RGEN-seq for highly sensitive amplification-free screen of off-target sites of gene editors.

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 gap

SUBMITTER: Kuzin A 

PROVIDER: S-EPMC8654851 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature