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

aBIOTECH 2020年第2期1卷 106-118页

作者： Kutubuddin A.Molla Justin Shih Yinong Yang Department of plant Pathology and Environmental Microbiology and the Huck Institute of the Life Sciences The Pennsylvania State UniversityUniversity ParkPA 16802USA ICAR-National Rice Research Institute Cuttack 753006India

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 *** precisely engineered point mutations were stably inherited to subsequent *** 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,*** selfing and genetic segregation,transgene-free,base edited wsl5 and z3 mutants were obtained in a short period of *** 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 *** 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.

关键词： Adenine base editor CRISPR/Cas9 genome editing plant base editing Precise point mutation Transgene-free rice

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