DNA-Free Genome Editing in Lettuce with Preassembled CRISPR-Cas9 Ribonucleoproteins

As an important fresh vegetable crop, lettuce awaits various trait enhancements. We have sought alternative ways of molecular breeding based on CRISPR/Cas9 technology. Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of lettuce and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.