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Antisense RNA Interference-Enhanced CRISPR/Cas9 Base Editing Method for Improving Base Editing Efficiency in
Yue Zhang1,2, Kaiyue Yun1,2,3, Huamei Huang1,2,3
1Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
ACS Synthetic Biology
|March 15, 2021
Summary
CRISPR/Cas9 base editing in Streptomyces lividans 66 was improved by transiently inhibiting uracil DNA glycosidase (UDG) using antisense RNA interference-enhanced base editing (asRNA-BE). This method enhances editing efficiency and maintains genome stability, offering a superior tool for Streptomyces genetic manipulation.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR/Cas9 base editors offer precise genomic manipulation with lower toxicity than traditional methods.
- Base editing has been applied in various Streptomyces species, but efficiency varies.
- Uracil DNA glycosidase (UDG) activity can reduce base editing efficiency by removing key intermediates.
Purpose of the Study:
- To evaluate base editing tools (BE2 and BE3) in Streptomyces lividans 66.
- To investigate the impact of uracil DNA glycosidase (UDG) on base editing efficiency.
- To develop an improved base editing strategy for enhanced efficiency and genome stability in S. lividans 66.
Main Methods:
- Functional assessment of BE2 and BE3 base editors in Streptomyces lividans 66.
- Gene knockout of endogenous uracil DNA glycosidases (UDG1 and UDG2) to enhance base editing.
- Development and application of antisense RNA interference-enhanced CRISPR/Cas9 Base Editing (asRNA-BE) for transient UDG inhibition.
Main Results:
- BE2 and BE3 showed lower base editing efficiency in S. lividans 66 compared to S. coelicolor.
- Knocking out UDG1 and UDG2 improved base editing efficiency by 3.4-67.4-fold.
- The developed asRNA-BE method achieved a 2.8-65.8-fold increase in editing efficiency with improved genome stability.
Conclusions:
- asRNA-BE is a highly effective base editing tool for Streptomyces lividans 66, significantly improving editing efficiency and genome stability.
- Transient inhibition of uracil DNA glycosidases is a viable strategy to enhance CRISPR/Cas9 base editing.
- The asRNA-BE method holds promise for advancing genetic engineering in other Streptomyces species.
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