Knockout of microRNAs Using the CRISPR-Cas9 System with Paired Synthetic crRNAs

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9) system derived from Streptococcus pyogenes uses a Cas9 nuclease directed by a guide RNA (gRNA) to create a DNA double-strand break (DSB) at the target site. The gRNAs can be dual synthetic molecules, like the native bacterial system containing a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) (Figure 1), or a single synthetic guide RNA (sgRNA). The DSB is most often repaired by either nonhomologous end joining (NHEJ) or homology directed repair (HDR) through endogenous mechanisms within mammalian cells. NHEJ can result in insertions or deletions (indels) that produce functional gene knockouts through nonsense mutations or introduction of a stop codon. When using CRISPR-Cas9 components targeting coding genes, there are typically multiple protospacer adjacent motif (PAM) sequences (NGG for S. pyogenes) to choose from along the gene to design a gRNA. For most CRISPR Cas9 genome engineering experiments, one targeting gRNA is sufficient to generate the desired functional gene knockout. However, for some applications, it may be advantageous to use two gRNAs to generate a larger deletion and ensure gene knockout or to remove an exon, long non-coding RNA (lncRNA), or transcriptional regulatory element.