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Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions
- Source :
- BMC Biology, BMC Biology, Vol 18, Iss 1, Pp 1-14 (2020)
- Publication Year :
- 2020
- Publisher :
- Springer Science and Business Media LLC, 2020.
-
Abstract
- BackgroundCytidine base editors (CBEs), composed of a cytidine deaminase fused to Cas9 nickase (nCas9), enable efficient C-to-T conversion in various organisms. However, current base editors can induce unwanted bystander C-to-T conversions when multiple Cs are present in the ~ 5-nucleotide activity window of cytidine deaminase, which negatively affects their precision. Here, we develop a new base editor which significantly reduces unwanted bystander activities.ResultsWe used an engineered human APOBEC3G (eA3G) C-terminal catalytic domain with preferential cytidine-deaminase activity in motifs with a hierarchy CCC>CCC>CC(where the preferentially deaminated C is underlined), to develop an eA3G-BE with distinctive CCcontext-specificity and reduced generation of bystander mutations. Targeted editing efficiencies of 18.3–58.0% and 54.5–92.2% with excellent CCcontext-specificity were generated in human cells and rabbit embryos, respectively. In addition, a base editor that can further recognize relaxed NG PAMs is achieved by combining hA3G with an engineered SpCas9-NG variant. The A3G-BEs were used to induce accurate single-base substitutions which led to nonsense mutation with an efficiency of 83–100% and few bystander mutations in Founder (F0) rabbits atTyrloci.ConclusionsThese novel base editors with improved precision and CCcontext-specificity will expand the toolset for precise gene modification in organisms.
- Subjects :
- Physiology
Nonsense mutation
APOBEC-3G Deaminase
Plant Science
Computational biology
Biology
General Biochemistry, Genetics and Molecular Biology
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
Structural Biology
CRISPR-Associated Protein 9
Cytidine Deaminase
Bystander effect
Animals
Humans
CRISPR
lcsh:QH301-705.5
CRISPR/Cas9
APOBEC3G
Ecology, Evolution, Behavior and Systematics
030304 developmental biology
Gene Editing
0303 health sciences
Cas9
Cytidine
Cell Biology
Cytidine deaminase
Precision
Base (topology)
lcsh:Biology (General)
chemistry
eA3G
Rabbits
Base editor
General Agricultural and Biological Sciences
030217 neurology & neurosurgery
Research Article
Developmental Biology
Biotechnology
Subjects
Details
- ISSN :
- 17417007
- Volume :
- 18
- Database :
- OpenAIRE
- Journal :
- BMC Biology
- Accession number :
- edsair.doi.dedup.....81e3a3b7f0aa9a75095120bd1bd83072