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

  • Ashley N. Turner
  • Reagan S. Andersen
  • Ivy E. Bookout
  • Lauren N. Brashear
  • James C. Davis
  • David M. Gahan
  • John P. Gotham
  • Baraa A. Hijaz
  • Ashish S. Kaushik
  • Jordan B. Mcgill
  • Victoria L. Miller
  • Zachariah P. Moseley
  • Cerissa L. Nowell
  • Riddhi K. Patel
  • MIA C. Rodgers
  • Yazen A. Shihab
  • Austin P. Walker
  • Sarah R. Glover
  • Samantha D. Foster
  • Anil K. Challa
Research Article
  • 5 Downloads

Abstract

Nodal-related protein (ndr2) is a member of the transforming growth factor type \(\upbeta \) 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 (\(\hbox {G}_{0}\)) embryos. We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (\(\hbox {G}_{0}\)) embryos. Targeted polymerase chain reaction 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 the 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 the ndr2 gene.

Keywords

zebrafish nodal-related protein ndr2 gene  cyclops gene CRISPR-Cas9 ribonucleoprotein complex ribonucleoprotein 

Notes

Acknowledgements

The authors thank the staff of the UAB Zebrafish Research Facility for providing care and maintenance of the facility, Dr Robert Kesterson and the UAB Transgenic & Genetically Engineered Models (TGEMs) Core facility for support, Dr Brad Yoder (CDIB, UAB) for providing laboratory space, Dr Michael Miller (CDIB, UAB) for help with microscopy, Dr Ben Johnson (VAI) and Dr David Crossman (Genetics, UAB) for help with sequence analysis, Kartik Manne for advice on structure prediction using PyMol, Ansuya Jogi and Dr Kiranam Chatti for editing and proofreading and the administrative support of the Science and Technology Honors (STH) Program. The authors also thank Dr Oreoluwa Adedoyin (MERIT Postdoctoral Scholar) and Lindsay Jenkins (STH undergraduate student) for assistance in the course. AKC thanks Dr Diane Tucker (STH Program) for advice on designing the CURE. This work was supported by a Teaching Innovation Grant to AKC by the Quality Enhancement Program (QEP) in the Center for Teaching & Learning, and the Department of Genetics, University of Alabama at Birmingham (UAB). This study was part of a Course-based Undergraduate Research Experience (CURE) for first year undergraduate students in the Science and Technology Honors (STH) Program at UAB. The STH Program provided support for materials and reagents.

Supplementary material

12041_2018_1033_MOESM1_ESM.docx (4.4 mb)
Supplementary material 1 (docx 4547 KB)

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Copyright information

© Indian Academy of Sciences 2018

Authors and Affiliations

  • Ashley N. Turner
    • 1
    • 2
  • Reagan S. Andersen
    • 3
  • Ivy E. Bookout
    • 3
  • Lauren N. Brashear
    • 3
  • James C. Davis
    • 3
  • David M. Gahan
    • 3
  • John P. Gotham
    • 3
  • Baraa A. Hijaz
    • 3
  • Ashish S. Kaushik
    • 3
  • Jordan B. Mcgill
    • 3
  • Victoria L. Miller
    • 3
  • Zachariah P. Moseley
    • 3
  • Cerissa L. Nowell
    • 3
  • Riddhi K. Patel
    • 3
  • MIA C. Rodgers
    • 3
  • Yazen A. Shihab
    • 3
  • Austin P. Walker
    • 3
  • Sarah R. Glover
    • 3
  • Samantha D. Foster
    • 3
  • Anil K. Challa
    • 1
  1. 1.Department of GeneticsUniversity of Alabama at BirminghamBirminghamUSA
  2. 2.Graduate Biomedical Sciences Program (Genetics, Genomics and Bioinformatics Theme)BirminghamUSA
  3. 3.Science and Technology Honors Program, University of Alabama at BirminghamBirminghamUSA

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