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

Godden, Alice M. ORCID:, Antonaci, Marco and Wheeler, Grant N. ORCID: (2023) An Efficient CRISPR-Cas9 Method to Knock Out MiRNA Expression in Xenopus Tropicalis. In: MicroRNA Detection and Target Identification. Methods in Molecular Biology . Humana Press, pp. 231-241. ISBN 978-1-0716-2981-9

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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.

Item Type: Book Section
Additional Information: Funding: This work was supported by UKRI Biotechnology and Biological Sciences Research Council Norwich Research Park Biosciences Doctoral Training Partnership grants to AMG (BB/M011216/1) and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 860635 to MA.
Uncontrolled Keywords: crispr-cas9,knock-out,micro rna,rescue,xenopus tropicalis,molecular biology,genetics ,/dk/atira/pure/subjectarea/asjc/1300/1312
Faculty \ School: Faculty of Science > School of Biological Sciences
UEA Research Groups: Faculty of Science > Research Groups > Wheeler Group
Faculty of Science > Research Groups > Cells and Tissues
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Depositing User: LivePure Connector
Date Deposited: 03 Feb 2023 17:30
Last Modified: 21 Apr 2023 01:49
DOI: 10.1007/978-1-0716-2982-6_15

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