Genome-wide investigation of multiplexed CRISPR-Cas12a-mediated editing in rice

We previously reported highly specific genome editing in rice by CRISPR-Cas9 and Cas12a with single DNA double strand break (DSB) (Tang et al., 2018). Two concurrent DSBs by CRISPR-Cas9 could generate defined deletions (Zhou et al., 2014), inversions (Schmidt et al., 2020), and translocations (Beying et al., 2020). Off-target effects of many simultaneous DSBs are unknown in plants. Here, we used whole-genome sequencing (WGS) to investigate off-target effects in rice plants edited by highly multiplexable CRISPR-Cas12a systems (Zhang et al., 2021). Our WGS study revealed highly specific multiplexed genome editing by CRISPR-Cas12a. We found low-frequency large chromosomal deletion and duplication events in plants that endured many (e.g., >50) simultaneous DSBs, but not in plants that endured a lower order DSBs (e.g., <10). While our short reads sequencing may not capture all chromosomal rearrangements, our results nevertheless shed important light on the analysis and regulation of engineered crops derived from multiplexed genome editing.