Back to Search
Start Over
Antisense RNA Interference-Enhanced CRISPR/Cas9 Base Editing Method for Improving Base Editing Efficiency in Streptomyces lividans 66.
- Source :
-
ACS synthetic biology [ACS Synth Biol] 2021 May 21; Vol. 10 (5), pp. 1053-1063. Date of Electronic Publication: 2021 Mar 15. - Publication Year :
- 2021
-
Abstract
- CRISPR/Cas9-mediated base editors, based on cytidine deaminase or adenosine deaminase, are emerging genetic technologies that facilitate genomic manipulation in many organisms. Since base editing is free from DNA double-strand breaks (DSBs), it has certain advantages, such as a lower toxicity, compared to the traditional DSB-based genome engineering technologies. In terms of Streptomyces , a base editing method has been successfully applied in several model and non-model species, such as Streptomyces coelicolor and Streptomyces griseofuscus . In this study, we first proved that BE2 (rAPOBEC1-dCas9-UGI) and BE3 (rAPOBEC1-nCas9-UGI) were functional base editing tools in Streptomyces lividans 66, albeit with a much lower editing efficiency compared to that of S. coelicolor . Uracil generated in deamination is a key intermediate in the base editing process, and it can be hydrolyzed by uracil DNA glycosidase (UDG) involved in the intracellular base excision repair, resulting in a low base editing efficiency. By knocking out two endogenous UDGs (UDG1 and UDG2), we managed to improve the base editing efficiency by 3.4-67.4-fold among different loci. However, the inactivation of UDG is detrimental to the genome stability and future application of engineered strains. Therefore, we finally developed a nti s ense RNA interference-enhanced CRISPR/Cas9 B ase E diting method (asRNA-BE) to transiently disrupt the expression of uracil DNA glycosidases during base editing, leading to a 2.8-65.8-fold enhanced editing efficiency and better genome stability. Our results demonstrate that asRNA-BE is a much better editing tool for base editing in S. lividans 66 and might be beneficial for improving the base editing efficiency and genome stability in other Streptomyces strains.
- Subjects :
- DNA Repair genetics
Down-Regulation genetics
Gene Knockout Techniques
Genetic Loci
Genome, Bacterial
Genomic Instability genetics
Microorganisms, Genetically-Modified
Streptomyces coelicolor genetics
Streptomyces coelicolor metabolism
Uracil metabolism
Uracil-DNA Glycosidase genetics
Uracil-DNA Glycosidase metabolism
CRISPR-Associated Protein 9 genetics
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats genetics
Gene Editing methods
RNA Interference
RNA, Antisense genetics
Streptomyces lividans enzymology
Streptomyces lividans genetics
Subjects
Details
- Language :
- English
- ISSN :
- 2161-5063
- Volume :
- 10
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- ACS synthetic biology
- Publication Type :
- Academic Journal
- Accession number :
- 33720688
- Full Text :
- https://doi.org/10.1021/acssynbio.0c00563