Multiplex genome editing in Arabidopsis thaliana using Mb3Cas12a

The use of CRISPR‐Cas proteins for the creation of multiplex genome engineering represents an important avenue for crop improvement, and further improvements for creation of knock‐in plant lines via CRISPR‐based technologies may enable the high‐throughput creation of designer alleles. To circumvent limitations of the commonly used CRISPR‐Cas9 system for multiplex genome engineering, we explored the use of Moraxella bovoculi 3 Cas12a (Mb3Cas12a) for multiplex genome editing in Arabidopsis thaliana. We identified optimized cis‐regulatory sequences for driving expression of single‐transcript multiplex crRNA arrays in A. thaliana, resulting in stable germline transmission of Mb3Cas12a‐edited alleles at multiple target sites. By utilizing this system, we demonstrate single‐transcript multiplexed genome engineering using of up to 13 crRNA targets. We further show high target specificity of Mb3Cas12a‐based genome editing via whole‐genome sequencing. Taken together, our method provides a simplified platform for efficient multiplex genome engineering in plant‐based systems.