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PLoS One. 2014 May 29;9(5):e98186. doi: 10.1371/journal.pone.0098186. eCollection 2014.

Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

Author information

1
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America.
2
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.
3
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.

Erratum in

  • PLoS One. 2014;9(8):e106396. Ahkmetova, Laila [corrected to Akhmetova, Laila].

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.

PMID:
24873830
PMCID:
PMC4038517
DOI:
10.1371/journal.pone.0098186
[Indexed for MEDLINE]
Free PMC Article
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