title: Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System.
abstract: In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ (PPAR-γ), ATP synthase epsilon subunit (ATP5E), and ovalbumin (OVA) genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the Puro(R) gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing.
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors: Yichun
authors: Y
authors: Bai
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors: Linjie
authors: L
authors: He
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors: Pengcheng
authors: P
authors: Li
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors: Kun
authors: K
authors: Xu
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors: Simin
authors: S
authors: Shao
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors: Chonghua
authors: C
authors: Ren
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors: Zhongtian
authors: Z
authors: Liu
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China Zhangzhy@nwsuaf.edu.cn 553527211@126.com.
authors: Zehui
authors: Z
authors: Wei
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China Zhangzhy@nwsuaf.edu.cn 553527211@126.com.
authors: Zhiying
authors: Z
authors: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D002645:Chickens; D000072669:Gene Editing; D015870:Gene Expression; D023061:Gene Order; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D009154:Mutation
keywords: CRISPR/Cas9 system; GenPred; Genomic Selection; Shared data resource; chicken DF-1 cell; mutant efficiency; off-target; surrogate reporter