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Should I use single gRNA or dual gRNA for CRISPR-mediated knockout?

For CRISPR-mediated genome editing, Cas9 nuclease is directed to the target site of site-specific guide RNA (gRNA) in the genome to create DNA cleavage. In most cases, to generate simple gene knockout, a single gRNA can be used together with Cas9 to generate a double-strand break (DSB), which is then inefficiently repaired by the non-homologous end joining (NHEJ), resulting in permanent mutations, such as small insertions or deletions, at the site of repair. A subset of these mutations will result in loss of function of the gene of interest due to frame-shifts, premature stop codons, etc.

Dual gRNAs can be used if Cas9_D10A nickase is being used to target the two opposite strands of a single target site. In this approach, the nickase enzyme will generate single strand cuts on both strands, one guided by each of the two gRNAs, resulting in DSBs at the target site. Generally, this method reduces off-target effects of CRISPR/Cas9 expression because targeting by both gRNAs is necessary for DSBs to be generated.

Dual gRNAs can also be used when Cas9_D10A nickase and an exogenous donor DNA template are being used to introduce specific base-changes (e.g. knockins) into a gene of interest. In this approach, the two opposite strands would be targeted by the two gRNAs at two sites flanking the desired mutation site, and homology-directed repair (HDR) pathways make use of the exogenous donor template to repair the excised sequence.

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