Guidelines for C to T base editing in plants: base-editing window, guide RNA length, and efficient promoter
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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.
KeywordsBase editor CRISPR-Cas9 Brassica napus Glycine max Nicotiana tabacum Herbicide resistance
B-CK, JW, S-TK, J-SK, S-GK designed experiments; B-CK, JW, S-JB performed plant transformation and tissue culture; B-CK, MC made vector constructs; B-CK, S-TK analyzed deep sequencing data; B-CK and S-GK mainly wrote the manuscript; J-SK, S-GK contributed to the revision of the manuscript and oversaw the project. This work was supported by Institute for Basic Science (IBS-R021-D1) and the Next-Generation BioGreen 21 Program (PJ01322603 and PJ01311601) provided by the Rural Development Administration to S-GK. We thank Emily Wheeler, Boston, for editorial assistance and Hyeran Kim for critical discussion.
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Conflict of interest
The authors declare that they have no competing interests.
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