different target sites. In some embodiments, the method detects or modifies multiple target nucleic acids or target nucleic acid sequences. In some embodiments, the method includes contacting the target nucleic acid with a guide RNA comprising multiple (e.g., 2, 3, 4, 5, 6, 8, 10 or more) crRNA sequences, wherein each crRNA comprises different target sequences.

[0284] Also provided are engineered cells comprising a modified target nucleic acid, the cells being produced using any of the methods described herein. The engineered cells can be used for cell therapy. Autologous or allogeneic cells can be used to prepare engineered cells using the methods described herein for cell therapy.

[0285] The methods described herein can also be used to generate isogenic lines of cells (eg, mammalian cells) to study genetic variants.

[0286] Also provided are engineered non-human animals comprising the engineered cells described herein. In some embodiments, the engineered non-human animals are genome-edited non-human animals. The engineered non-human animals can be used as disease models.

[0287] The technology of producing non-human genome editing or transgenic animals is well known in the art, including but not limited to: pronuclear microinjection, viral infection, transformation of embryonic stem cells and induced pluripotent stem (iPS) cells. The detailed methods that can be used include but are not limited to the methods described by Sundberg and Ichiki (2006, Genetically Engineered Mice Handbook, CRC Press) and Gibson (2004, APrimer Of Genome Science 2nded. Sunderland, Mass.: Sinauer).

[0288] The engineered animal can be any suitable species, including but not limited to: cattle, horses, sheep, dogs, deer, felines, goats, pigs, primates, and lesser known mammals such as elephants, deer, zebras, or camels.

[0289] Treatment

[0290] In some embodiments, there is provided a use of the aforementioned engineered CRISPR-Cas12i system in the preparation of a medicament for treating a disease or disorder associated with a target nucleic acid in a cell of an individual.

[0291] Further provided is a method of treating using any of the methods for modifying the target nucleic acid in the cell as described herein. In some embodiments, the application provides a method for treating a disease or condition associated with a target nucleic acid in an individual cell, comprising contacting the target nucleic acid with any of the CRISPR-Cas12i systems of the engineering approaches described herein, wherein the guide sequence of the guide RNA is complementary to the target sequence of the target nucleic acid, wherein the engineered Cas12i effector protein and the guide RNA are associated with each other to bind to the target nucleic acid to modify the target nucleic acid, so that the disease or condition is treated. In some embodiments, mutations (e.g., knockout or knock-in mutations) are introduced into the target nucleic acid. In some embodiments, the expression of the target nucleic acid is enhanced. In some embodiments, the expression of the target nucleic acid is suppressed. In some embodiments, the present application provides a method for treating a disease or condition in an individual, comprising administering to the individual an effective amount of any one of the engineered CRISPR-Cas12i systems described herein and a donor DNA encoding a therapeutic agent, wherein the guide sequence of the guide RNA is complementary to the target sequence of the target nucleic acid of the individual, wherein the engineered Cas12i effector protein and the guide RNA bind to each other to bind to the target nucleic acid and insert the donor DNA into the target sequence, thereby treating the disease or condition.

[0292] In some embodiments, the application provides a method for treating a disease or condition of an individual, comprising administering to the individual an effective amount of an engineered cell comprising a modified target nucleic acid, wherein the engineered cell is prepared by contacting the cell with any one of the engineered CRISPR-Cas12i systems described herein, wherein the guide sequence of the guide RNA is complementary to the target sequence of the target nucleic acid, wherein the engineered Cas12i effector protein and the guide RNA are mutually associated to bind to the target nucleic acid to modify the target nucleic acid. In some embodiments, the engineered cell is an immune cell. In some embodiments, the individual is a human. In some embodiments, the individual is an animal, such as a model animal such as a rodent, a pet, or a farm animal. In some embodiments, the individual is a mammal.

[0293] In some embodiments, the disease or condition is selected from the group consisting of cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection. In some embodiments, the target nucleic acid is PCSK9. In some embodiments, the disease or condition is cardiovascular disease. In some embodiments, the disease or condition is coronary artery disease. In some embodiments, the method reduces cholesterol levels in an individual. In some embodiments, the method treats diabetes in an individual.

[0294] Delivery Method

[0295] In some embodiments, the engineered CRISPR-Cas12i system described herein or its components, nucleic acid molecules thereof, or nucleic acid molecules encoding or providing its components can be delivered to a host cell (e.g., any of the vectors described in the "Constructs and Vectors" section above) via a variety of delivery systems such as plasmids or viruses. In some embodiments or methods, the engineered CRISPR-Cas12i system can be delivered by other methods, such as nuclear transfection or electroporation of a ribonucleoprotein complex consisting of the engineered Cas12i effector protein and one or more cognate RNA guide sequences thereof.

