Analysis of novel domain-specific mutations in the zebrafishndr2/cyclopsgene generated using CRISPR-Cas9 RNPs

AbstractNodal-related protein (ndr2) is a member of the transforming growth factor type β superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in thendr2gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development, and cyclopia. Alleles of thendr2gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen differentndr2alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced, and the remaining alleles were obtained via random insertion, gene targeting (TALEN), or unknown methods. Therefore, most mutation sites were random and could not be predicteda priori. Using the CRISPR-Cas9 system fromStreptococcus pyogenes, we targeted distinct regions in all three exons of zebrafishndr2and observed cyclopia in the injected (G0) embryos. We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted PCR amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using anin vitronuclease assay andin vivoanalysis using embryos. We describe one specific mutant allele resulting in loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain- specific mutations and provides further insights into the structure-function of thendr2gene.