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An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.

Alice M Godden1, Marco Antonaci1, Nicole J Ward1

  • 1School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, United Kingdom.

Developmental Biology
|December 30, 2021
PubMed
Summary

CRISPR-Cas9 gene knockouts effectively disabled microRNAs (miRNAs) miR-196a and miR-219 in Xenopus, revealing their crucial roles in neural crest development and craniofacial formation.

Keywords:
CRISPR-Cas9CraniofacialNeural crestPigmentXenopusmiRNAs

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Area of Science:

  • Developmental Biology
  • Molecular Genetics
  • Stem Cell Biology

Background:

  • MicroRNAs (miRNAs) regulate gene expression post-transcriptionally.
  • Neural crest (NC) cells are vital for developing peripheral nervous system, pigment cells, and craniofacial structures.
  • Dysregulation of NC development leads to neurocristopathies.

Purpose of the Study:

  • To develop and validate a novel CRISPR-Cas9 approach for reliable miRNA knockout (KO).
  • To investigate the roles of miR-196a and miR-219 in Xenopus neural crest development.
  • To compare CRISPR-Cas9 KO with morpholino knockdown (KD) of these miRNAs.

Main Methods:

  • Designed a dual-guide RNA CRISPR-Cas9 strategy for miRNA KO.
  • Generated miR-196a and miR-219 KO in Xenopus embryos.
  • Validated KO efficiency and analyzed phenotypes using in situ hybridization, q-RT-PCR, and Sanger sequencing.
  • Performed rescue experiments with miRNA mimics to confirm specificity.

Main Results:

  • CRISPR-Cas9 KO of miR-196a and miR-219 resulted in loss of neural crest cells.
  • KO embryos exhibited altered neural plate and hatching gland development.
  • Significant craniofacial and pigment abnormalities were observed in tadpoles.

Conclusions:

  • The dual-guide RNA CRISPR-Cas9 method provides an effective means for miRNA KO.
  • miR-196a and miR-219 are essential for normal Xenopus neural crest development.
  • These miRNAs play critical roles in craniofacial and pigment cell formation.