Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors.
Nat Methods
; 18(6): 643-651, 2021 06.
Article
en En
| MEDLINE
| ID: mdl-34099937
ABSTRACT
Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, their genome-wide specificity remains poorly understood. Here we report Detect-seq for the evaluation of CBE specificity. It enables sensitive detection of CBE-induced off-target sites at the genome-wide level. Detect-seq leverages chemical labeling and biotin pulldown to trace the editing intermediate deoxyuridine, thereby revealing the editome of CBE. In addition to Cas9-independent and typical Cas9-dependent off-target sites, we discovered edits outside the protospacer sequence (that is, out-of-protospacer) and on the target strand (which pairs with the single-guide RNA). Such unexpected off-target edits are prevalent and can exhibit a high editing ratio, while their occurrences exhibit cell-type dependency and cannot be predicted based on the sgRNA sequence. Moreover, we found out-of-protospacer and target-strand edits nearby the on-target sites tested, challenging the general knowledge that CBEs do not induce proximal off-target mutations. Collectively, our approaches allow unbiased analysis of the CBE editome and provide a widely applicable tool for specificity evaluation of various emerging genome editing tools.
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Banco de datos:
MEDLINE
Asunto principal:
Citosina
/
Edición Génica
Límite:
Humans
Idioma:
En
Año:
2021
Tipo del documento:
Article