Targeted genome modification in protoplasts of a tea cultivar Kolkhida using RNA-guided Cas9 endonuclease

Gene-editing tools enable precise, targeted genome modifications, providing new approach for the rapid and sustainable improvement of tea plant (Camellia sinensis (L.) Kuntze). Developing such an approach is especially important due to the perennial nature and complex genetics of the tea plant, which make traditional breeding slow and inefficient. To validate a gene editing protocol in the elite local tea cultivar Kolkhida three candidate genes were selected. Two guide RNAs (gRNAs) were designed for each gene, and corresponding constructs for targeted genome modification in tea were generated. Successful modifications of the target sequences in cv. Kolkhida tea protoplasts were achieved for all three target genes. The high mutagenic efficiency of the selected gRNAs was observed for two out of three genes, including induction of precise deletions between target motifs. gRNAs were delivered in protoplasts via co-transfection technique, and combined gRNA activity was observed when transfection efficiency exceeded 28%. The genome modification method for tea protoplasts established in this study can serve as a screening protocol to evaluate the in vivo efficiency of different genome editing approaches in the tea plant.