title: An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.
abstract: The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.
authors.affiliation: 1] Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2] University of Chinese Academy of Sciences, Beijing 100080, China [3].
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Zhu
authors.affiliation: 1] Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2].
authors.forename: Yajie
authors.initials: Y
authors.lastname: Xu
authors.affiliation: 1] Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2].
authors.forename: Shanshan
authors.initials: S
authors.lastname: Yu
authors.affiliation: 1] Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2] University of Chinese Academy of Sciences, Beijing 100080, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Lu
authors.affiliation: 1] Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2] University of Chinese Academy of Sciences, Beijing 100080, China.
authors.forename: Mingqin
authors.initials: M
authors.lastname: Ding
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Cheng
authors.affiliation: 1] Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2] University of Chinese Academy of Sciences, Beijing 100080, China.
authors.forename: Guoxu
authors.initials: G
authors.lastname: Song
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Gao
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Liangming
authors.initials: L
authors.lastname: Yao
authors.affiliation: 1] Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2] University of Chinese Academy of Sciences, Beijing 100080, China.
authors.forename: Dongdong
authors.initials: D
authors.lastname: Fan
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Shu
authors.initials: S
authors.lastname: Meng
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xuewen
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Shengdi
authors.initials: S
authors.lastname: Hu
authors.affiliation: Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tian
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems; D002460:Cell Line; D016678:Genome; D060005:Genotyping Techniques; D006801:Humans; D054643:INDEL Mutation / Q000235:genetics*; D051379:Mice