Heritable Virus-Induced Germline Editing in Tomato

We report the successful implementation of heritable virus-induced genome editing (VIGE) in tomato (Solanum lycopersicum) 1 .We generated three transgenic tomato lines expressing Streptococcus pyogenes Cas9 (SpCas9) under the control of Cauliflower mosaic virus 35S, S. lycopersicum ribosomal protein S5A, or S. lycopersicum YAO promoters.These three lines were tested for somatic and heritable editing using the tobacco rattle virus (TRV)-based system carrying guide RNAs (gRNAs) fused with mobile RNA sequences.TRV with gRNA targeted to Phytoene desaturase (SlPDS) and Downy mildew resistance 6 (SlDMR6) genes fused to mobile RNA sequences showed significant somatic editing efficiency in all three tomato lines expressing SpCas9.However, the progenies from SlYAO promoter-driven SpCas9 tomato infected with TRV with gRNA targeted to SlDMR6 fused to mobile RNA sequence resulted in monoallelic mutations with a frequency of 3%.Optimization of environmental conditions, such as reduced light intensity, significantly increased heritable editing frequencies, from 0% to 86% at the SlPDS and 3% to 100% at the SlDMR6, including biallelic mutations.These findings underscore the use of appropriate promoters to express Cas nucleases and optimized environmental conditions to enhance heritable genome editing efficiency in tomato using VIGE.Furthermore, our method enables the generation of mutants without additional tissue culture or transformation once a SpCas9-expressing tomato line is established.