Expanding plant gene editing scope with highly multiplexable Cas12a systemsg

Yiping QiM.1

** Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA.

yiping@umd.edu

Abstract

CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Compared to Cas9, Cas12a has shown higher targeting specificity in plants. Unlike Cas9 that usually generates small deletions, Cas12a cleavage generates staggered ends resulting in larger deletions, making it a suitable nuclease for gene knockout. Moreover, Cas12a only requires a short CRISPR RNA (crRNA) for each target and possesses RNase activity for crRNA array processing, making it an ideal platform for multiplexed editing. Cas12a has been widely applied in plants and achieved high editing efficiencies for single gene targets. However, comprehensive genome engineering using CRISPR usually requires simultaneous targeting of multiple genes at defined locations, which cannot be achieved easily and efficiently with current Cas12a systems, due to their strict PAM (protospacer adjacent motif) requirements and limited multiplex capacity. To expand the targeting scope of Cas12a, we screened nine new Cas12a orthologs and identified six that possess high editing activity and specificity in rice. Among them, Mb2Cas12a stands out with high editing efficiency, relaxed PAM requirements and tolerance to low temperatures. Engineered Mb2Cas12a can also target altered PAMs. These new Cas12a systems have greatly expanded the targeting scope of Cas12a in major crops (rice, maize and wheat). To further enable large-scale genome engineering, we compared 12 multiplexed Cas12a systems and identified a potent system that exhibited nearly 100% biallelic editing efficiency with the ability to target as many as 14 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems were also developed for multi-gene repression in plants. This study has greatly expanded the targeting scope of Cas12a for crop genome engineering.

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Keywords: CRISPR-Cas12a, PAM, multiplexed editing, plant gene editing