title: Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System.
abstract: The CRISPR/Cas9 system has enabled highly efficient genome targeted editing for various organisms. However, few studies have focused on CRISPR/Cas9 nuclease-mediated chicken genome editing compared with mammalian genomes. The current study combined CRISPR with yeast Rad52 (yRad52) to enhance targeted genomic DNA editing in chicken DF-1 cells. The efficiency of CRISPR/Cas9 nuclease-induced targeted mutations in the chicken genome was increased to 41.9% via the enrichment of the dual-reporter surrogate system. In addition, the combined effect of CRISPR nuclease and yRad52 dramatically increased the efficiency of the targeted substitution in the myostatin gene using 50-mer oligodeoxynucleotides (ssODN) as the donor DNA, resulting in a 36.7% editing efficiency after puromycin selection. Furthermore, based on the effect of yRad52, the frequency of exogenous gene integration in the chicken genome was more than 3-fold higher than that without yRad52. Collectively, these results suggest that ssODN is an ideal donor DNA for targeted substitution and that CRISPR/Cas9 combined with yRad52 significantly enhances chicken genome editing. These findings could be extensively applied in other organisms.
authors.affiliation: School of Biological Science and Engineering, Shaanxi SCI-TECH University, Hanzhong, Shaanxi, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.affiliation: School of Biological Science and Engineering, Shaanxi SCI-TECH University, Hanzhong, Shaanxi, China.
authors.forename: Likai
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Pathogenic Microbiology and Immunology, School of Basic Medical Sciences, Xi'an Jitong University, Xi'an, Shaanxi, China.
authors.forename: Yijie
authors.initials: Y
authors.lastname: Guo
authors.affiliation: School of Biological Science and Engineering, Shaanxi SCI-TECH University, Hanzhong, Shaanxi, China.
authors.forename: Weili
authors.initials: W
authors.lastname: Du
authors.affiliation: School of Biological Science and Engineering, Shaanxi SCI-TECH University, Hanzhong, Shaanxi, China.
authors.forename: Yajun
authors.initials: Y
authors.lastname: Yin
authors.affiliation: School of Biological Science and Engineering, Shaanxi SCI-TECH University, Hanzhong, Shaanxi, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: School of Biological Science and Engineering, Shaanxi SCI-TECH University, Hanzhong, Shaanxi, China.
authors.forename: Hongzhao
authors.initials: H
authors.lastname: Lu
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D064113:CRISPR-Cas Systems; D002460:Cell Line; D002645:Chickens; D000072669:Gene Editing; D015870:Gene Expression; D055879:Gene Knock-In Techniques; D023061:Gene Order; D018390:Gene Targeting; D017930:Genes, Reporter; D016678:Genome; D006801:Humans; D016296:Mutagenesis; D055435:Myostatin / Q000235:genetics; D011485:Protein Binding; D015203:Reproducibility of Results
chemical_list: C525911:MSTN protein, human; D055435:Myostatin