	US 12,473,559 B2
	Cas9/RNA complexes for inducing modifications of target endogenous nucleic acid sequences in nucleuses of eukaryotic cells
Jin-Soo Kim, Seoul (KR); Seung Woo Cho, Seoul (KR); and Sojung Kim, Seoul (KR)
Assigned to ToolGen Incorporated, Seoul (KR)
Filed by TOOLGEN INCORPORATED, Seoul (KR)
Filed on Sep. 15, 2023, as Appl. No. 18/467,952.
Application 18/467,952 is a continuation of application No. 18/313,946, filed on May 8, 2023.
Application 18/467,952 is a continuation of application No. 18/314,050, filed on May 8, 2023.
Application 18/313,946 is a continuation of application No. 17/004,338, filed on Aug. 27, 2020.
Application 18/313,946 is a continuation of application No. 14/685,568, filed on Apr. 13, 2015, granted, now 10,851,380, issued on Dec. 1, 2020.
Application 14/685,568 is a continuation of application No. PCT/KR2013/009488, filed on Oct. 23, 2013.
Claims priority of provisional application 61/837,481, filed on Jun. 20, 2013.
Claims priority of provisional application 61/803,599, filed on Mar. 20, 2013.
Claims priority of provisional application 61/717,324, filed on Oct. 23, 2012.
Prior Publication US 2024/0052356 A1, Feb. 15, 2024 Prior Publication US 2024/0240192 A9, Jul. 18, 2024
Int. Cl. C12N 15/00 (2006.01); C12N 9/16 (2006.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/11 (2006.01); C12N 15/52 (2006.01); C12N 15/63 (2006.01); C12N 15/82 (2006.01); C12N 15/85 (2006.01); C12N 15/90 (2006.01)

CPC C12N 15/52 (2013.01) [C12N 9/16 (2013.01); C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/111 (2013.01); C12N 15/63 (2013.01); C12N 15/8216 (2013.01); C12N 15/85 (2013.01); C12N 15/907 (2013.01); C12Y 301/21 (2013.01); C12N 2310/10 (2013.01); C12N 2310/20 (2017.05); C12N 2310/531 (2013.01)]

8 Claims

1. A method of inducing a modification of a target endogenous nucleic acid sequence in a nucleus of a human cell, comprising:

preparing a Cas9 protein, wherein the Cas9 protein comprises a nuclear localization signal (NLS);

preparing a single-guide RNA (sgRNA), wherein the sgRNA comprises a crRNA and a tracrRNA, wherein the sgRNA is transcribed in vitro or synthesized chemically, and wherein the target endogenous nucleic acid sequence includes a portion complementary to the crRNA of the sgRNA;

providing a buffer in an in vitro environment;

disposing the Cas9 protein into the buffer;

disposing the sgRNA into the buffer, wherein the sgRNA is disposed in at least a two-fold molar excess over the Cas9 protein in the buffer,

allowing the Cas9 protein and the sgRNA to complex in the in vitro environment to form a Cas9/sgRNA complex;

transfecting the Cas9/sgRNA complex into the human cell by electroporation, whereby the Cas9/sgRNA complex induces the modification of the target endogenous nucleic acid sequence in the nucleus of the human cell.