[0296] In some embodiments, delivery is via nanoparticles or exosomes.

[0297] In some embodiments, paired Cas12i nickase complexes can be directly delivered using nanoparticles or other direct protein delivery methods so that the complex comprising two paired crRNA elements is delivered together. In addition, the protein can be delivered to cells by viral vectors or directly, and then directly delivers the CRISPR array comprising two paired spacers for double nicks. In some cases, for direct RNA delivery, RNA can be conjugated to at least one sugar moiety such as N-acetylgalactosamine (GalNAc) (particularly triantennary GalNAc).

[0298] IV. Kits and Articles

[0299] Also provided are compositions, kits, unit doses, and articles of manufacture comprising one or more components of any of the engineered Cas12i nucleases, engineered Cas12i effector proteins, or engineered CRISPR-Cas12i systems described herein.

[0300] In some embodiments, a kit is provided, comprising: one or more AAV vectors encoding any of the engineered Cas12i nucleases, engineered Cas12i effector proteins, or engineered CRISPR-Cas12i systems described herein. In some embodiments, the kit further comprises one or more guide RNAs. In some embodiments, the kit further comprises donor DNA. In some embodiments, the kit further comprises cells such as human cells.

[0301] The kit may include one or more additional components, such as containers, reagents, culture media, cytokines, buffers, antibodies, etc., to allow propagation of the engineered cells. The kit may also include a device for administering the composition.

[0302] The kit may also include instructions for using the engineered CRISPR-Cas12i system described herein, such as methods for detecting or modifying target nucleic acids. In some embodiments, the kit includes instructions for treating or diagnosing a disease or condition. Instructions for use related to the kit components generally include information on the dosage, dosing schedule, and route of administration for the deliberate treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. For example, such a kit (which includes a sufficient dose of the composition disclosed herein) may be provided to provide effective treatment of an individual over an extended period of time. The kit may also include a plurality of unit doses of the composition and instructions for use, the packaging quantity of which is sufficient for storage and use in pharmacies (e.g., hospital pharmacies and compounding pharmacies).

[0303] The kit of the present invention is in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed polyester film or plastic bags), etc. The kit may optionally provide additional components, such as buffers and explanatory information. Therefore, the present application also provides an article, which includes a vial (e.g., a sealed vial), a bottle, a jar, a flexible package, etc.

[0304] The article can include a container and a label or package insert bonded to or on the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials (such as glass or plastic). Typically, the container holds a composition that effectively treats a disease or condition as described herein, and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition in an individual. The label or package insert will further include instructions for administering the composition to an individual.

[0305] Package insert refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products.

[0306] In addition, the article of manufacture may further include a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. From a commercial and user perspective, it may also include other materials, including other buffers, diluents, filters, needles, and syringes.

[0307] Examples

[0308] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0309] Example 1: The amino acids interacting with PAM in the reference Cas12i2 enzyme were replaced with positively charged amino acids, and their gene editing efficiency was verified.

[0310] Plasmid construction

[0311] The coding sequence of Cas12i2 is codon optimized (human) and synthesized. The variant of Cas protein is produced by site-directed mutagenesis based on PCR. The specific method is to divide the DNA sequence design of Cas12i2 protein into two parts with the mutated site as the center, design two pairs of primers to amplify the two parts of DNA sequence respectively, introduce the sequence that needs to be mutated on the primer at the same time, and finally load the two fragments onto the pCAG-2A-eGFP vector by Gibson cloning. The combination of mutants is achieved by splitting the DNA of Cas12i2 protein into multiple segments, and constructing using PCR and Gibson clone. The position of the mutant is determined by analyzing the structural information of Cas12i2 using protein structure visualization software commonly used in the art (such as PyMol, Chimera and other software can be selected). The structural information of Cas12i2 is obtained by referring to PDB:6LTU, 6LTR, 6LU0, 6LTP). Cas12i2 effector protein is expressed in human 293T cells by pCAG-2A-eGFP vector. The DNA encoding Cas12i2 protein is inserted between XmaI and NheI. A vector expressing crRNA of Cas12i2 in 293T was constructed by ligating annealed oligonucleotides containing the target sequence into the BasI-digested pUC19-U6-i2-crRNA backbone.

[0312] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0313] HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the degree of confluence reached 70%. By using Lipofectamine 3000 (Invitrogen), 600ng of plasmid encoding Cas12i2 protein and 3000ng of plasmid encoding crRNA were transfected into each 24-cell culture dish. After 68h of transfection, HEK293T cells for fluorescence-activated cell sorting (FACS) were digested with trypsin-EDTA (0.05%) (Gibco). Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP signal.

