Analysis of novel domain-specific mutations in the zebrafish ndr2/cyclops gene generated using CRISPR-Cas9 RNPs

• Published in: bioRxiv
• Main Authors: Turner, Ashley N, Andersen, Reagan S, Bookout, Ivy E, Brashear, Lauren N, Davis, James C, Gahan, David M, Gotham, John P, Hijaz, Baraa A, Kaushik, Ashish S, Mcgill, Jordan B, Miller, Victoria L, Moseley, Zachariah P, Nowell, Cerissa L, Patel, Riddhi K, Rodgers, Mia C, Shihab, Yazen A, Walker, Austin P, Glover, Sarah R, Foster, Samantha D, Challa, Anil Kumar
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 14.03.2018
• Subjects: Alleles; Central nervous system; CRISPR; Cyclops gene; Embryogenesis; Exons; Floor plate; Forebrain; Gene targeting; Holoprosencephaly; Mutagenesis; Mutation; Nervous system; Nuclease; Pattern formation; Phenotypes; Structure-function relationships; Transcription activator-like effector nucleases; Zebrafish
• DOI: 10.1101/277715

Nodal-related protein (ndr2) is a member of the transforming growth factor type 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 the zebrafish, Danio rerio, ndr2 gene lead to similar phenotypes, including loss of medial floor plate, severe deficits in ventral forebrain development, and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, eight of which are from chemical mutagenesis, four are radiation-induced, and the remaining alleles were obtained by random insertion, gene targeting (TALEN) or unknown. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of ndr2 and 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 have small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele in detail that results in the loss of the conserved terminal cysteine coding sequences. This study is another demonstration of the utility of CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into structure-function of ndr2 gene.