Patent ID: 11939571
Assignee: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method of generating a library of carrier plasmids in which the carrier plasmids carry a mutation in a nucleic acid segment of a target cell, wherein carrier plasmids within the library carry different mutations in the nucleic acid segment, the method comprising:
synthesizing on an array a library of oligonucleotides, each oligonucleotide comprising:
(a) a segment encoding a guide nucleic acid which is complementary to a target site in a nucleic acid in the target cell, and
(b) a donor DNA comprising the target site which is flanked by two sequences which are complementary to two regions of the nucleic acid of the target cell, wherein the two regions are on opposite sides of the target site, wherein the donor DNA consists of a single nucleotide mutation or more within the target site; and
recombining the library of oligonucleotides with a backbone plasmid,
wherein the oligonucleotides and the backbone plasmid recombine to form a circularized plasmid in which the segment encoding the guide nucleic acid is operably linked downstream from a promoter, wherein the donor DNA in the library of oligonucleotides comprises at least 24 distinct mutations each on a separate molecule of donor DNA.