[0314] Targeted deep sequencing analysis for genome modification

[0315] FACS-sorted GFP-positive 293FT cells were lysed with buffer L and incubated at 55°C for 3 hours and then at 95°C for 10 minutes. dsDNA fragments containing target sites in different genomic sites were PCR amplified using the corresponding primers. For targeted deep sequencing, cell lysate was used directly as a template to directly amplify the target site by barcoded PCR. The PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency of insertions and deletions (%) was analyzed using CRISPResso2 software by calculating the ratio of reads containing insertions or deletions. In this application, the frequency of insertions and deletions (%) is uniformly used as an indicator for comparison and analysis of gene editing efficiency. Reads with a number less than 0.05% of the complete reads were discarded.

[0316] Example 1-A selected four engineered Cas12i2 with single amino acid substitutions

[0317] According to the method described in Example 1, engineered Cas12i2 enzymes with single mutations in amino acid sequences were expressed respectively. The preferred amino acid substitution patterns and their corresponding gene editing efficiencies are shown in Figure 1 and Table 1. In Figure 1, we first selected the distance between Cas12i2 and PAM DNA. The 10 amino acids within the range of arginine (R) were tested for point mutations: E176, E178, Y226, A227, N229, E237, K238, K264, T447, and E563. By comparing the gene editing efficiency of these mutants with that of wild-type Cas12i2 at two genomic sites: CCR5-3 and RNF2-7 in 293T cells, we found that mutants with the following amino acid substitutions: E176R, K238R, T447R, and E563R can effectively improve the gene editing efficiency (Figure 1 shows that four amino acid substitutions have obtained insertion and deletion rates higher than about 10% (this is the gene editing efficiency of the reference enzyme for CCR5-3) and higher than about 12% (this is the gene editing efficiency of the reference enzyme for RNF2-7), which is significantly better than other single amino acid substitution schemes), and the remaining 6 mutants have no help in improving the gene editing efficiency, and even have serious negative effects.

[0318] Example 1-B Comparison of engineered Cas12i2 with multiple preferred amino acid substitutions

[0319] According to the method described in Example 1, the engineered Cas12i2 enzymes whose amino acid sequences have two or more preferred amino acid substitutions are expressed respectively, and the comparison of their combination methods and gene editing efficiencies is shown in Tables 1 and Figure 2 . Figure 2 It is shown that we combined the point mutations in the four mutants that can improve efficiency, namely E176R, K238R, T447R and E563R, obtained by screening in Example 1-A. By comparing the gene editing efficiency of these mutants with wild-type Cas12i2 at three genomic sites: CCR5-3, CCR5-5, and RNF2-7 in 293T cells, we found that after combining point mutations, mutants with further improved efficiency can be obtained. In particular, when the four mutations are combined together (E176R+K238R+T447R+E563R), the combined mutation with the best efficiency can be obtained.

[0320] Summary of experimental results of Example 1

[0321]

[0322] Table 1 Summary of results of Example 1 (gene editing efficiency)

[0323] Example 2: The amino acids involved in opening the DNA double strand in the reference Cas12i2 enzyme were replaced with amino acids with aromatic rings, and their gene editing efficiencies were verified respectively

[0324] Plasmid construction

[0325] The variant of Cas12i2 protein is produced by site-directed mutagenesis based on PCR. The specific method is to divide the DNA sequence design of Cas12i2 protein into two parts with the mutated site as the center, design two pairs of primers to amplify the two parts of DNA sequence respectively, introduce the sequence that needs to be mutated on the primer at the same time, and finally load the two fragments into pCAG-2A-eGFP vector by Gibson clone. The determination of the amino acid replacement position can be obtained by analyzing the structural information of Cas12i2 using common protein structure visualization software (for example, software such as PyMol and Chimera can be used). The structural information of Cas12i2 is obtained by referring to PDB:6LTU, 6LTR, 6LU0, 6LTP). Cas12i2 effector protein is expressed in human 293T cells by pCAG-2A-eGFP vector. The DNA encoding Cas12i2 protein is inserted between XmaI and NheI. The vector for expressing Cas12i2 crRNA in 293T was constructed by ligating annealed oligonucleotides containing the target sequence into the BasI-digested pUC19-U6-i2-crRNA backbone.

[0326] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0327] HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the degree of confluence reached 70%. By using Lipofectamine 3000 (Invitrogen), 600ng of plasmid encoding Cas12i2 protein and 3000ng of plasmid encoding crRNA were transfected into each 24-cell culture dish. After 68h of transfection, HEK293T cells for fluorescence-activated cell sorting (FACS) were digested with trypsin-EDTA (0.05%) (Gibco). Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP signal.

