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Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions
- Source :
- BMC Biology, Vol 18, Iss 1, Pp 1-14 (2020)
- Publication Year :
- 2020
- Publisher :
- BMC, 2020.
-
Abstract
- Abstract Background Cytidine 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. Results We 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 CC context-specificity and reduced generation of bystander mutations. Targeted editing efficiencies of 18.3–58.0% and 54.5–92.2% with excellent CC context-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 at Tyr loci. Conclusions These novel base editors with improved precision and CC context-specificity will expand the toolset for precise gene modification in organisms.
- Subjects :
- CRISPR/Cas9
Base editor
eA3G
Precision
Biology (General)
QH301-705.5
Subjects
Details
- Language :
- English
- ISSN :
- 17417007
- Volume :
- 18
- Issue :
- 1
- Database :
- Directory of Open Access Journals
- Journal :
- BMC Biology
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.9e565a1f40e481092a9fb73ed252cee
- Document Type :
- article
- Full Text :
- https://doi.org/10.1186/s12915-020-00849-6