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Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor
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Journal Article
Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor
2024
Overview
Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.
Single-stranded DNA donors using current design parameters remain inefficient for precise gene editing. Here, the authors engineer RAD51-preferred sequences into HDR-boosting modules for donors, enhancing HDR efficiency across various genomic loci and cell types.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
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/ 13/31
/ 13/44
/ 13/89
/ 38/35
/ 38/5
/ 38/77
/ 38/88
/ 38/90
/ 42/109
/ 45/22
/ 45/23
/ 45/29
/ Bacterial Proteins - genetics
/ Bacterial Proteins - metabolism
/ CRISPR-Associated Protein 9 - genetics
/ CRISPR-Associated Protein 9 - metabolism
/ CRISPR-Associated Proteins - genetics
/ CRISPR-Associated Proteins - metabolism
/ DNA
/ DNA, Single-Stranded - genetics
/ DNA, Single-Stranded - metabolism
/ Editing
/ Endodeoxyribonucleases - genetics
/ Endodeoxyribonucleases - metabolism
/ Genomics
/ Homology
/ Humanities and Social Sciences
/ Humans
/ Modules
/ Proteins
/ Rad51 Recombinase - genetics
/ Rad51 Recombinase - metabolism
/ Repair
/ Science