[0328] Targeted deep sequencing analysis for genome modification

[0329] FACS sorted GFP-positive 293FT cells were lysed with buffer L and incubated at 55°C for 3 hours and then at 95°C for 10 minutes. dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers. For targeted deep sequencing, cell lysate was used directly as a template to directly amplify the target site by barcoded PCR. PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of insertions and deletions was analyzed using CRISPResso2 software by calculating the ratio of reads containing insertions or deletions. Reads with a number less than 0.05% of the complete reads were discarded.

[0330] from Figure 2 It can be seen that first, we selected the amino acids involved in opening the DNA double helix in the Cas12i2 enzyme: Q163 and N164 for point mutation tests of amino acids with aromatic rings (Y, F, W). By comparing the gene editing efficiency of these mutants with that of wild-type Cas12i2 at three genomic sites: CCR5-3, CCR5-5, and RNF2-7 in 293T cells, we found that there are five mutants: Q163W, Q163Y, Q163F, N164Y, and N164F that can effectively improve the gene editing efficiency (at least at one genomic site). It can be concluded that N164Y and N164F both showed excellent gene editing efficiency at three genomic sites; while N164W had no effect on improving the gene editing efficiency compared to the reference enzyme.

[0331] Summary of experimental results of Example 2

[0332]

[0333] Table 2 Summary of results of Example 2 (gene editing efficiency)

[0334] Example 3: Replacing the amino acids in the RuvC domain of the reference Cas12i2 enzyme that interact with the single-stranded DNA substrate with positively charged amino acids and verifying the efficiency of its gene editing

[0335] Plasmid construction

[0336] The variant of Cas12i2 protein is produced by site-directed mutagenesis based on PCR. The specific method is to divide the DNA sequence design of Cas12i2 protein into two parts centered on the mutated site, design two pairs of primers to amplify the two parts of DNA sequence respectively, introduce the sequence that needs mutation on the primer at the same time, and finally load the two fragments onto the pCAG-2A-eGFP vector by Gibson clone. The combination of mutants is achieved by splitting the DNA of Cas12i2 protein into multiple sections, and constructing using PCR and Gibsonclone. The position of the mutant is determined by analyzing the structural information of Cas12i2 using commonly used protein structure visualization software (for example, software such as PyMol and Chimera can be used). The structural information of Cas12i2 is referenced to PDBID:6LTU,6LTR,6LU0,6LTP. The ssDNA substrates shown in these Cas12i2 structures have only 5nt. In order to obtain information about the interaction between longer ssDNA and Cas12i2, we made a homology comparison between the structure of Cas12i1 (PDB ID: 6W5C, 6W62 and 6W64; Zhang H. et al. Nature Structural & Molecular Biology 27, 1069-1076 (2020)) and the structure of Cas12i2, so that the ssDNA substrate (9nt) in the Cas12i1 structure was placed in the RuvC catalytic pocket of Cas12i2, and the amino acids within 9A were further searched through the model. The Cas12i2 effector protein was expressed in human 293T cells via the pCAG-2A-eGFP vector. The DNA encoding the Cas12i2 protein was inserted between XmaI and NheI. A vector for expressing Cas12i2crRNA in 293T was constructed by connecting the annealed oligonucleotide containing the target sequence to the pUC19-U6-i2-crRNA backbone digested with BasI.

[0337] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0338] HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the degree of confluence reached 70%. By using Lipofectamine 3000 (Invitrogen), 600ng of plasmid encoding Cas12i2 protein and 3000ng of plasmid encoding crRNA were transfected into each 24-cell culture dish. After 68h of transfection, HEK293T cells for fluorescence-activated cell sorting (FACS) were digested with trypsin-EDTA (0.05%) (Gibco). Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP signal.

[0339] Targeted deep sequencing analysis for genome modification

[0340] FACS sorted GFP-positive 293FT cells were lysed with buffer L and incubated at 55°C for 3 hours and then at 95°C for 10 minutes. dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers. For targeted deep sequencing, cell lysate was used directly as a template to directly amplify the target site by barcoded PCR. PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of insertions and deletions was analyzed using CRISPResso2 software by calculating the ratio of reads containing insertions or deletions. Reads with a number less than 0.05% of the complete reads were discarded.

[0341] exist Figure 3a In the study, we replaced the amino acids in the reference Cas12i2 enzyme located in the RuvC domain and interacting with the single-stranded DNA substrate with positively charged amino acids. By comparing the gene editing efficiency of these mutants with that of wild-type Cas12i2 at two genomic sites: CCR5-3 and RNF2-7 in 293T cells, we found that three mutants: N391R, I926R, and G929R can effectively improve the gene editing efficiency (at least at one genomic site). Among them, I926R showed excellent gene editing efficiency at both genomic sites.

