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Cas9 enzyme is mediated by the coordinated functions of two catalytic domains. Alanine substitution in these domains creates two Cas9 nickase variants:
- Variant D10A produces a nick on the targeting strand,
H840A nicks the non-targeting strand.
This double nicking strategy can help reduce off-target effect.
However, the need for two guide RNAs to function simultaneously can make nickase experiments more complicated than standard CRISPR-Cas9 editing.
In this webinar, Shuqi Yan and Mollie Schubert characterize factors that affect the efficiency of cooperative nicking in cell cultures. They also summarize key design and planning considerations for achieving efficient gene editing or homology directed repair (HDR).
The cleavage activity of the