Protospacer adjacent motif sequence and method for modifying target nucleic acid in genome of cell by using same
View Patent ↗Provided are: a method of modifying a target nucleic acid in the genome of a cell by using a novel PAM sequence; and a cell in which a target nucleic acid of the genome of the cell is modified by the method. Accordingly, genome editing may be performed by targeting a position, which has not been previously targeted, as a target for genome editing, and thus the range of applications of genome editing may be expanded.
1 . A method of 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 variant,
wherein the Cas nuclease variant is Streptococcus pyogenes Cas9-HF1 (SpCas9-HF1); and a guide RNA, 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 nucleotide sequence selected from the group consisting of 5′-RAGN-3′, 5′-CAGH-3′, 5′-TAGB-3′, 5′-VGAG-3′, and 5′-NGGN-3′,
the target nucleic acid can be recognized by a complex comprising: the SpCas9-HF1; and the guide RNA, and
the complex comprising the SpCas9-HF1 and 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 2 to 24 consecutive nucleotide sequences in the 5′- or 3′-direction of the PAM in the target nucleic acid.
3 . The method of claim 1 , the length of the guide RNA is 17 to 24 nucleotides.
4 . The method of claim 1 , the modification is cleavage, insertion, ligation, deamination, or a combination thereof.