![]() ![]() This concern drives developing strategies for improving targeted nuclease specificities 14– 17 as well as methods for genome-wide off-target sites detection. One continuing concern for recruiting RGENs, ZFN, or TALENs for genome engineering is the potential for off-target DSB activity at non-consensus sites within the genome 11– 13. Nevertheless, high-efficiency genome editing by all platforms is based on the ability to make a targeted DNA double-strand break (DSB) in the chromosomal sequence of interest, which can be repaired by nonhomologous end-joining (NHEJ) 9 or homology-directed repair (HDR) 10. The rapid pace with which RGENs have been adopted for gene editing, is due to their perceived simplicity compared to other platforms. RNA-guided Cas9 endonuclease complexes (RGENs) became a third wave of programmable endonucleases for targeted genome editing, along with previously known zinc-finger nucleases (ZFNs) 7 and transcription activator–like effector nucleases (TALENs) 7, 8. The CRISPR–Cas9 system is a potent genome editing tool for biology 1– 3, and has a great potential for gene therapy 4– 6. A detailed comparison of RGEN-seq with other off-target detection methods is provided using a previously characterized set of guide RNAs. 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. 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. 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. Of note, existing methods rely on PCR amplification, tagging, and affinity purification which can introduce bias, contaminants, sample loss through handling, etc. Although several biochemical assays for the characterization of nuclease off-target effects have been recently published, significant technical and methodological issues still remain. ![]() Sensitive detection of off-target sites produced by gene editing nucleases is crucial for developing reliable gene therapy platforms. ![]()
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