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• W4200402520 abstract "Genome editing using CRISPR/Cas is rapidly being developed for gene targeting in eukaryotes including plants. However, gene targeting by homology-directed DNA recombination (HDR) is an infrequent event compared to the dominant DNA repair by non-homologous end-joining. Another bottleneck is the ineffective delivery of CRISPR/Cas components into plant cells. To overcome these constraints, here a geminiviral replicon from Beet curly top virus (BCTV) has been produced with a wide host range and high DNA accumulation capacity for efficient delivery of CRISPR/Cas12a components into plant cells. Initially, a BCTV replicon was prepared after removing the virion sense genes from an infectious full-length clone for agrobacterium mediated infection. This replicon expressed a green fluorescent protein (GFP) marker gene at a high level compared to T-DNA binary vector. In transient assay, the BCTV replicon produced a higher rate of mutagenesis and HDR in the GFP transgene in Nicotiana benthamiana through efficient delivery of CRISPR/Cas12a components compared to the cognate T-DNA control. This was through a range of complete or partial HDR for conversion of GFP into YFP after exchange of a single amino acid (Thr224Tyr) in the target gene. In addition, induced mutagenesis and HDR in the target gene were heritable. Thus, the BCTV replicon provides a new tool for efficient delivery of CRISPR/Cas12a components that could be used in a wide range of dicotyledonous plants. The established GFP to YFP system and the GFP mutant line produced also enable further optimization and understanding of HDR in plants via CRISPR/Cas12a system using geminiviral replicons." @default.
• W4200402520 created "2021-12-31" @default.
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• W4200402520 date "2022-03-01" @default.
• W4200402520 modified "2023-09-25" @default.
• W4200402520 title "Targeted mutagenesis in plants using Beet curly top virus for efficient delivery of CRISPR/Cas12a components" @default.
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• W4200402520 doi "https://doi.org/10.1016/j.nbt.2021.12.002" @default.
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