Wang Ling, Yang Likai, Guo Yijie, Du Weili, Yin Yajun, Zhang Tao, Lu Hongzhao
School of Biological Science and Engineering, Shaanxi SCI-TECH University, Hanzhong, Shaanxi, China.
Department of Pathogenic Microbiology and Immunology, School of Basic Medical Sciences, Xi'an Jitong University, Xi'an, Shaanxi, China.
PLoS One. 2017 Jan 9;12(1):e0169768. doi: 10.1371/journal.pone.0169768. eCollection 2017.
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.
CRISPR/Cas9系统已实现对各种生物体进行高效的基因组靶向编辑。然而，与哺乳动物基因组相比，很少有研究聚焦于CRISPR/Cas9核酸酶介导的鸡基因组编辑。当前研究将CRISPR与酵母Rad52（yRad52）相结合，以增强鸡DF-1细胞中的靶向基因组DNA编辑。通过双报告替代系统的富集，CRISPR/Cas9核酸酶诱导的鸡基因组靶向突变效率提高到了41.9%。此外，使用50聚体寡脱氧核苷酸（ssODN）作为供体DNA时，CRISPR核酸酶和yRad52的联合作用显著提高了肌肉生长抑制素基因靶向替换的效率，在嘌呤霉素筛选后编辑效率达到36.7%。此外，基于yRad52的作用，鸡基因组中外源基因整合的频率比没有yRad52时高出3倍多。总体而言，这些结果表明ssODN是靶向替换的理想供体DNA，并且CRISPR/Cas9与yRad52相结合可显著增强鸡基因组编辑。这些发现可广泛应用于其他生物体。
