Functional knockout of a microRNA using CRISPR-Cas9
CRISPR-Cas9 can be used to knockout specific microRNAs in cell lines
CRISPR-Cas9 utilizes guide RNA to target the Cas9 nuclease to a specific sequence of DNA and induce a double-strand break (DSB) at the desired site. Repair of these DSBs can result in random insertions, deletions or changes in the DNA sequence, and this process has been widely utilized to facilitate functional knockout of protein coding genes. However, when targeting CRISPR-Cas9 to microRNA genes, a single site of gene editing may not be sufficient to disrupt microRNA stem-loop structure and function. To overcome this potential limitation, paired guide RNAs targeting both 5’ and 3’ of the precursor (pre)microRNA region have been shown to result in larger deletions, thus resulting in removal of the stem-loop that contains the mature microRNA necessary for function.
In an application note on knockout of microRNA, we utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but our goal was to use two crRNAs to remove the entire stem-loop.
The CRISPR Design Tool was used to rapidly design and order crRNAs targeting the genomic sites flanking the pre-miR-hsa-122. Transfection with paired crRNAs resulted in deletion of the pre-hsa-miR-221 DNA as assessed by Sanger sequencing and consequently multiple clonal cell lines were generated. Critically, cell lines containing deletions or alterations in the DNA region encoding hsa-mir-221 displayed decreased function as assessed by a luciferase reporter assay.
Therefore, this approach utilizing two crRNAs to generate deletions of the pre-microRNA genomic DNA is an effective strategy for functional microRNA knockout. Importantly, this strategy yields a high percentage of clonal cell lines containing the desired deletion.
- A paired guide RNA approach resulted in functional knockout of hsa-miR-221 in U2OS cells
- Multiple guide RNA pairs showed similar editing efficiency without displaying toxicity
- Seven of nine randomly tested clonal cell lines showed a disruption of the targeted genomic region, five of which contained confirmed deletions of the pre-hsa-miR-221 genomic DNA
These data show that using the CRISPR-Cas9 system with two crRNAs which target 5’ and 3’ of the pre-microRNA genomic sequence results in functional microRNA knockout.