title: Highly Efficient and Rapid Detection of the Cleavage Activity of Cas9/gRNA via a Fluorescent Reporter.
abstract: The RNA-guided endonuclease clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) derived from CRISPR systems is a simple and efficient genome-editing technology applied to various cell types and organisms. So far, the extensive approach to detect the cleavage activity of customized Cas9/guide RNA (gRNA) is T7 endonuclease I (T7EI) assay, which is time and labor consuming. In this study, we developed a visualized fluorescent reporter system to detect the specificity and cleavage activity of gRNA. Two gRNAs were designed to target porcine immunoglobulin M and nephrosis 1 genes. The cleavage activity was measured by using the traditional homology-directed repair (HDR)-based fluorescent reporter and the single-strand annealing (SSA)-based fluorescent reporter we established in this study. Compared with the HDR assay, the SSA-based fluorescent reporter approach was a more efficient and dependable strategy for testing the cleavage activity of Cas9/gRNA, thereby providing a universal and efficient approach for the application of CRISPR/Cas9 in generating gene-modified cells and organisms.
authors.affiliation: College of Animal Science, Jilin University, Changchun, 130062, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: College of Animal Science, Jilin University, Changchun, 130062, China.
authors.forename: Songcai
authors.initials: S
authors.lastname: Liu
authors.affiliation: College of Animal Science, Jilin University, Changchun, 130062, China.
authors.forename: Yunyun
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: College of Animal Science, Jilin University, Changchun, 130062, China.
authors.forename: Linyan
authors.initials: L
authors.lastname: Nie
authors.affiliation: College of Animal Science, Jilin University, Changchun, 130062, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Lv
authors.affiliation: College of Animal Science, Jilin University, Changchun, 130062, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.affiliation: College of Animal Science, Jilin University, Changchun, 130062, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of Animal Science, Jilin University, Changchun, 130062, China. haolinlin@jlu.edu.cn.
authors.forename: Linlin
authors.initials: L
authors.lastname: Hao
mesh_terms: D000818:Animals; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D004260:DNA Repair; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D018390:Gene Targeting; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism*; D034421:Sus scrofa
chemical_list: D064130:CRISPR-Associated Proteins; D005456:Fluorescent Dyes; D000094704:RNA, Guide, CRISPR-Cas Systems; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Cas9/gRNA; HR reporter assay; SSA reporter assay; T7EI