Improving the efficiency of CRISPR-Cas12a-based genome editing with site-specific covalent Cas12a-crRNA conjugates.

The CRISPR-Cas12a system shows unique features compared with widely used Cas9, making it an attractive and potentially more precise alternative. However, the adoption of this system has been hindered by its relatively low editing efficiency. Guided by physical chemical principles, we covalently conjugated 5' terminal modified CRISPR RNA (crRNA) to a site-specifically modified Cas12a through biorthogonal chemical reaction. The genome editing efficiency of the resulting conjugated Cas12a complex (cCas12a) was substantially higher than that of the wild-type complex. We also demonstrated that cCas12a could be used for precise gene knockin and multiplex gene editing in a chimeric antigen receptor T cell preparation with efficiency much higher than that of the wild-type system. Overall, our findings indicate that covalently linking Cas nuclease and crRNA is an effective approach to improve the Cas12a-based genome editing system and could potentially provide an insight into engineering other Cas family members with low efficiency as well.