In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites

Jeffry D Sander,David R Liu,Vikram Pattanayak,Manching Ku,J Keith Joung,Jennifer A Foden,Samantha J Linder,Cherie L Ramirez,Deepak Reyon,Bradley E Bernstein,Noam Shoresh

doi:10.1093/nar/gkt716

Abstract

Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described–one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.

Highlights

Gene-editing nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) nucleases, can be used to create targeted sequence alterations with high efficiencies in numerous cell types and organisms [1,2,3,4,5,6,7]
We performed an initial test of our approach by developing a classifier based on in vitro site selection data previously obtained for ZFNs targeted to a site in the human CCR5 gene
Our results show in silico abstraction of in vitro cleavage data provides a strategy that more broadly identifies all potential off-target sites of ZFN activity in human cells than previously described methods

Summary

Introduction

Gene-editing nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) nucleases, can be used to create targeted sequence alterations with high efficiencies in numerous cell types and organisms [1,2,3,4,5,6,7]. In vitro selections with a CCR5-targeted ZFN pair identified 36 potential off-target cleavage sites that occur in the sequence of the human genome; analysis of these sites in human cells in which CCR5-targeted ZFNs had been expressed revealed nine bona fide off-target sites [8] Another approach, described by von Kalle and colleagues, exploited the incorporation of integrase-deficient lentivirus (IDLV) DNAs into nuclease-induced doublestranded breaks to map ZFN cleavage sites in human cells [9]. Application of this approach to the same CCR5-targeted ZFNs characterized with the in vitro selection approach identified four off-target genomic sites

Methods

Results

Conclusion