A meta-analysis of gRNA library screens enables an improved understanding of the impact of gRNA folding and structural stability on CRISPR-Cas9 activity
CRISPR systems are known to be inhibited by unwanted secondary structures that form within the guide RNA (gRNA). The minimum free energy of predicted secondary structures has been used in prediction algorithms. However, the types of structures as well as the degree to which a predicted structure can inhibit Cas9/gRNA activity is not well characterized. Here we perform a meta-analysis of published CRISPR-Cas9 datasets to better understand the role of secondary structures in inhibiting gRNA activity. We identify two inhibitory structures and provide estimated free energy cutoffs at which they become impactful. Further, we identify the prevalence of these structures in existing datasets. The cutoffs provided help to explain conflicting impacts of free energy values in different datasets as well as providing a guideline for future gRNA designs. Clearly define two secondary structures that inhibit CRISPR-Cas9 activity Provide free energy calculations and cutoffs at which each structure begins to inhibit activity Evaluate impact of these structures in published datasets
