Generating Zebrafish RNA-Less Mutant Alleles by Deleting Gene Promoters with CRISPR/Cas9

Danio rerio (zebrafish), traditionally used in forward genetic screens, has in the last decade become a popular model for reverse genetic studies with the introduction of TALENS, zinc finger nucleases, and CRISPR/Cas9. Unexpectedly, homozygous frameshift mutations generated by these tools frequently result in phenotypes that are less penetrant than those seen in embryos injected with antisense morpholino oligonucleotides targeting the same gene. One explanation for the difference is that some frameshift mutations result in nonsense-mediated decay of the gene transcript, a process which can induce expression of homologous genes. This form of genetic compensation, called transcriptional adaptation, does not occur when the mutant allele results in no RNA transcripts being produced from the targeted gene. Such RNA-less mutants can be generated by deleting a gene's promoter using a pair of guide RNAs and Cas9 protein. Here, we present a protocol and use it to generate alleles of arhgap29b and slc41a1 that lack detectable zygotic transcription. In the case of the arhgap29b mutant, an emerging phenotype did not segregate with the promoter deletion mutation, highlighting the potential for off-target mutagenesis with these tools. In summary, this chapter describes a method to generate zebrafish mutants that avoid a form of genetic compensation that occurs in many frameshift mutants.