An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus

• Published in: Developmental biology Vol. 483; pp. 66 - 75
• Main Authors: Godden, Alice M., Antonaci, Marco, Ward, Nicole J., van der Lee, Michael, Abu-Daya, Anita, Guille, Matthew, Wheeler, Grant N.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2022; Elsevier
• Subjects: Animals; Craniofacial; CRISPR-Cas Systems; CRISPR-Cas9; Gene Expression Regulation, Developmental; Gene Knockdown Techniques - methods; Gene Knockout Techniques - methods; In Situ Hybridization - methods; MicroRNAs - genetics; miRNAs; Morpholinos - genetics; Neural crest; Neural Crest - embryology; Neural Crest - metabolism; Neural Plate - embryology; Neural Plate - metabolism; Neurulation - genetics; Phenotype; Pigment; RNA, Guide, CRISPR-Cas Systems - genetics; Transcription Factors - genetics; Transcription Factors - metabolism; Transcriptome - genetics; Xenopus; Xenopus laevis - embryology; Xenopus laevis - genetics; Xenopus Proteins - genetics; Xenopus Proteins - metabolism; Xenopus; MO; miRNAs; KO; Neural crest; INDEL; Pigment; NC; sgRNA; CRISPR-Cas9; NF; KD; Craniofacial
• ISSN: 0012-1606; 1095-564X
• DOI: 10.1016/j.ydbio.2021.12.015

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20–22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and −219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively ‘drop out’ a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO’s both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes. [Display omitted] •We have developed a novel CRISPR-Cas9 approach to KO miRNAs.•Loss of miR-196a leads to loss of pigment phenotypes.•Loss of miR-219 leads to craniofacial phenotypes.•MiRNAs can affect the development of Xenopus neural crest.