AUTHOR=Pan Wenbo , Liu Xue , Li Dayong , Zhang Huawei 

TITLE=Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.930592

DOI=10.3389/fpls.2022.930592

ISSN=1664-462X

ABSTRACT=<p>The efficiency of the CRISPR/Cas9 genome editing system remains limited in many crops. Utilizing strong promoters to boost the expression level of <italic>Cas9</italic> are commonly used to improve the editing efficiency. However, these strategies also increase the risk of off-target mutation. Here, we developed a new strategy to utilize intron-mediated enhancement (IME)-assisted <italic>35S</italic> promoter to drive <italic>Cas9</italic> and sgRNA in a single transcript, which escalates the editing efficiency by moderately enhancing the expression of both <italic>Cas9</italic> and sgRNA. In addition, we developed another strategy to enrich cells highly expressing <italic>Cas9</italic>/sgRNA by co-expressing the developmental regulator gene <italic>GRF5</italic>, which has been proved to ameliorate the transformation efficiency, and the transgenic plants from these cells also exhibited enhanced editing efficiency. This system elevated the genome editing efficiency from 14–28% to 54–81% on three targets tested in lettuce (<italic>Lactuca sativa</italic>) without increasing the off-target editing efficiency. Thus, we established a new genome editing system with highly improved on-target editing efficiency and without obvious increasement in off-target effects, which can be used to characterize genes of interest in lettuce and other crops.</p>