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.
Competing Interest Statement
The authors have declared no competing interest.
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