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.
|Title of host publication||MicroRNA Detection and Target Identification|
|Subtitle of host publication||Methods and Protocols|
|Number of pages||11|
|Publication status||Published - 24 Jan 2023|
|Name||Methods in Molecular Biology|
- Micro RNA
- Xenopus tropicalis