title: Improving the efficiency for generation of genome-edited zebrafish by labeling primordial germ cells.
abstract: Although CRISPR/Cas, a new versatile genome-editing tool, has been widely used in a variety of species including zebrafish, an important vertebrate model animal for biomedical research, the low efficiency of germline transmission of induced mutations and particularly knockin alleles made subsequent screening for heritable offspring tedious, time-consuming, expensive and at times impossible. In this study, we reported a method for improving the efficiency of germline transmission screening for generation of genome-edited zebrafish mutants. Co-microinjecting yfp-nanos3 mRNA with Cas9 mRNA, sgRNA and single strand DNA donor to label the distribution of microinjected nucleotides in PGCs (primordial germ cells), we demonstrated that founders carrying labeled PGCs produced much higher numbers of knockin and knockout progeny. In comparison with the common practice of selecting founders by genotyping fin clips, our new strategy of selecting founders with tentatively fluorescent-labeled PGCs significantly increase the ease and speed of generating heritable knocking and knockout animals with CRISPR/Cas9.
authors.affiliation: MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, Nanjing 210061, China.
authors.forename: Zhangji
authors.initials: Z
authors.lastname: Dong
authors.affiliation: MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, Nanjing 210061, China.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Dong
authors.affiliation: MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, Nanjing 210061, China.
authors.forename: Wenshang
authors.initials: W
authors.lastname: Jia
authors.affiliation: MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, Nanjing 210061, China.
authors.forename: Shasha
authors.initials: S
authors.lastname: Cao
authors.affiliation: MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, Nanjing 210061, China. Electronic address: qingshun@nju.edu.cn.
authors.forename: Qingshun
authors.initials: Q
authors.lastname: Zhao
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D018703:Founder Effect; D018390:Gene Targeting / Q000379:methods*; D016678:Genome / Q000235:genetics*; D005854:Germ Cells / Q000166:cytology / Q000378:metabolism*; D054643:INDEL Mutation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D059645:Mutation Rate; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D008164:Luminescent Proteins
keywords: CRISPR/Cas9; Gene targeting; Germline transmission; Homologous recombination; Zebrafish