Expanding Cas12a Utility in Plants

CRISPR-based genome editing tools enable a major leap forward in precision plant breeding because they provide extensive control of sequence variation at native trait genes. Cas12a (formerly Cpf1) enzymes have unique advantages compared to Cas9, including shorter guide sequences and the ability to mature their own CRISPR RNAs (crRNAs) from various transcript structures. To maximize access to important trait targets it is important to minimize sequence requirements inherent in the editing tool. However, Cas12a protospacer adjacent motif (PAM) requirements limit the ability to access trait targets and most orthologues exhibit poor activity in plants. Here we expand Cas12a PAM accessibility in plants with an Acidaminococcus sp. BV3L6 Cas12a engineered variant, enAsCas12a. Using this variant, we enhance total corn and soybean genome coding region accessibilities to 8.5 and 5.9 times higher compared to wild type LbCas12a, bringing average target density with Cas12a variants to 1.2 and 2.1 times higher than wild type SpCas9, respectively. This enables broad use of Cas12a-based tools for editing trait targets in plant product and trait discovery pipelines.