Toward optimizing diversifying base editors for high-throughput mutational scanning studies

Abstract Base editors, including diversifying base editors that create C>N mutations, are potent tools for systematically installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little informa...

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Vydané v:	Nucleic acids research Ročník 53; číslo 12
Hlavní autori:	Schwartz, Carley I, Abell, Nathan S, Li, Amy, Aradhana, Tycko, Josh, Truong, Alisa, Montgomery, Stephen B, Hess, Gaelen T
Médium:	Journal Article
Jazyk:	English
Vydavateľské údaje:	England Oxford University Press 20.06.2025
Predmet:	AICDA (Activation-Induced Cytidine Deaminase) CRISPR-Associated Protein 9 - genetics CRISPR-Associated Protein 9 - metabolism CRISPR-Cas Systems Cytidine Deaminase - genetics Cytidine Deaminase - metabolism DNA Mutational Analysis - methods Gene Editing - methods HEK293 Cells Humans Methods Mutation Point Mutation RNA, Guide, CRISPR-Cas Systems - genetics
ISSN:	0305-1048, 1362-4962, 1362-4962
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Popis
Shrnutí:	Abstract Base editors, including diversifying base editors that create C>N mutations, are potent tools for systematically installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little information exists on how the composition of the editor (deaminase, recruitment method, and fusion architecture) affects editing. To address this knowledge gap, the effect of various design features, such as deaminase recruitment and delivery method (electroporation or lentiviral transduction), on editing was assessed across ∼200 synthetic target sites. The direct fusion of a hyperactive variant of activation-induced cytidine deaminase to the N-terminus of dCas9 (DivA-BE) produced the highest editing efficiency, ∼4-fold better than the previous CRISPR-X method. Additionally, DivA-BE mutagenized the DNA strand that anneals to the targeting sgRNA (target strand) to create complementary C>N mutations, which were absent when the deaminase was fused to the C-terminus of dCas9. Based on these studies that comprehensively analyze the editing patterns of several popular base editors, DivA-BE editors efficiently diversified their target sites, albeit with increased indel frequencies. Overall, the improved editing efficiency makes the DivA-BE editors ideal for discovering functional variants in mutational scanning assays. Graphical Abstract Graphical Abstract
Bibliografia:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Carley I Schwartz Nathan S Abell should be regarded as Joint First Authors.
ISSN:	0305-1048 1362-4962 1362-4962
DOI:	10.1093/nar/gkaf620