title: Highly efficient enrichment of porcine cells with deletions induced by CRISPR/Cas9 using dual fluorescence selection.
abstract: Production of nuclear donor cells with a high percentage of desired modifications is a critical step in the successful generation of genetically modified pigs through somatic cell nuclear transfer (SCNT). The CRISPR/Cas9 system has been used for efficient modification of the nuclear DNA in eukaryotic cells, including porcine cells. However, in vitro modified cells are often phenotypically indistinguishable from unmodified cells, hampering their enrichment. Here we investigate a dual fluorescence selection system for the efficient enrichment of porcine embryonic fibroblasts (PEFs) with CRISPR/Cas9-induced chromosomal deletions. Enrichment of cells with 170 bp deletions reached a frequency of 74%, whilst enrichment of cells with a larger 5 kb deletions achieved a frequency of 46%. This demonstrates the utility of a dual fluorescence reporter as an attractive tool for improving the efficiency of generating genome edited pigs.
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China.
authors.forename: Zuyong
authors.initials: Z
authors.lastname: He
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China.
authors.forename: Baozhu
authors.initials: B
authors.lastname: Du
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China.
authors.forename: Peiqing
authors.initials: P
authors.lastname: Cong
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China. Electronic address: chyaosh@mail.sysu.edu.cn.
authors.forename: Yaosheng
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000818:Animals; D001483:Base Sequence; D055430:Bone Morphogenetic Protein 15 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D005347:Fibroblasts; D005434:Flow Cytometry / Q000379:methods*; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D013552:Swine
chemical_list: D055430:Bone Morphogenetic Protein 15; D049452:Green Fluorescent Proteins; D004247:DNA
keywords: BMP15; CRISPR/Cas9; FACS; Genomic deletion