Patent ID: 11905549
Assignee: GENAHEAD BIO, INC.
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method of modifying a target site in the genome of a eukaryotic cell, the method comprising:
(1) introducing into the cell, four separate introduction nucleic acids consisting of:
(a) a template nucleic acid comprising a nucleic acid sequence encoding an RNA-guided nuclease,
(b) a template nucleic acid comprising a nucleic acid sequence encoding a guide RNA, or a guide RNA,
(c) a template nucleic acid comprising a nucleic acid sequence encoding a selectable marker, wherein the selectable marker is a drug resistance gene, and
(d) a targeting template nucleic acid comprising an exogenous nucleic acid sequence; and

(2) selecting a cell expressing the selectable marker by culturing the cell after said introducing in a presence of a drug and thereby, obtaining a living cell,
wherein
the number of moles (C) of (c) the template nucleic acid comprising a nucleic acid sequence encoding a selectable marker is smaller than any of the number of moles (A) of (a) the template nucleic acid comprising a nucleic acid sequence encoding an RNA-guided nuclease, the number of moles (B) of (b) the template nucleic acid comprising a nucleic acid sequence encoding a guide RNA, or the guide RNA, and the number of moles (D) of (d) the targeting template nucleic acid comprising an exogenous nucleic acid sequence, and
wherein the ratio of the number obtained by multiplying the number of moles (C) of (c) the template nucleic acid comprising a nucleic acid sequence encoding a selectable marker by the number of types (4) of the introduction nucleic acids to the total of the number of moles (A) of (a) the template nucleic acid comprising a nucleic acid sequence encoding an RNA-guided nuclease, the number of moles (B) of (b) the template nucleic acid comprising a nucleic acid sequence encoding a guide RNA, or the guide RNA, the number of moles (C) of (c) the template nucleic acid comprising a nucleic acid sequence encoding a selectable marker, and the number of moles (D) of (d) the template nucleic acid comprising an exogenous nucleic acid sequence (C×4/(A+B+C+D)) is 0.01 to 0.04.