CRISPR-Cas systems provide powerful tools for genome editing in both eukaryotic and bacterial organisms. Cas9 cleavage is dependent on a guide RNA (gRNA) that matches a target site adjacent to a protospacer adjacent motif (PAM). The result is a relatively easy method for directing Cas9 cleavage to new target sites by changing the targeting sequence in the gRNA. However, Cas9 activity is highly variable in a gRNA-specific manner. Algorithms have been developed to predict gRNA activity and while often successful in their training context, these algorithms are not good at predicting gRNA activity in new species or contexts. We therefore collect and re-analyze 20 gRNA library datasets and highlight context specific differences to explain why current predictions do not generalize well. We then provide potential strategies for understanding the impact of new contexts in sequence specific gRNA activity and/or mitigating the effects of these contexts.

Highlighting the sequence specific impact of context on CRISPR-Cas9 gRNA activity

ACS Fall 2021

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