title: Rapid reverse genetic screening using CRISPR in zebrafish.
abstract: Identifying genes involved in biological processes is critical for understanding the molecular building blocks of life. We used engineered CRISPR (clustered regularly interspaced short palindromic repeats) to efficiently mutate specific loci in zebrafish (Danio rerio) and screen for genes involved in vertebrate biological processes. We found that increasing CRISPR efficiency by injecting optimized amounts of Cas9-encoding mRNA and multiplexing single guide RNAs (sgRNAs) allowed for phenocopy of known mutants across many phenotypes in embryos. We performed a proof-of-concept screen in which we used intersecting, multiplexed pool injections to examine 48 loci and identified two new genes involved in electrical-synapse formation. By deep sequencing target loci, we found that 90% of the genes were effectively screened. We conclude that CRISPR can be used as a powerful reverse genetic screening strategy in vivo in a vertebrate system.
authors: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors: Arish N
authors: AN
authors: Shah
authors: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors: Crystal F
authors: CF
authors: Davey
authors: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors: Alex C
authors: AC
authors: Whitebirch
authors: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors: Adam C
authors: AC
authors: Miller
authors: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors: Cecilia B
authors: CB
authors: Moens
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D004625:Embryo, Nonmammalian / Q000502:physiology*; D005786:Gene Expression Regulation; D018507:Gene Expression Regulation, Developmental; D056426:Genetic Loci; D005820:Genetic Testing / Q000379:methods*; D010858:Pigmentation / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000196:embryology; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D029961:Zebrafish Proteins