Single-nucleotide editing for zebra3 and wsl5 phenotypes in rice using CRISPR/Cas9-mediated adenine base editors

The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break (DSB) or supplying a DNA donor template for homology-directed repair (HDR). In this study, adenine base editors (ABEs) were used for rapid generation of precise point mutations in two distinct genes, OsWSL5, and OsZEBRA3 (Z3), in both rice protoplasts and regenerated plants. The precisely engineered point mutations were stably inherited to subsequent generations. These single nucleotide alterations resulted in single amino acid changes and associated wsl5 and z3 phenotypes as evidenced by white stripe leaf and light green/dark green leaf pattern, respectively. Through selfing and genetic segregation, transgene-free, base edited wsl5 and z3 mutants were obtained in a short period of time. We noticed a novel mutation (V540A) in Z3 locus could also mimic the phenotype of Z3 mutation (S542P). Furthermore, we observed unexpected non- A/G or T/C mutations in the ABE editing window in a few of the edited plants. The ABE vectors and the method from this study could be used to simultaneously generate point mutations in multiple target genes in a single transformation and serve as a useful base editing tool for crop improvement as well as basic studies in plant biology.