JP-2022530981-A5 -

Dates

Publication Date

20230510

Application Date

20200501

Description

In some aspects, the target nucleic acid includes single-stranded or double-stranded DNA. In some aspects, the programmable nickase exhibits PAM-independent nicking and cleavage. In some aspects, the programmable nickase nicks a single strand of double-stranded DNA. In some aspects, the programmable nickase cleaves single-stranded DNA. In some aspects, the programmable nickase contains the Cas14 protein. In further aspects, the Cas14 protein contains the Cas14e protein. In further aspects, the Cas14 protein contains 400–800 amino acid residues. [Invention 1001] A method for introducing cleavage into a target nucleic acid, wherein the target nucleic acid (a) A first guide nucleic acid comprising a first region that binds to a first programmable nickas having a length of 900 amino acids or less; and (b) A second guide nucleic acid comprising a first region that binds to a second programmable nickas, having a length of 900 amino acids or less. The stage in which cutting is introduced by bringing it into contact with the object. Includes, The method comprising: a first guide nucleic acid including a second region that binds to a target nucleic acid; a second guide nucleic acid including a second region that binds to a target nucleic acid; and the second region of the first guide nucleic acid and the second region of the second guide nucleic acid binding to the opposing strand of the target nucleic acid. [Invention 1002] The method of the present invention 1001, wherein the first programmable nickase and the second programmable nickase have a length of 350 to 900 amino acids. [Invention 1003] The method of the present invention 1001 or 1002, wherein the first programmable nickase and the second programmable nickase have a length of 480 to 550 amino acids. [Invention 1004] A method according to any one of the present invention 1001 to 1003, wherein the first programmable nickase and the second programmable nickase are type V CRISPR/Cas enzymes. [Invention 1005] The method of the present invention 1004, wherein the type V CRISPR/Cas enzyme contains three partial RuvC domains. [Invention 1006] The method of the present invention 1005, wherein the three partial RuvC domains are RuvC-I, RuvC-II, and RuvC-III subdomains. [Invention 1007] The method according to any one of the invention 1001 to 1006, wherein the first programmable nickase and the second programmable nickase are Cas14 proteins. [Invention 1008] The method of the present invention 1007, wherein the Cas14 protein is Cas14a protein, Cas14b protein, Cas14c protein, Cas14d protein, or Cas14e protein. [Invention 1009] The method of the present invention 1007 or 1008, wherein the Cas14 protein is the Cas14a protein. [Invention 1010] The method of the present invention 1007 or 1008, wherein the Cas14 protein is the Cas14b protein. [Invention 1011] The method of the present invention 1007 or 1008, wherein the Cas14 protein is the Cas14e protein. [Invention 1012] A method according to any one of the invention 1001 to 1011, wherein a first programmable nickase, a second programmable nickase, or both, have at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 91 or SEQ ID NO: 170. [Invention 1013] A method according to any one of the invention 1001 to 1012, wherein the first programmable nickase, the second programmable nickase, or both are one of the following: SEQ ID NO: 1 to SEQ ID NO: 91 or SEQ ID NO: 170. [Invention 1014] A method according to any one of the invention 1001 to 1013, wherein the first programmable nickase, the second programmable nickase, or both, have sequence identity with SEQ ID NO: 1 of at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99%. [Invention 1015] A method according to any one of the invention 1001 to 1014, wherein the first programmable nickase, the second programmable nickase, or both are SEQ ID NO: 1. [Invention 1016] A method according to any one of the invention 1001 to 1013, wherein the first programmable nickase, the second programmable nickase, or both, have sequence identity with SEQ ID NO: 10 of at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99%. [Invention 1017] A method according to any one of the invention 1001-1013 or 1016, wherein the first programmable nickase, the second programmable nickase, or both are SEQ ID NO: 10. [Invention 1018] A method according to any one of the invention 1001 to 1013, wherein the first programmable nickase, the second programmable nickase, or both, have sequence identity with SEQ ID NO: 11 of at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99%. [Invention 1019] A method according to any one of the invention 1001-1013 or 1018, wherein the first programmable nickase, the second programmable nickase, or both are SEQ ID NO: 11. [Invention 1020] A