US 11,898,270 B2
Pig genome-wide specific sgRNA library, preparation method therefor and application thereof
Shuhong Zhao, Hubei (CN); Shengsong Xie, Hubei (CN); Changzhi Zhao, Hubei (CN); Xinyun Li, Hubei (CN); Xiangdong Liu, Hubei (CN); Xiaosong Han, Hubei (CN); Gaojuan Yang, Hubei (CN); Yang Gao, Hubei (CN); Yilong Chen, Hubei (CN); Xiaoyong Du, Hubei (CN); Yiliang Miao, Hubei (CN); Yunlong Ma, Hubei (CN); and Xiaolei Liu, Hubei (CN)
Assigned to HUAZHONG AGRICULTURAL UNIVERSITY, Hubei (CN)
Appl. No. 16/628,481
Filed by HUAZHONG AGRICULTURAL UNIVERSITY, Hubei (CN)
PCT Filed Aug. 21, 2017, PCT No. PCT/CN2017/098324
§ 371(c)(1), (2) Date Apr. 3, 2020,
PCT Pub. No. WO2019/006833, PCT Pub. Date Jan. 10, 2019.
Claims priority of application No. 201710533398.9 (CN), filed on Jul. 3, 2017.
Prior Publication US 2020/0325597 A1, Oct. 15, 2020
Int. Cl. C12N 15/90 (2006.01); C40B 40/02 (2006.01); C12N 9/22 (2006.01); C12N 15/113 (2010.01); C12N 15/85 (2006.01); C12Q 1/6869 (2018.01); C40B 40/06 (2006.01); C40B 50/06 (2006.01)
CPC C40B 40/02 (2013.01) [C12N 9/22 (2013.01); C12N 15/113 (2013.01); C12N 15/85 (2013.01); C12N 15/907 (2013.01); C12Q 1/6869 (2013.01); C40B 40/06 (2013.01); C40B 50/06 (2013.01); C12N 2810/10 (2013.01)] 8 Claims
 
1. A porcine whole genome-specific sgRNA library, wherein the library includes:
(i) N kinds of vectors expressing porcine-specific sgRNA, wherein N is a positive integer ≥20,000, and the porcine-specific sgRNA is a target gene for the porcine whole genome and the target gene is selected from the group consisting of: (a) a protein encoding gene, (b) a lincRNA, (c) a miRNA and (d) a combination of (a), (b) and (c);
in addition, the porcine-specific sgRNA has the following structural characteristics:
(1) the target gene locus targeted by the sgRNA contains PAM as NGG (wherein N is any base of A, T, C, G);
(2) the length of sgRNA is 19 or 20 nt;
(3) the GC content of sgRNA is 40-60%;
(4) a whole-genome off-target evaluation of sgRNA selects off-target sites that do not contain 1 and 2 base mismatches, compared with other sgRNAs, sgRNAs with a smallest number of off-targets are selected;
(5) for a porcine-specific sgRNA that targets a protein-coding gene, the binding position thereof is located in a region 500 bp downstream of the start codon ATG of the coding gene; for a porcine-specific sgRNA that targets a lincRNA gene, the binding position thereof is located in a 500 bp region downstream of the lincRNA transcription start point; for a porcine-specific sgRNA that targets a miRNA gene, the binding position thereof is a miRNA precursor sequence or a mature sequence; and (ii) optionally, M kinds of negative control vectors, which are the vectors expressing unrelated sgRNA, wherein M is a positive integer.