[0342] exist Figure 3b , 3cIn the study, we further replaced the amino acids in the reference Cas12i2 enzyme located in the RuvC domain and interacting with the single-stranded DNA substrate with positively charged amino acids. By comparing the gene editing efficiency of these mutants with that of wild-type Cas12i2 in 293T cells at two genomic sites: CCR5-3 and RNF2-7, we found that many mutants can effectively improve the gene editing efficiency (at least at one genomic site). The ranking of single amino acid replacement schemes that improve gene editing efficiency is: D362R>E323R>Q425R>N925R>other mutants with improved efficiency.

[0343] exist Figure 3d In Figure 3a , 3b , 3c screened four mutants that can improve efficiency, namely E323R, D362R, Q425R and I926R, and combined them. By comparing the gene editing efficiency of these mutants with that of wild-type Cas12i2 in 293T cells at two genomic sites: CCR5-3 and RNF2-7, we found that the combination of point mutations can obtain mutants with further improved efficiency.

[0344] exist Figure 3e In Figure 3a , 3b 3c The point mutations in some mutants that can improve efficiency were combined with I926G and 439GG. By comparing the gene editing efficiency of these mutants with that of wild-type Cas12i2 at two genomic sites: CCR5-3 and RNF2-7 in 293T cells, we found that the combination of point mutations can produce mutants with further improved efficiency.

[0345] Summary of experimental results of Example 3

[0346]

[0347]

[0348]

[0349] Table 3 Summary of the results (gene editing efficiency) of Example 3. *439GG means that two glycines are inserted after amino acid No. 439.

[0350] Example 4: Replacing the amino acids that interact with the DNA-RNA double helix in the reference Cas12i2 enzyme with positively charged amino acids and verifying its gene editing efficiency

[0351] Plasmid construction

[0352] The variant of Cas12i2 protein is produced by site-directed mutagenesis based on PCR. The specific method is to divide the DNA sequence design of Cas12i2 protein into two parts with the mutated site as the center, design two pairs of primers to amplify the two parts of DNA sequence respectively, introduce the sequence that needs to be mutated on the primer at the same time, and finally load the two fragments into pCAG-2A-eGFP vector by Gibson clone. The combination of mutants is achieved by splitting the DNA of Cas12i2 protein into multiple segments, and constructing using PCR and Gibsonclone. The position of the mutant is determined by analyzing the structural information of Cas12i2 using commonly used protein structure visualization software (for example, software such as PyMol and Chimera can be used). The structural information of Cas12i2 is obtained by referring to PDB:6LTU, 6LTR, 6LU0, 6LTP). Cas12i2 effector protein is expressed in human 293T cells by pCAG-2A-eGFP vector. The DNA encoding Cas12i2 protein is inserted between XmaI and NheI. The vector for expressing Cas12i2 crRNA in 293T was constructed by ligating annealed oligonucleotides containing the target sequence into the BasI-digested pUC19-U6-i2-crRNA backbone.

[0353] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0354] HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the degree of confluence reached 70%. By using Lipofectamine 3000 (Invitrogen), 600ng of plasmid encoding Cas12i2 protein and 3000ng of plasmid encoding crRNA were transfected into each 24-cell culture dish. After 68h of transfection, HEK293T cells for fluorescence-activated cell sorting (FACS) were digested with trypsin-EDTA (0.05%) (Gibco). Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP signal.

[0355] Targeted deep sequencing analysis for genome modification

[0356] FACS sorted GFP-positive 293FT cells were lysed with buffer L and incubated at 55°C for 3 hours and then at 95°C for 10 minutes. dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers. For targeted deep sequencing, cell lysate was used directly as a template to directly amplify the target site by barcoded PCR. PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of insertions and deletions was analyzed using CRISPResso2 software by calculating the ratio of reads containing insertions or deletions. Reads with a number less than 0.05% of the complete reads were discarded.

[0357] Figure 4 Table 4 summarizes the gene editing efficiency of Cas12i2 mutants and wild-type Cas12i2 in 293T cells at 2 genomic sites: CCR5-3 and RNF2-7. We found that there are 7 mutants: G116R, E117R, T159R, S161R, E319R, E343R, and D958R can effectively improve gene editing efficiency (at least at one genomic site). Among them, D958R showed excellent gene editing efficiency at 2 genomic sites.

[0358] Summary of experimental results of Example 4

[0359]

[0360]

[0361]

[0362] Table 4 Summary of the results of Example 4 (gene editing efficiency).

[0363] Example 5: The engineered amino acid mutations of Cas12i2 that partially improve the gene editing efficiency screened in Examples 1-4 are combined, and their gene editing efficiency is verified.

