Efficiency and Inheritance Patterns of CRISPR/CAS9-Induced Gene Modifications in Lettuce (L. sativa)

The efficiency of CRISPR/CAS9 targeted mutagenesis in lettuce through Agrobacterium-mediated stable transformation was evaluated by targeting a gene (LsNCED4) essential for thermo-inhibition of seed germination in lettuce. Three constructs, each containing a single gRNA targeting LsNCED4, were transformed into lettuce cv. Salinas and cv. Cobham Green. Analysis of 50 T1 plants by deep amplicon sequencing on an Illumina platform resulted in an overall efficiency of 54% (27) plants with editing at the target site; 28% (14) plants showed mono- or bi-allelic editing in the T1 primary transformants. Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. The phenotype/genotype correlation was assayed by germinating T2 families of 38 T1 lines at high temperature and subsequently genotyping thermosensitive and thermotolerant T2 plants by amplicon sequencing. In both Cobham Green and Salinas, virtually no germination (<3%) occurred in T2 families derived from wildtype T1 plants, whereas germination was high (>50%) in T2 families derived from bi-allelic T1 plants. Germination at a temperature gradient (28-39°C) revealed a genotype-specific temperature sensitivity. Cobham Green wildtype seeds are completely inhibited at 32°C, while NCED4 mutants can germinate up to 80% at 39°C. Salinas wildtype seeds are more tolerant to high temperature and are only completely inhibited completely at 37°C, while NCED4 mutants can germinate up to 90%. In conclusion, the CRISPR/CAS9 system was highly efficient in generating mono- and bi-allelic mutants in one generation using Agrobacterium-mediated stable integration of an expression vector constitutively expressing CAS9 and one sgRNA.