Chapter title |
An Efficient CRISPR-Cas9 Method to Knock Out MiRNA Expression in Xenopus Tropicalis.
|
---|---|
Chapter number | 15 |
Book title |
MicroRNA Detection and Target Identification
|
Published in |
Methods in molecular biology, January 2023
|
DOI | 10.1007/978-1-0716-2982-6_15 |
Pubmed ID | |
Book ISBNs |
978-1-07-162981-9, 978-1-07-162982-6
|
Authors |
Godden, Alice M, Antonaci, Marco, Wheeler, Grant N, Godden, Alice M., Wheeler, Grant N. |
Abstract |
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. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 1 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 1 | 100% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 1 | 100% |