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This content will become publicly available on February 1, 2023

Title: dCas9-based gene editing for cleavage-free genomic knock-in of long sequences
Abstract Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR–Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9–SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9–SSAP editor has low on-target errors and minimal off-target effects, showing higher accuracy than canonical Cas9 methods. It is effective for inserting kilobase-scale sequences, with an efficiency of up to approximately 20% and robust performance across donor designs and cell types, including human stem cells. We show that dCas9–SSAP is less sensitive to inhibition of DNA repair enzymes than Cas9 references. We further performed truncation and aptamer engineering to minimize its size to fit into a single adeno-associated-virus vector for future application. Together, this tool opens opportunities towards safer long-sequence genome engineering.
Authors:
; ; ; ; ; ;
Award ID(s):
1953686
Publication Date:
NSF-PAR ID:
10338388
Journal Name:
Nature Cell Biology
Volume:
24
Issue:
2
Page Range or eLocation-ID:
268 to 278
ISSN:
1465-7392
Sponsoring Org:
National Science Foundation
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