Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs

The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas9 (CRISPR associated protein 9) system has been successfully adopted as a versatile genetic tool for functional manipulations, due to its convenience and effectiveness. Genetics lesions induced by single guide RNA (gRNA) are usually small indel (insertion-deletion) DNA mutations. The impact of this type of CRISPR-induced DNA mutation on the coded mRNA transcription processing and protein translation can be complex. Unexpected or unknown transcripts, generated through alternative splicing, may impede the generation of successful loss-of-function mutants. To create null or null-like loss-of-function mutant zebrafish, we employed simultaneous multiple gRNA injection into single-cell stage embryos. We demonstrated that DNA composed of multiple exons, up to 78kb in length, can be deleted in the smarca2 gene locus. Additionally, two different genes ( rnf185 and rnf215 ) were successfully mutated in F 1 fish with multiple exon deletions using this multiplex gRNA injection strategy. We expect this approach will be useful for knock-out studies in zebrafish and other vertebrate organisms, especially when the phenotype of a single gRNA-induced mutant is not clear.