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Background The CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of  Acidaminococcus  sp. Cas12a ( As Cas12a) and  Lachnospiraceae bacterium  Cas12a ( Lb Cas12a). Results We created new  As Cas12a variant termed “ As Cas12a-Plus” with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAF V600E  mutant,  As Cas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAF V600E  cancer cell killing. By introducing the corresponding substitutions into  Lb Cas12a, we also generated  Lb Cas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity. Conclusions Our discoveries provide alternative  As Cas12a and  Lb Cas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.

Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity