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

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

Zupeng Wang^1,2,3, Xiaoying Liu^1,2,3, Xiaodong Xie^1,2,3

¹Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China.

International Journal of Molecular Sciences|June 2, 2021

View abstract on PubMed

Summary

Adenine base editor 8e (ABE8e) shows higher A-to-G editing efficiency in Nicotiana benthamiana compared to ABE7.10. Optimized ABE8e systems demonstrated specific editing with no off-target effects, making it suitable for plant genome engineering.

Area of Science:

Plant biotechnology
Genome editing technologies
Molecular biology

Background:

Adenine base editors (ABEs) enable precise A-to-G base conversions.
ABE8e has shown promise in rice, but its utility and comparison to ABE7.10 in other plant species are underexplored.

Purpose of the Study:

To evaluate and compare the A-to-G editing efficiency of ABE8e and ABE7.10 in Nicotiana benthamiana.
To optimize ABE8e editing efficiency using different expression cassettes.
To assess the specificity and potential off-target effects of ABE8e in plants.

Main Methods:

Development of polycistronic tRNA-gRNA (PTG) expression cassettes for ABE8e and ABE7.10.
Transient and stable transformation of Nicotiana benthamiana.
Comparison of A-to-G editing efficiencies between PTG-ABE8e and PTG-ABE7.10.
Optimization of ABE8e by altering sgRNA expression cassettes (PTG and single transcript unit - STU).
In silico prediction and experimental assessment of off-target editing.

Main Results:

PTG-ABE8e exhibited significantly higher A-to-G editing efficiency than PTG-ABE7.10 in N. benthamiana.
Both PTG and STU expression systems enhanced ABE8e efficiency for A-to-G conversion.
No off-target editing events were detected at predicted potential off-targeting sites using PTG-ABE8e, indicating high specificity.

Conclusions:

ABE8e, particularly with the PTG expression structure, is more efficient and suitable for A-to-G base editing in Nicotiana benthamiana.
The findings provide valuable insights for optimizing adenine base editor tools for applications in diverse plant species.