Poster Presentation International Plant Molecular Biology Conference 2024

RNA-derived DNA-free base editing in plants (#52)

Eunbin Lee 1 , Yunsun Kim 1 , Min ju Kim 1 , Donghui Lee 1 , Beum-Chang Kang 1
  1. Horticulture, Jeonbuk National University, Jeonju-si, Jeollabuk-do, Republic of Korea

 Clustered regularly interspaced short palindromic repeats (CRISPR)-based base editing systems precisely substitute targeted point mutations without DNA double-strand breaks (DSBs). A rapid and precise method for evaluating their substitutions is necessary to observe the desired conversions. In this study, we constructed a cytosine base editor (CBE) and adenine base editor (ABE) containing nCas9 fused with human APOBEC3A (hA3A) and engineered deoxyadenosine deaminase (TadA-8e), respectively. We targeted the lettuce acetolactate synthase (ALS) gene and flowering locus T (FT) gene, and base editor mRNAs and target sgRNAs were synthesized by in vitro transcription. Each RNA was co-transfected to protoplasts via polyethylene glycol-mediated delivery, and base editing efficiencies were measured using targeted deep sequencing on day three post-transfection. Our system induced base editing in protoplast at frequencies up to 21% and 5.1%. Base edits induced RNA-base the CBE and ABE specific to the ALS and FT genes were detected in lettuce calli with frequencies of up to 26% and 7.3%, respectively. Furthermore, we generated herbicide-resistant plants using chlorsulfuron selection by introducing a point mutation in the ALS gene.