Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs

By James A. Gagnon, Eivind Valen, Summer B. Thyme, P. Huang, Laila Ahkmetova, A. Pauli, Tessa G. Montague, Steven R. Zimmerman, Constance Richter, A. Schier. PLoS ONE, 2014

873 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/47e756260eb9b4392b14cce1027dbc37a4330aa7

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Citations in the corpus

Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR (2016, background): “This is consistent with previous reports that the Doench score is not accurate in zebrafish [31, 40].”
WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system (2015, background): “The importance of gRNA efficacy has only been appreciated very recently, with multiple studies attempting to identify sequence features that are relevant to functionally active sgRNAs [21, 25–28].”
CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering (2016, background): “To address these issues, research has focused on identifying the sequence features that contribute to effective (and ineffective) sgRNAs (11–16), as well as the development of new CRISPR variants that expand the targeting range and specificity of the nuclease (17–20).”
Reverse genetic screening reveals poor correlation between Morpholino-induced and mutant phenotypes in zebrafish (2014, methodology): “The sgRNA was synthesized and used to generate mutant founders based on published protocols (Gagnon et al., 2014).”
Whole organism lineage tracing by combinatorial and cumulative genome editing (2016, methodology): “sgRNAs specific to each site of the v6 or v7 array were generated as previously described (54), 736 except that sgRNAs were isolated after transcription by column purification (Zymogen).”
CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing (2019, background): “Surprisingly, it has been shown that double-strand break (DSB) repairs are not random: Cas9-induced DSBs using the same sgRNA often give rise to the same mutations (23).”
CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing (2014, background): “In addition, for CRISPR/Cas9 mode, the results are ranked by: (iii) GC-content, and (iv) the presence of a guanine at position 20 in the sgRNA target site (14,15).”