Off-Target Specificity Overview

Targeted genome editing is a broadly applicable technology that enables precise disruption or modification of genes through the use of site-specific nucleases (e.g. Cas9). These nucleases are programmed to cut DNA at a specific sequence, leaving behind a double-stranded break (DSB). These DSBs are typically repaired by the endogenous cellular machinery via the non-homologous end joining (NHEJ) or homology directed repair (HDR) pathways. 

Imperfect specificity of engineered nucleases can result in DSBs at genomic locations other than the intended on-target site and a key concern for industrial and therapeutic applications has been identifying these genome editing "off-target" effects. Unintended edits may have unpredictable or unknown cellular consequences or can confound experimental results. One specific concern regarding off-target mutagenesis is that they can result in cells with a selective growth advantage or increase oncogenic potential.

The field has moved toward utilizing empirical sensitive, unbiased, genome-scale assays to evaluate off-target activity. Tsai et al. outlines the advantages and limitations of the various published methods.

GUIDE-Seq (Genome-wide Unbiased Identification of DSBs Enabled by Sequencing) is based on the efficient integration of an end-protected double-stranded oligodeoxynucleotide (dsODN) tag into the sites of nuclease-induced DSBs in live human cells. Tag-specific amplification, high-throughput sequencing, and mapping enables the identification of sites of double-strand break activity in the genome.

CIRCLE-Seq (Comprehensive In vitro Reporting of Cleavage Events by Sequencing) is an unpublished method developed in the lab of J. Keith Joung.

Beacon has exclusively licensed the GUIDE-Seq and CIRCLE-Seq assays from Massachusetts General Hospital.