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

Abstract

Nodal-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 the ndr2 gene 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 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, 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 predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish 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 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 an in vitro nuclease assay and in vivo analysis 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 domainspecific mutations and provides further insights into the structure-function of the ndr2 gene..

Student Perceptions of Authoring a Publication Stemming from a Course-Based Undergraduate Research Experience (CURE)

“…The manuscript was submitted to bioRxiv as a preprint article in March 2018 ( Turner et al. , 2018b ), submitted for peer review in April 2018, accepted in June 2018, and published online in December 2018 ( Turner et al. , 2018a ).…”

“…Specifically, it is unknown to what extent students perceive that they benefit from CURE publications and how they view authorship, especially in the context of a CURE. To begin to address these gaps in the literature, we conducted an in-depth interview study of students enrolled in a CURE that resulted in a CURE publication ( Turner et al. , 2018a ).…”

Generation of a GLO-2 deficient mouse reveals its effects on liver carbonyl and glutathione levels

“…DNA oligonucleotide primers used for PCR and the expected amplicon sizes are provided in Table 1 . The PCR amplicons were analyzed by polyacrylamide gel electrophoresis and products with heteroduplexes were cloned and sequenced using the Sanger method to obtain the mutant allele information (previously described in detail [ 15 ]). Pronuclear injections of zygotes and their transfer into pseudo-pregnant female mice resulted in a litter of 19 pups.…”

Physiological and metabolic characteristics of novel double‐mutant female mice with targeted disruption of both growth hormone‐releasing hormone and growth hormone receptor

“…we aimed at identifying a high scoring (low off-target) site in one of the early exons so as to generate indels that can create a loss-offunction allele; we found a target site in exon 4. Single guide RNA (sgRNA) molecules were generated using a cloning-free method as described earlier (Turner et al, 2018). Cas9 protein was obtained from MacroLabs at UC Berkeley.…”