TadA reprogramming to generate potent miniature base editors with high precision

Abstract

Although miniature CRISPR-Cas12f systems were recently developed, the editing efficacy and targeting range of derived miniature cytosine and adenine base editors (miniCBEs and miniABEs) have not been comprehensively addressed. Moreover, functional miniCBEs have not yet be established. Here we generate various Cas12f-derived miniCBEs and miniABEs with improved editing activities and diversified targeting scopes. We reveal that miniCBEs generated with traditional cytidine deaminases exhibit wide editing windows and high off-targeting effects. To improve the editing signatures of classical CBEs and derived miniCBEs, we engineer TadA deaminase with mutagenesis screening to generate potent miniCBEs with high precision and minimized off-target effects. We show that newly designed miniCBEs and miniABEs are able to correct pathogenic mutations in cell lines and introduce genetic mutations efficiently via adeno-associated virus delivery in the brain in vivo. Together, this study provides alternative strategies for CBE development, expands the toolkits of miniCBEs and miniABEs and offers promising therapeutic tools for clinical applications.