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

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

Cas9 generates minimal allele diversity.

<p>Target sites and alleles are shown for four genes for pools of embryos or single embryos (n = 12 per target with Cas9 mRNA, n = 12 per target with Cas9 protein). For each target site, the top panel is from a pool of embryos while the bottom panel represents alleles from single embryos. Each plot indicates the observed mutant alleles using arches that connect the bases surrounding the deletion. In the top panel, the y-axis and color of the arch indicate allele abundance; for ease of visualization, the bottom panel indicates allele abundance only with color. PAM is indicated on the DNA sequence in uppercase. g<i>ria3a</i> sgRNA induces only low indel frequencies when injected with Cas9 mRNA.</p

5′GG dinucleotide pair and promoter choice influence sgRNA activity.

<p><b>A.</b> Indel frequencies of 5′GG sgRNAs modified with the addition of a 5′adenine. <b>B.</b> Indel frequencies of sgRNAs modified by substituting the 5′ dinucleotide as indicated. <b>C.</b> Indel frequencies of sgRNAs transcribed using either T7 (repeated data from above) or SP6 polymerase. Altered bases to the template are indicated in bold, mismatched bases to the genome are underlined.</p

Overcoming allele biases by inserting stop codon oligonucleotide with injected Cas9/sgRNA complexes.

<p><b>A.</b> Diagram of Cas9/sgRNA complex injection. <b>B.</b> Purified His-tagged Cas9 protein complexed with sgRNA (n = 12 embryos per target) or Cas9 mRNA and sgRNA (n = 12 embryos per target) were injected and indel frequency measured in single embryos at 1 dpf. <b>C.</b> Diagram of a stop codon oligonucleotide and genomic target site for <i>mezzo</i>. <b>D.</b> Clutches of embryos from adult injected fish were screened by PCR for germline transmission using PCR with a gene-specific primer and a primer specific to the inserted sequence (top panel), or two primers specific to the genome (bottom panel) as a positive control. Specific products indicated with arrows, non-specific product indicated with an asterix. <b>E.</b> DNA sequence of a <i>mezzo</i> insertion allele transmitted through the germline, 6/16 embryos in this clutch contained this mutation.</p