• Home
  • Search Results
  • Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity.

Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity.

Molecular cell (2020-07-30)
Shuai Jin, Hongyuan Fei, Zixu Zhu, Yingfeng Luo, Jinxing Liu, Shenghan Gao, Feng Zhang, Yu-Hang Chen, Yanpeng Wang, Caixia Gao
ABSTRACT

Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Potassium chloride, ACS reagent, 99.0-100.5%
Sigma-Aldrich
D-Mannitol, ≥98%
Sigma-Aldrich
MES hydrate, ≥99.5% (titration)
Sigma-Aldrich
Potassium hydroxide, reagent grade, 90%, flakes
Sigma-Aldrich
Magnesium chloride hexahydrate, ACS reagent, 99.0-102.0%
Sigma-Aldrich
Calcium chloride dihydrate, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
Sigma-Aldrich
1-Naphthaleneacetic acid, suitable for plant cell culture, BioReagent, ≥95%, crystalline
Sigma-Aldrich
2,4-Dichlorophenoxyacetic acid, ≥95%, crystalline
Sigma-Aldrich
Poly(ethylene glycol), BioUltra, 4,000
Sigma-Aldrich
Copper(II) sulfate pentahydrate, suitable for plant cell culture, ≥98%