Genome editing encompasses the powerful concept of directly correcting mutations in endogenous genes to cure or prevent diseases, particularly to cure or prevent inherited genetic disorders. An emerging example of this approach is the clinical trial of a zinc finger nuclease (ZFN) therapeutic engineered to disrupt CCR5, a co-receptor for HIV. Four main classes of engineered nucleases have been implicated in genome editing: 1) meganucleases, 2) zinc-finger nucleases, 3) transcription activator effector-like nucleases (TALEN), and 4) Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas RNA-guided nucleases (RGN).
However, the potential therapeutic applications of these engineered nucleases will require a comprehensive knowledge of their off-target effects to minimize the risk of deleterious outcomes. Several in vivo or in vitro methods have been developed to detect off-target (Wang, et al., (2015), Nat. Biotechnol. 33:175-179); Crosetto, et al., (2013), Nature Methods 10:361-368); Frock, et al. (2016), Nat. Biotechnol. 33:179-187). Most of methods rely on an integration of viral DNA or short double strand DNA tags into the double strand break (DSB) followed by polymerase chain reaction (PCR) amplification and next generation sequencing (NGS). However, these methods can only detect partial off-targets due to DNA tag's degradation in cells, leading to the low efficiency of tag integration in DSB. A recent development of “Genome-wide, Unbiased Identification of DSBs Enabled by sequencing” (GUIDE-seq) method used phosphorothioate-modified double strand DNA as tag that prevented degradation and increased integration efficiency of tag into DSB in cells. However, one major issue of this method is non-specific PCR amplification that causes low specificity and low sensitivity due to a high-background. The invention provided herein addresses these and other shortcomings in the art.