title: Heritable and precise zebrafish genome editing using a CRISPR-Cas system.
abstract: We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications.
authors.affiliation: Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America.
authors.forename: Woong Y
authors.initials: WY
authors.lastname: Hwang
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Fu
authors.forename: Deepak
authors.initials: D
authors.lastname: Reyon
authors.forename: Morgan L
authors.initials: ML
authors.lastname: Maeder
authors.forename: Prakriti
authors.initials: P
authors.lastname: Kaini
authors.forename: Jeffry D
authors.initials: JD
authors.lastname: Sander
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.forename: Randall T
authors.initials: RT
authors.lastname: Peterson
authors.forename: Jing-Ruey Joanna
authors.initials: JR
authors.lastname: Yeh
mesh_terms: D000818:Animals; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000235:genetics; D064113:CRISPR-Cas Systems; D005818:Genetic Engineering; D016678:Genome; D018095:Germ-Line Mutation; D054643:INDEL Mutation; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D059645:Mutation Rate; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D015027:Zebrafish / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D009841:Oligonucleotides