[0364] Plasmid construction

[0365] The combination of mutants is constructed by splitting the DNA of Cas12i2 protein into multiple segments and using PCR and Gibson clone. The position of the mutant is determined by analyzing the structural information of Cas12i2 using common protein structure visualization software (for example, PyMol, Chimera and other software can be used). The structural information of Cas12i2 is obtained by referring to PDB: 6LTU, 6LTR, 6LU0, 6LTP). Cas12i2 effector protein is expressed in human 293T cells via pCAG-2A-eGFP vector. The DNA encoding Cas12i2 protein is inserted between XmaI and NheI. A vector expressing Cas12i2crRNA in 293T is constructed by connecting the annealed oligonucleotide containing the target sequence to the pUC19-U6-i2-crRNA backbone digested with BasI.

[0366] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0367] HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the degree of confluence reached 70%. By using Lipofectamine 3000 (Invitrogen), 600ng of plasmid encoding Cas12i2 protein and 3000ng of plasmid encoding crRNA were transfected into each 24-cell culture dish. After 68h of transfection, HEK293T cells for fluorescence-activated cell sorting (FACS) were digested with trypsin-EDTA (0.05%) (Gibco). Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP signal.

[0368] Targeted deep sequencing analysis for genome modification

[0369] FACS sorted GFP-positive 293FT cells were lysed with buffer L and incubated at 55°C for 3 hours and then at 95°C for 10 minutes. dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers. For targeted deep sequencing, cell lysate was used directly as a template to directly amplify the target site by barcoded PCR. PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of insertions and deletions was analyzed using CRISPResso2 software by calculating the ratio of reads containing insertions or deletions. Reads with a number less than 0.05% of the complete reads were discarded.

[0370] exist Figure 5 In the example 1, 2, and 3, we further combined the amino acid mutations or amino acid mutation combinations obtained by screening: E176R+K238R+T447R+E563R, N164Y, E323R+D362R, I926R, E323R+D362R+I926R, E323R+D362R+I926G, E323R+D362R+I926G+439G, E323R+D362R+I926G+439GG. By comparing the gene editing efficiency of these mutants with wild-type Cas12i at 5 genomic sites: CCR5-3, CCR5-5, CD34-8, CD34-9, and RNF2-14 in 293T cells, we found that after the point mutation combination, mutants with further improved efficiency can be obtained. At the same time, the mutant that we believe to be the most efficient (E176R+K238R+T447R+E563R+N164Y+E323R+D362R) was named CasXX.

[0371] Summary of results of Example 5 (taking the gene editing efficiency data reflected at RNF2-14 as an example)

[0372]

[0373]

[0374] Table 5 Summary of the results of Example 5 (gene editing efficiency)

[0375] *439G means that a glycine is inserted after amino acid 439.

[0376] In addition, we tested the gene editing efficiency of the following mutation combinations by T7 endonuclease 1 (T7E1) assay and targeted deep sequencing: E176R+K238R+T447R+E563R+N164Y+D958R; E176R+K238R+T447R+E563R+I926R+D958R; E176R+K238R+T447R+E563R+E323R+D362R+D958R; N164Y+I926R+D958R; E176R+K238R+T447R+E563R+N164Y+I926R+D958R. 7R+E563R+N164Y+E323R+D362R+D958R; E176R+K238R+T447R+E563R+N164Y+I9 26R+E323R+D362R+D958R; E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I 926G+D958R; E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+439GG+D958R; and E176R+K238R+T447R+E563R+N164Y+E323R+D362R+I926G+439G+D958R

[0377] Example 6: Comparison of CasXX with conventional gene editing tools to verify its gene editing efficiency.

[0378] Plasmid construction

[0379] The coding sequences of AsCas12a, BhCas12b v4, SpCas9, SaCas9, and SaCas9-KKH were codon-optimized (human) and synthesized. Cas effector proteins were expressed in human 293T cells using the pCAG-2A-eGFP vector. DNA encoding Cas proteins was inserted between XmaI and NheI. Vectors expressing sgRNA or crRNA of AsCas12a, BhCas12b v4, SpCas9, SaCas9, SaCas9-KKH, and Cas12i2 in 293T were constructed by ligating annealed oligonucleotides containing target sequences into the BasI-digested pUC19-U6-i2-crRNA backbone.

[0380] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0381] HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the degree of confluence reached 70%. By using Lipofectamine 3000 (Invitrogen), 600ng of plasmids encoding Cas proteins and 3000ng of plasmids encoding crRNA were transfected into each 24-cell culture dish. After 68h of transfection, HEK293T cells for fluorescence-activated cell sorting (FACS) were digested with trypsin-EDTA (0.05%) (Gibco). Cell sorting was performed using MoFlo XDP (BeckmanCoulter) with GFP signal.

[0382] Targeted deep sequencing analysis for genome modification

[0383] FACS sorted GFP-positive 293FT cells were lysed with buffer L and incubated at 55°C for 3 hours and then at 95°C for 10 minutes. dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers. For targeted deep sequencing, cell lysate was used directly as a template to directly amplify the target site by barcoded PCR. PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of insertions and deletions was analyzed using CRISPResso2 software by calculating the ratio of reads containing insertions or deletions. Reads with a number less than 0.05% of the complete reads were discarded.

