Genome editing in plants with MAD7 nuclease

Abstract MAD7 is an engineered nuclease of the class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) family with a low level of homology to canonical Cas12a nucleases. It has been publicly released as a royalty-free nuclease for both academic and commercial use. Here, we demonstrate that the CRISPR-MAD7 system can be used for genome editing, and recognizes T-rich PAM sequences (YTTN) in plants. Its editing efficiency in rice and wheat is comparable to that of the widely-used CRISPR-LbCas12a system. We developed two variants, MAD7-RR and MAD7-RVR, that increase the target range of MAD7, as well as an M-AFID (a MAD7-APOBEC fusion-induced deletion) system that creates predictable deletions from 5'-deaminated Cs to the MAD7-cleavage site. Moreover, we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs. Using the CRISPR-MAD7 system, we have obtained regenerated mutant rice and wheat plants with up to 65.6% efficiency.