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.
Highlights
There is an urgent need for precise, predictable, inexpensive, and easy-to-use genome engineering tools that are applicable to a wide range of model organisms and cell types
We used the previously described 59GG-N18-NGG single-guide RNAs (sgRNAs) architecture, while relaxing the first two bases to allow 59AG-N18NGG or 59GA-N18-NGG target sites for some loci, since these were previously suggested to be acceptable as initiating bases for T7 RNA polymerase [9]
Cas9 mRNA and sgRNA were co-injected into zebrafish zygotes, survival was scored at 24–30 hours postfertilization and genomic DNA was prepared from injected and uninjected embryos
Summary
There is an urgent need for precise, predictable, inexpensive, and easy-to-use genome engineering tools that are applicable to a wide range of model organisms and cell types. The CRISPR RNA and the trans-activating RNA can be fused to generate a single-guide RNA (sgRNA) sufficient for site-directed cleavage of target DNA [4] This simplified system has been implemented in a variety of in vivo settings, resulting in efficient mutagenesis by NHEJ-mediated small insertions or deletions [5,6,7,8,9]. A primary advantage of the Cas method over existing methods is that mutagenesis can be directed to diverse genomic locations by exchanging the sgRNA, without the need to reengineer the Cas enzyme This flexibility and the published rates of mutagenesis (comparable or superior to ZFNs and TALENs) make Cas an attractive system for site-directed mutagenesis. These questions by targeting a large number of genomic loci in zebrafish using an optimized Cas system and detecting mutations with next-generation sequencing
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