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- W4225996919 abstract "Sensitive detection of off-target sites produced by gene editing nucleases is crucial for developing reliable gene therapy platforms. Although several biochemical assays for the characterization of nuclease off-target effects have been recently published, significant technical and methodological issues still remain. Of note, existing methods rely on PCR amplification, tagging, and affinity purification which can introduce bias, contaminants, sample loss through handling, etc. Here we describe a sensitive, PCR-free next-generation sequencing method (RGEN-seq) for unbiased detection of double-stranded breaks generated by RNA-guided CRISPR-Cas9 endonuclease. Through use of novel sequencing adapters, the RGEN-Seq method saves time, simplifies workflow, and removes genomic coverage bias and gaps associated with PCR and/or other enrichment procedures. RGEN-seq is fully compatible with existing off-target detection software; moreover, the unbiased nature of RGEN-seq offers a robust foundation for relating assigned DNA cleavage scores to propensity for off-target mutations in cells. A detailed comparison of RGEN-seq with other off-target detection methods is provided using a previously characterized set of guide RNAs." @default.
- W4225996919 created "2022-05-05" @default.
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- W4225996919 date "2021-12-08" @default.
- W4225996919 modified "2023-09-29" @default.
- W4225996919 title "RGEN-seq for highly sensitive amplification-free screen of off-target sites of gene editors" @default.
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- W4225996919 doi "https://doi.org/10.1038/s41598-021-03160-8" @default.
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