An Efficient CRISPR-Cas9 Method to Knock Out MiRNA Expression in Xenopus Tropicalis

Research output: Chapter in Book/Report/Conference proceedingChapter


In recent years CRISPR-Cas9 knockouts (KO) have become increasingly utilized to study gene function. MicroRNAs (miRNAs) are short noncoding RNAs, 20–25 nucleotides long, which affect gene expression through posttranscriptional repression. As miRNAs are so small and due to the limitations of known PAM sequences, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore developed a novel approach using two guide RNAs to effectively “drop out” a miRNA. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of q-RT-PCR and Sanger sequencing. To show specificity of the phenotype, we provide a protocol to use miRNA mimics to rescue the KO phenotype.
Original languageEnglish
Title of host publicationMicroRNA Detection and Target Identification
Subtitle of host publicationMethods and Protocols
EditorsTamas Dalmay
PublisherHumana Press
Number of pages11
ISBN (Print)978-1-0716-2981-9
Publication statusPublished - 24 Jan 2023

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
ISSN (Print)1064-3745


  • CRISPR-Cas9
  • Knock-out
  • Micro RNA
  • Rescue
  • Xenopus tropicalis

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