[0384] exist Figure 6a In this study, we first tested the gene editing efficiency of CasXX at 62 human genome sites. CasXX showed extremely strong gene editing capabilities, with an average gene editing efficiency of more than 60%, and gene editing efficiencies of more than 50% at almost all sites tested. In addition, it showed high gene editing efficiency for any NTTN PAM (N=A,T,G,C). This is something that wild-type Cas12i cannot do.

[0385] exist Figure 6b In order to further demonstrate the gene editing ability of our engineered CasXX, we compared CasXX with AsCas12a at the TTTN PAM site. Our CasXX showed a higher average gene editing efficiency. At the same time, compared with BhCas12b v4 at the TTN PAM site, our CasXX showed a higher average gene editing efficiency.

[0386] exist Figure 6cIn order to further demonstrate the gene editing ability of our engineered CasXX, we compared CasXX with SpCas9 at the same site. Our CasXX showed a higher average gene editing efficiency. At the same time, compared with SaCas9 and SaCas9-KKH at the same site, our CasXX showed a higher average gene editing efficiency.

[0387] exist Figure 6d In the data, we found that the gene editing efficiency of CasXX in the mouse Hepa1-6 cell line was statistically significant. It can be seen that CasXX exhibited strong gene editing capabilities at 65 sites, with an average gene editing efficiency of over 60%.

[0388] Although the embodiments of the present invention are described above in conjunction with the accompanying drawings, the present invention is not limited to the above specific embodiments and application fields, and the above specific embodiments are only illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can also make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present invention, all of which belong to the protection of the present invention.

[0389] Sequence Listing

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Claims

1. An engineered Cas12i nuclease; comprising the following amino acid replacements: The amino acids that interact with PAM in the reference Cas12i nuclease are replaced with positively charged amino acids, where SEQ ID NO.1 is the amino acid sequence of the reference Cas12i nuclease on which the above-mentioned engineering modification is based; wherein the amino acid that interacts with PAM is the amino acid at position 176; wherein the amino acid position numbering is as defined in SEQ ID NO.1, wherein the positively charged amino acid is R or K.

2. The engineered Cas12i nuclease according to claim 1, wherein The amino acid interacting with PAM is further selected from the 238th, 447th, and / or 563rd amino acids; wherein the amino acid position numbering is as defined in SEQ ID NO.

1.

3. The engineered Cas12i nuclease according to claim 1, wherein The Cas12i nuclease comprises any one or combination of the following mutations: (1) E176R; (2) E176R, K238R and T447R; wherein the amino acid position numbering is as defined in SEQ ID NO.

1.

4. The engineered Cas12i nuclease according to claim 1, wherein the engineered Cas12i nuclease further comprises replacing one or more amino acids involved in opening a double-stranded DNA in a reference Cas12i nuclease with amino acids with aromatic rings; in, The one or more amino acids involved in opening the DNA double helix are one or more amino acids at the following positions: 163 and / or 164; wherein the amino acid position numbering is as defined in SEQ ID NO.

1.

5. The engineered Cas12i nuclease of claim 4, wherein The one or more amino acids involved in opening the DNA double helix are replaced by amino acids with aromatic rings, and the amino acids with aromatic rings are F, Y or W.

6. The engineered Cas12i nuclease of claim 5, wherein The replacement of one or more amino acids involved in opening the DNA double strand in the reference Cas12i nuclease with amino acids with aromatic rings refers to: Q163F, Q163Y, Q163W, and / or N164F or N164Y.

7. The engineered Cas12i nuclease of claim 1, further comprising replacing one or more amino acids in the reference Cas12i nuclease that are located in the RuvC domain and interact with a single-stranded DNA substrate with positively charged amino acids; in, The one or more amino acids located in the RuvC domain and interacting with the single-stranded DNA substrate are one or more amino acids at the following positions: 323, 362, 425, 925, 926, 391, 424 and / or 929; wherein the amino acid position numbering is as defined in SEQ ID NO.

1.

8. The engineered Cas12i nuclease of claim 7, wherein One or more amino acids involved in the cleavage of double-stranded DNA in the reference Cas12i nuclease are replaced with positively charged amino acids, wherein the positively charged amino acids are R or K.

9. The engineered Cas12i nuclease of claim 8, wherein The Cas12i nuclease also comprises any one of the following mutations or combinations of mutations: 1) E323R; (2) D362R; (3) Q425R; (4) N925R; (5) I926R; (6) E323R and D362R; (7) E323R and Q425R; (8) E323R and I926R; (9) Q425R and I926R; (10) D362R and I926R; (11) N925R and I926R; (12) E323R, D362R and Q425R; (13) E323R, D362R and I926R; (14) E323R, Q425R and I926R; (15) D362R, N925R and I926R; and / or (16) E323R, D362R, Q425R and I926R; wherein the amino acid position numbers are as defined in SEQ ID NO.

