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ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana.

ABSTRACT: Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the polycistronic tRNA-gRNA expression cassette (PTG-ABE8e) and PTG-ABE7.10 and compared their A-to-G editing efficiencies using both transient and stable transformation in the allotetraploid Nicotiana benthamiana. We found that the editing efficiency of PTG-ABE8e was significantly higher than that of PTG-ABE7.10, indicating that ABE8e was more efficient for A-to-G conversion in N. benthamiana. We further optimized the ABE8e editing efficiency by changing the sgRNA expression cassette and demonstrated that both PTG and single transcript unit (STU) enhanced ABE8e efficiency for A-to-G conversion in N. benthamiana. We also estimated the potential off-target effect of PTG-ABE8e at potential off-targeting sites predicted using an online tool in transgenic plants, and no off-target editing event was found for potential off-targeting sites selected, indicating that ABE8e could specifically facilitate A-to-G conversion. Our results showed that ABE8e with PTG structure was more suitable for A-to-G conversion in N. benthamiana and provided valuable clues for optimizing ABE tools in other plants.

SUBMITTER: Wang Z

PROVIDER: S-EPMC8198424 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cytosine base editors (CBEs) for inducing targeted DNA base editing in Nicotiana benthamiana.

Project description:BackgroundThe base editors can introduce point mutations accurately without causing double-stranded DNA breaks or requiring donor DNA templates. Previously, cytosine base editors (CBEs) containing different deaminases are reported for precise and accurate base editing in plants. However, the knowledge of CBEs in polyploid plants is inadequate and needs further exploration.ResultsIn the present study, we constructed three polycistronic tRNA-gRNA expression cassettes CBEs containing A3A, A3A (Y130F), and rAPOBEC1(R33A) to compare their base editing efficiency in allotetraploid N. benthamiana (n = 4x). We used 14 target sites to compare their editing efficiency using transient transformation in tobacco plants. The sanger sequencing and deep sequencing results showed that A3A-CBE was the most efficient base editor. In addition, the results showed that A3A-CBE provided most comprehensive editing window (C1 ~ C17 could be edited) and had a better editing efficiency under the base background of TC. The target sites (T2 and T6) analysis in transformed N. benthamiana showed that only A3A-CBE can have C-to-T editing events and the editing efficiency of T2 was higher than T6. Additionally, no off-target events were found in transformed N. benthamiana.ConclusionsAll in all, we conclude that A3A-CBE is the most suitable vector for specific C to T conversion in N. benthamiana. Current findings will provide valuable insights into selecting an appropriate base editor for breeding polyploid plants.

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Simplified adenine base editors improve adenine base editing efficiency in rice.

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