title: Targeted genome editing in a quail cell line using a customized CRISPR/Cas9 system.
abstract: Soon after RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease opened a new era of targeted genome editing, the CRISPR/Cas9 platform began to be extensively used to modify genes in various types of cells and organisms. However, successful CRISPR/Cas9-mediated insertion/deletion (indel) mutation remains to be demonstrated in avian cell lines. The objective of this study was to design a poultry-specific CRISPR/Cas9 system to efficiently introduce targeted deletion mutation in chromosomes of the quail muscle clone 7 (QM7) cell line using a customized quail CRISPR vector. In this study, two avian-specific promoters, quail 7SK (q7SK) promoter and CBh promoter, the hybrid form of cytomegalovirus and chicken β-actin promoters, were cloned into a CRISPR vector for the expression of guide RNA and Cas9 protein, respectively. Then, guide RNA, which was designed to target 20-base pair (bp) nucleotides in the quail melanophilin (MLPH) locus, was ligated to the modified CRISPR vector and transfected to QM7 cells. Our results showed multiple indel mutations in the quail MLPH locus in nearly half of the alleles being tested, suggesting the high efficiency of the system for targeted gene modification. The new CRISPR vector developed from this study has the potential application to generate knockout avian cell lines and knockout poultry.
authors.affiliation: Department of Animal Sciences, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Jinsoo
authors.initials: J
authors.lastname: Ahn
authors.affiliation: Department of Animal Sciences, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Joonbum
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Ju Yeon
authors.initials: JY
authors.lastname: Park
authors.affiliation: Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju-gun, Jeonbuk 55365, Republic of Korea.
authors.forename: Keon Bong
authors.initials: KB
authors.lastname: Oh
authors.affiliation: Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju-gun, Jeonbuk 55365, Republic of Korea.
authors.forename: Seongsoo
authors.initials: S
authors.lastname: Hwang
authors.affiliation: Department of Food Animal Health Research Program, Ohio Agricultural and Research Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691, United States.
authors.forename: Chang-Won
authors.initials: CW
authors.lastname: Lee
authors.affiliation: Department of Animal Sciences, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Kichoon
authors.initials: K
authors.lastname: Lee
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D000072669:Gene Editing / Q000379:methods / Q000662:veterinary*; D018390:Gene Targeting / Q000379:methods*; D054643:INDEL Mutation; D011784:Quail / Q000235:genetics*; D017393:RNA Editing / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: 7SK promoter; CBh promoter; CRISPR/Cas9; QM7 (quail muscle clone 7) cell line; melanophilin (MLPH) locus