CRISPR-Cas9 is a powerful tool for making targeted mutagenesis. To provide more insights into the efficiency of CRISPR in creating stable homozygous mutants, the team of Lien Bertier from the University of California targeted LsNCED4, a Ggene controlling thermoinhibition of seed germination in lettuce (Lactuca sativa).
Three constructs, each harboring a different single gRNA targeting a different site in LsNCED4, were transformed into lettuce cultivars Salinas and Cobham Green. Analysis of the transformants revealed that 57% of transformants contained mutations at the target site. Editing efficiency was similar in both cultivars. Analysis of T1 and T2 plants for each of the three gRNAs showed that the mutations developed were reproducible and characteristic for each gRNA.
Knockouts of NCED4 resulted in significant increases in the maximum temperature for seed germination, with seeds capable of germinating at 37°C. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal if not zero consequences.
Targeting NCED4 could be used to enrich for germline-edited lettuce events by germinating seeds at high temperature.

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