Development of a CRISPR-Cas9-Based Multiplex Genome-Editing Vector and Stay-Green Lettuce

Lettuce (Lactuca sativa) is one of the most popular vegetables in the Asteraceae family. We recently succeeded in modifying the lettuce genome using the CRISPR-Cas9 system, targeting two chlorophyll metabolism-related genes: STAY-GREEN (SGR) and BALANCE of CHLOROPHYLL METABOLISM (BCM). SGR encodes an Mg-dechelatase, which catalyzes chlorophyll a (Chla) degradation. BCM acts as a scaffold protein to control Chl levels by regulating both Chl synthesis and degradation. Lettuce genome contains a single copy of both SGR (LsSGR) and BCM (LsBCM). We developed a set of plasmid vectors for multiplex genome editing using CRISPR-Cas9 system, which involves the tandem cloning of guide RNA cassettes using restriction enzymes and Gateway-based transfer of the cassettes to binary vectors. Using this genome-editing system, we obtained single mutants of lssgr or lsbcm and lssgr lsbcm double mutant in a single transformation with high efficiency, indicating that this system works in lettuce very well. Food waste is a serious problem worldwide, and approximately half of such waste is comprised of fruits and vegetables. Our lssgr lettuce mutant exhibited a stay-green phenotype, which could help reduce postharvest food waste. In addition, our genome-editing system worked efficiently in two distantly related species, lettuce and Arabidopsis, suggesting that it may be applicable to a wide range of plant species across families.