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

Site-directed mutagenesis has been widely employed in plants due to their crucial role in gene-function studies. To expedite societal use of genome-edited crops, we aimed to develop an alternative way of plant genome editing without introducing foreign DNA into cells. DNA-free methods may alleviate regulatory concerns related to genetically modified plants. To this end, we transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice 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. Therefore, it should be better to treat the crop plants engineered with DNA-free genome editing methods as the varieties generated after conventional mutagenesis and even natural spontaneous mutations.