Method for modifying target nucleic acid in genome of cell
View Patent ↗Provided are: a method for modifying a target nucleic acid in the genome of a cell using a novel PAM sequence; and a cell in which a target nucleic acid in the genome is modified thereby. Accordingly, genome editing can be performed by targeting a position that could not be previously targeted as a target for genome editing, and thus the range of applications of genome editing can be expanded.
1. A method for modifying a target nucleic acid in the genome of a cell, the method comprising:
incubating a cell comprising a target nucleic acid; a polynucleotide encoding a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease or a variant thereof, and a guide RNA,
wherein the Cas nuclease or a variant thereof is Streptococcus pyogenes Cas9-NG (SpCas9-NG),
wherein the target nucleic acid comprises a protospacer adjacent motif (PAM) and a target sequence complementary to the guide RNA,
the PAM consists of a sequence selected from the group consisting of 5′-NTG-3′, 5′-GTH-3′, 5′-VCGD-3′, and 5-TCGDG-3′,
the target nucleic acid can be recognized by a complex of the Cas nuclease or a variant thereof with the guide RNA, and
the complex of the Cas nuclease or a variant thereof with the guide RNA modifies the target nucleic acid sequence-specifically.
2. The method of claim 1 , wherein the guide RNA is a polynucleotide complementary to a nucleotide sequence having consecutive 2 to 24 nucleotides in the 5′- or 3′-direction of the PAM in the target nucleic acid.
3. The method of claim 1 , wherein the length of the guide RNA is 17 to 24 nucleotides.
4. The method of claim 1 , wherein the modification is cleavage, insertion, ligation, deamination, or combinations thereof.