Engineered Cas12a/Cpf1-Plus nuclease enables gene editing with enhanced activity and specificity

The widespread use of the Cas12a (formerly Cpf1) nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity. Currently reported Cpf1 variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting specificity of Acidaminococcus sp. Cpf1 (AsCpf1) and Lachnospiraceae bacterium Cpf1 (LbCpf1). We discovered that the engineered AsCpf1-Plus was a high-active and high-specific variant, and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCpf1-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCpf1, we also generated LbCpf1-Plus, suggesting this strategy may be generally applicable across Cpf1 orthologs. We compared Cpf1-Plus, other variants described in this study, and the reported Cpf1-HF, enCpf1, and Cpf1-ultra, and found that Cpf1-Plus outperformed other variants with a good balance for enhanced activity and improved specificity. Thus, our discoveries provide alternative AsCpf1 and LbCpf1 variants with high specificity and activity and expand the toolbox for gene editing.