Guidelines for C to T base editing in plants: base-editing window, guide RNA length, and efficient promoter

The Cas9 protein fused with a cytidine deaminase can induce C to T substitutions at a specific site when directed by a guide RNA. Here, we compared the substitution activity and the substitution range of two base-editing systems, APOBEC1-nCas9 and nCas9-PmCDA1, in the protoplasts of Glycine max, Brassica napus, and Nicotiana tabacum. To prevent unwanted nucleotide substitution, we manipulated the length of guide RNA and found the change of nucleotide substitution activity in the target window of nCAS9-PmCDA1. Based on these results, the specific C to T conversion in the acetolactate synthase gene of N. tabacum was induced to generate herbicide-resistant plants. During the screening of herbicide-resistant plants, we found that ubiquitin promoter-driven base-editor system was much efficient than 35S promoter-driven base-editor system. This study provides guidelines on which a base editor to use and describes how to fine-tune a guide RNA for precise substitutions in plants.