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PLoS One DOI:10.1371/journal.pone.0098186

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

Publication TypeJournal Article
Year of Publication2014
AuthorsGagnon, JA, Valen, E, Thyme, SB, Huang, P, Akhmetova, L, Ahkmetova, L, Pauli, A, Montague, TG, Zimmerman, S, Richter, C, Schier, AF
JournalPLoS One
Date Published2014
KeywordsAlleles, Animals, Gene Frequency, Humans, INDEL Mutation, Mutagenesis, Mutation Rate, Oligonucleotides, RNA, Guide, Zebrafish

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.


Alternate JournalPLoS ONE
PubMed ID24873830
PubMed Central IDPMC4038517
Grant ListR01 HG005111 / HG / NHGRI NIH HHS / United States
R01 HL109525 / HL / NHLBI NIH HHS / United States
K99 HD076935 / HD / NICHD NIH HHS / United States
R21 HD072733 / HD / NICHD NIH HHS / United States
R01 GM056211 / GM / NIGMS NIH HHS / United States