Production method for genome-edited cells
View Patent ↗A production method for a cell in which only one allele is genome-edited includes: a step of introducing, into a cell, (A) at least one selected from the group consisting of (a1) a guide RNA in which one or more nucleotide residues are added to a 5′-end of a spacer sequence, (a2) a guide RNA containing a spacer sequence having single-base or multiple-base mismatches with respect to a target sequence, and (a3) an expression vector that can cause the guide RNA of (a1) or (a2) to be expressed, and (B) at least one selected from the group consisting of a Cas protein and an expression vector that can cause the Cas protein to be expressed.
1 . A guide RNA comprising a spacer sequence,
wherein 15 or more nucleotide residues are added to the 5′-end of the spacer sequence, and
wherein said 15 or more nucleotide residues comprise a polycytosine or a polyguanine including 15 or more nucleotide residues.
2 . The guide RNA according to claim 1 ,
wherein the spacer sequence has single-base or multiple-base mismatches with respect to a target sequence.
3 . An expression vector comprising a sequence encoding the guide RNA according to claim 1 .
4 . The expression vector according to claim 3 , wherein the expression vector further comprises a Cas protein coding sequence and enables a Cas protein to be expressed.
5 . The expression vector according to claim 4 ,
wherein the Cas protein is a Cas9 protein.
6 . A production kit for a cell which is genome-edited, the production kit comprising:
(A) at least one guide RNA according to claim 1 and an expression vector comprising a sequence encoding the guide RNA.
7 . The production kit according to claim 6 further comprising:
(B) at least one selected from the group consisting of a Cas protein and an expression vector for the Cas protein.
8 . The guide RNA according to claim 1 ,
wherein the guide RNA further comprises a repeat sequence, and
wherein the spacer sequence targets a target region, and the repeat sequence forms base pairs with an anti-repeat sequence derived from a tracrRNA to form a complex with a Cas protein.
9 . The guide RNA according to claim 1 ,
wherein the guide RNA has at least one feature selected from the group consisting of:
(a) a suppressed cytotoxicity as compared with a guide RNA in which no nucleotide residues are added to the 5′ end of the spacer sequence;
(b) an increased homology directed repair frequency as compared with a guide RNA in which no nucleotide residues are added to the 5′ end of the spacer sequence; and
(c) an increased monoallelic frequency of genome editing as compared with a guide RNA in which no nucleotide residues are added to the 5′ end of the spacer sequence.
10 . A guide RNA, comprising a spacer sequence and a repeat sequence,
wherein the spacer sequence targets a target region, and the repeat sequence forms base pairs with an anti-repeat sequence derived from a tracrRNA to form a complex with a Cas protein,
wherein 15 or more nucleotide residues are added to a 5′-end of the spacer sequence, and wherein said 15 or more nucleotide residues comprise a polycytosine or a polyguanine of 15 or more nucleotide residues,
wherein the guide RNA suppresses cytotoxicity, increases homology directed repair frequency, or increases mono-allelic genome editing frequency in genome editing compared to a guide RNA which does not have a nucleotide residue added to the 5′ end of the spacer sequence.
11 . An expression vector comprising a sequence encoding the guide RNA according to claim 10 .
12 . The guide RNA according to claim 1 ,
wherein the 15 or more nucleotide residues added to the 5′-end of the spacer sequence are 20 or more nucleotide residues.
13 . The guide RNA according to claim 1 ,
wherein the 15 or more nucleotide residues added to the 5′-end of the spacer sequence comprises a guanine nucleotide at the 5′-end of the 15 or more nucleotide residues.
14 . The guide RNA according to claim 1 ,
wherein the 15 or more nucleotide residues added to the 5′-end of the spacer sequence comprise:
a polycytosine consisting of cytosine residues, wherein the polycytosine is at least 5 residues in length,
nucleotide residues consisting of 17 cytosine residues, an adenosine residue, and 2 cytosine residues, in order from the 5′ end,
nucleotide residues consisting of 22 cytosine residues, an adenosine residue, and 2 cytosine residues, in order from the 5′ end, or
nucleotide residues consisting of 27 cytosine residues, an adenosine residue, and 2 cytosine residues, in order from the 5′ end.