Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish

Inconsistent activity limits the use of CRISPR-Cas9 in zebrafish. We show supernumerary guanine nucleotides at the 5′ ends of single guide RNAs (sgRNAs) account for diminished CRISPR-Cas9 activity in zebrafish embryos. Genomic sequences can be targeted consistently with extremely high efficiency using Cas9 ribonucleoproteins (RNPs) containing either a sgRNA molecule or a synthetic crRNA:tracrRNA duplex that perfectly matches the protospacer target site. Following injection of zebrafish eggs with such RNPs, virtually every copy of a targeted locus harbors an induced indel mutation. Loss of gene function is often complete, as F0 embryos closely resemble true null mutants without detectable non-specific effects. Mosaicism is sufficiently low in F0 embryos that cell non-autonomous gene functions can be probed effectively and redundant activities of genes can be uncovered when two genes are targeted simultaneously. Finally, heritable deletion mutations of at least 50 kbp can be readily induced using pairs of duplex guide RNPs targeted to a single chromosome. •Cas9 RNPs with synthetic crRNA:tracrRNAs are highly mutagenic in zebrafish embryos•Mutagenized F0 embryos mimic null mutants and lack confounding non-specific traits•Redundant gene activity can be uncovered, and heritable deletions can be induced•5′ guanines of gRNAs that do not complement the protospacer diminish Cas9 RNP activity Cas9 RNP complexes consisting of synthetic crRNA:tracrRNA duplex guide RNAs consistently induce mutations in virtually all copies of a targeted gene in zebrafish embryos. Hoshijima et al. show these tools allow effective screening of individual or combinations of gene function in F0 embryos and the facile induction of deletion mutations.