US 12,391,941 B2
Extended single guide RNA and use thereof
Jin-Soo Kim, Seoul (KR); Ka Yeong Lim, Seoul (KR); Beum-Chang Kang, Busan (KR); and Seuk Min Ryu, Seoul (KR)
Assigned to INSTITUTE FOR BASIC SCIENCE, Daejeon (KR)
Appl. No. 16/964,277
Filed by INSTITUTE FOR BASIC SCIENCE, Daejeon (KR)
PCT Filed Jan. 23, 2019, PCT No. PCT/KR2019/000962
§ 371(c)(1), (2) Date Jul. 23, 2020,
PCT Pub. No. WO2019/147014, PCT Pub. Date Aug. 1, 2019.
Claims priority of application No. 10-2018-0008492 (KR), filed on Jan. 23, 2018.
Prior Publication US 2021/0032621 A1, Feb. 4, 2021
Int. Cl. C12N 15/11 (2006.01); C12N 9/22 (2006.01); C12N 15/113 (2010.01); C12N 15/90 (2006.01)
CPC C12N 15/11 (2013.01) [C12N 9/22 (2013.01); C12N 15/907 (2013.01); C12N 2310/20 (2017.05)] 3 Claims
 
1. A method of producing a genetically modified eukaryotic cell having a base substitution, comprising:
(a) preparing a composition for base editing comprising
(i) an adenosine base editor (ABE) or cytosine base editor (CBE),
wherein the ABE is selected from the group consisting of apolipoprotein B editing complex 1 (APOBEC1) and activation-induced deaminase (AID), the CBE is tRNA-specific adenosine deaminase (tadA);
(ii) Cas 9 nickase (nCas9); and
(iii) an extended guide RNA (ggX20) hybridizable with a target sequence,
wherein the ggX20 comprises a sequence of 20 nucleotides of the target sequence, and two additional non-complementary guanines (G) in front of 20 nucleotides in the 5′ direction from protospacer adjacent motif (PAM),
wherein the target sequence is selected from HEK2, HBB, and FANCF;
(b) introducing the composition into a eukaryotic cell,
wherein the extended guide RNA hybridizes with the target sequence,
wherein the nCas9 makes a single stranded nick,
wherein the ABE converts an adenosine to a guanine at position 18 of the target sequence in the 5′ direction from the PAM;
wherein the CBE converts a cytosine to a thymine at position 20, 21, or 23 of the target sequence in the 5′ direction from the PAM, and
wherein the base substitution does not cause a double stranded break.