Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity.
Mol Cell
; 79(5): 728-740.e6, 2020 09 03.
Article
em En
| MEDLINE
| ID: mdl-32721385
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.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Oryza
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Antígenos de Histocompatibilidade Menor
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Citidina Desaminase
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Citosina
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Edição de Genes
Limite:
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article