title: Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.
abstract: The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: James A
authors.initials: JA
authors.lastname: Gagnon
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Eivind
authors.initials: E
authors.lastname: Valen
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Summer B
authors.initials: SB
authors.lastname: Thyme
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America; Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Peng
authors.initials: P
authors.lastname: Huang
authors.forename: Laila
authors.initials: L
authors.lastname: Akhmetova
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Laila
authors.initials: L
authors.lastname: Ahkmetova
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Andrea
authors.initials: A
authors.lastname: Pauli
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Tessa G
authors.initials: TG
authors.lastname: Montague
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Steven
authors.initials: S
authors.lastname: Zimmerman
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Constance
authors.initials: C
authors.lastname: Richter
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America; Center for Brain Science, Harvard University, Cambridge, Massachusetts, United States of America; Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, United States of America; Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Schier
mesh_terms: D000483:Alleles; D000818:Animals; D005787:Gene Frequency; D006801:Humans; D054643:INDEL Mutation; D016296:Mutagenesis; D059645:Mutation Rate; D009841:Oligonucleotides / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism
chemical_list: D009841:Oligonucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems