Wang, X. et al. Nat. Biotechnol. 33, 175–178 (2015).

Genome-editing tools, particularly transcription activator–like effector nucleases (TALENs) and the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system, are becoming increasingly widely used. But despite their popularity, questions about their off-target activity remain. Wang et al. present an unbiased off-target screen by tagging nuclease-created DNA double-strand breaks with integrase-defective lentiviral vectors (IDLV), which can be amplified by PCR and detected by sequencing. Whereas TALENs did not show any off-target effects when targeted to four endogenous genes, the Cas9 nuclease showed several, but these could be eliminated by the use of a Cas9 nickase. The IDLV assay can detect cleavage frequencies down to 1%, and the findings of frequent off-target sites with single-nucleotide bulges should inform the design of more specific guide RNAs for Cas9.