The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells
- Author:
Jiawei LV
1
;
Shuang WU
;
Renyue WEI
;
Yan LI
;
Junxue JIN
;
Yanshuang MU
;
Yu ZHANG
;
Qingran KONG
;
Xiaogang WENG
;
Zhonghua LIU
Author Information
- Publication Type:Original Article
- Keywords: CRISPR-Cas systems; guide RNA; heteroduplex; genome editing; swine
- MeSH: Base Sequence; Complement System Proteins; CRISPR-Cas Systems; DNA; Genome; Nucleotides; RNA, Guide; Swine
- From:Journal of Veterinary Science 2019;20(3):e23-
- CountryRepublic of Korea
- Language:English
- Abstract: The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.