1.

10. The engineered Cas12i nuclease as described in claim 1, further comprising replacing one or more amino acids interacting with the DNA-RNA double helix in the reference Cas12i nuclease with positively charged amino acids; in, The one or more amino acids interacting with the DNA-RNA double helix are one or more of the following amino acids: 116, 117, 159, 161, 319, 343 and / or 958; wherein the amino acid position numbering is as defined in SEQ ID NO.

1.

11. The engineered Cas12i nuclease of claim 10, wherein One or more amino acids that interact with the DNA-RNA double helix in the reference Cas12i nuclease are replaced with positively charged amino acids, wherein the positively charged amino acids are R or K.

12. The engineered Cas12i nuclease of claim 11, wherein Replacing one or more amino acids interacting with the DNA-RNA double helix in the reference Cas12i nuclease with positively charged amino acids, wherein the positively charged amino acid is R or K refers to any one of the following mutations or combinations of mutations: G116R, E117R, T159R, S161R, E319R, E343R, and / or D958R; wherein the amino acid position numbering is as defined in SEQ ID NO.

1.

13. The engineered Cas12i nuclease according to any one of claims 1 to 12, comprising one or more flexible region mutations, wherein the flexible region mutations are located at one or more of the following positions: 439 and / or 926; wherein, The amino acid position numbers are as defined in SEQ ID NO.

1.

14. The engineered Cas12i nuclease of claim 13, wherein The one or more flexible region mutations are: I926G; and / or 439G or 439GG.

15. An engineered Cas12i nuclease; The engineered Cas12i nuclease comprises any one or more of the following mutations: (1) E176R and T447R; (2) E176R, K238R and T447R; wherein, The amino acid position numbers are as defined in SEQ ID NO.

1.

16. An engineered Cas12i nuclease comprising an amino acid sequence as shown in any one of SEQ ID NO.7-12.

17. An engineered Cas12i effector protein comprising the engineered Cas12i nuclease according to any one of claims 1 to 16.

18. The engineered Cas12i effector protein of claim 17, wherein the effector protein is capable of inducing double-strand breaks or single-strand breaks in a DNA molecule.

19. The engineered Cas12i effector protein of claim 17, wherein the engineered Cas12i nuclease is an enzyme-inactive mutant having D599A, E833A, S883A, H884A, R900A and / or D1019A.

20. The engineered Cas12i effector protein as described in claim 17, further comprising a functional domain fused to the engineered Cas12i nuclease.

21. The engineered Cas12i effector protein of claim 20, wherein the functional domain is selected from the group consisting of a translation initiation domain, a transcription repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain, a reverse transcriptase domain, a reporter domain, and a nuclease domain.

22. The engineered Cas12i effector protein of claim 17, comprising a first polypeptide and a second polypeptide, the first polypeptide comprising amino acid residues 1 to X of the N-terminal portion of the engineered Cas12i nuclease of any one of claims 1-16, and the second polypeptide comprising amino acid residues X+1 to the C-terminus of the engineered Cas12i nuclease of any one of claims 1-16, wherein the first polypeptide and the second polypeptide are capable of associating with each other in the presence of a guide RNA comprising a guide sequence to form a clustered regularly interspaced short palindromic repeats (CRISPR) complex that specifically binds to a target nucleic acid comprising a target sequence complementary to the guide sequence.

23. An engineered CRISPR-Cas12i system, comprising: (a) the engineered Cas12i effector protein of any one of claims 17-22; as well as (b) a guide RNA comprising a guide sequence complementary to the target sequence, or one or more nucleic acids encoding the guide RNA, The engineered Cas12i effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to a target nucleic acid comprising the target sequence and induces modification of the target nucleic acid.

24. A method for detecting a target nucleic acid in a sample for non-disease diagnosis purposes, comprising: (a) contacting the sample with the engineered CRISPR-Cas12i system of claim 23 and a tagged detection nucleic acid that is single-stranded and does not hybridize to the guide sequence of the guide RNA; as well as (b) measuring a detectable signal generated by cleavage of the tagged detection nucleic acid by the engineered Cas12i effector protein, thereby detecting the target nucleic acid.

25. Use of the engineered CRISPR-Cas12i system of claim 23 in the preparation of a medicament for treating a disease or disorder associated with a target nucleic acid in a cell of an individual.

26. A method for modifying a target nucleic acid comprising a target sequence, comprising contacting the target nucleic acid with the engineered CRISPR-Cas12i system described in claim 23.

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