Abstract
Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived tools recently developed to direct a C-to-T base conversion. Stable genomic integration of CRISPR/Cas9 components through Agrobacterium-mediated transformation is the most widely used approach in dicotyledonous plants. However, elimination of foreign DNA may be difficult to achieve, especially in vegetatively propagated plants. In this study, we targeted the acetolactate synthase (ALS) gene in tomato and potato by a CBE using Agrobacterium-mediated transformation. We successfully and efficiently edited the targeted cytidine bases, leading to chlorsulfuron-resistant plants with precise base edition efficiency up to 71% in tomato. More importantly, we produced 12.9% and 10% edited but transgene-free plants in the first generation in tomato and potato, respectively. Such an approach is expected to decrease deleterious effects due to the random integration of transgene(s) into the host genome. Our successful approach opens up new perspectives for genome engineering by the co-edition of the ALS with other gene(s), leading to transgene-free plants harboring new traits of interest.
Highlights
Genome editing tools, mostly based on the CRISPR/Cas9 system, have rapidly emerged in plants for gene function analysis and to improve agronomical traits
double-strand breaks (DSBs) can be repaired by an error-prone non-homologous end-joining (NHEJ) mechanism that may result in small insertions or deletions in the targeted sequence, leading to gene knock-out, an essential tool for gene function analysis
Because Agrobacterium is sometimes used for transient expression of transcriptional units located on the transfer DNA (T-DNA) [19], we developed a specific selection protocol in order to exploit this potentiality to obtain T-DNA-free events by transiently expressing the Cytidine base editors (CBEs)
Summary
Mostly based on the CRISPR/Cas system, have rapidly emerged in plants for gene function analysis and to improve agronomical traits This technology relies on the creation of DNA double-strand breaks (DSBs) at predefined targeted sites in the plant genome. A refinement of genome editing consists of the precise and predictable point mutations that are even more promising to decipher natural genomic variations and for crop breeding purposes This could be achieved via homologous recombination (HR), but this repair mechanism suffers from low efficiency in plants and the delivery of donor DNA templates in plant cells, needed to promote the process, is still challenging [1]. Base editors have been successfully used in a number of crops, including rice, tomato, potato, wheat, maize, rape and watermelon [3,4,5,6,7,8,9,10,11]
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