	US 12,215,334 B2
	Polynucleotide for modifying target sequence and use thereof
Kohji Kusano, Tokyo (JP); Takayuki Kitogo, Tokyo (JP); Makoto Inoue, Tokyo (JP); Tsugumine Shu, Tokyo (JP); and Toyotaka Mori, Tokyo (JP)
Assigned to I'ROM GROUP CO., LTD., Tokyo (JP)
Appl. No. 16/631,407
Filed by I'ROM GROUP CO., LTD., Tokyo (JP)
PCT Filed Jul. 19, 2018, PCT No. PCT/JP2018/027141 § 371(c)(1), (2) Date Jan. 15, 2020, PCT Pub. No. WO2019/017438, PCT Pub. Date Jan. 24, 2019.
Claims priority of application No. 2017-141691 (JP), filed on Jul. 21, 2017.
Prior Publication US 2020/0216858 A1, Jul. 9, 2020
Int. Cl. C12N 15/11 (2006.01); A61K 39/155 (2006.01); C12N 7/00 (2006.01); C12N 15/00 (2006.01); C12N 15/85 (2006.01); C12N 15/86 (2006.01)

CPC C12N 15/85 (2013.01) [A61K 39/155 (2013.01); C12N 7/00 (2013.01); C12N 15/00 (2013.01); C12N 15/11 (2013.01); C12N 15/86 (2013.01); C12N 2320/00 (2013.01); C12N 2710/16643 (2013.01); C12N 2760/18011 (2013.01); C12N 2760/18842 (2013.01); C12N 2800/10 (2013.01); C12N 2810/6072 (2013.01)]

10 Claims

1. An isolated cell comprising:

1) A circular donor polynucleotide for modifying a genomic sequence of the isolated cell, wherein the circular donor polynucleotide comprises:

a) a region homologous to the genomic sequence to be modified, wherein the homologous region comprises:

i) one or more modification positions, wherein each modification position corresponds to a position in the genomic sequence to be modified, and the modification position in the homologous region has a different sequence from the corresponding position in the genomic sequence to be modified; and

ii) a single cleavable site; and

b) a linker polynucleotide linking both ends of the homologous region to each other, wherein the linker polynucleotide comprises a positive selection marker gene and a negative selection marker gene; and

2) A vector that expresses a restriction enzyme capable of cleaving the single cleavable site of the circular donor polynucleotide.