Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection
By V. Pattanayak, C. L. Ramirez, J. Keith Joung, David R. Liu. 2011
469 citations recorded by the source corpus.
Engineered zinc-finger nucleases (ZFNs) are promising tools for genome manipulation, and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 1011 DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGFA genes, respectively. Analysis of identified sites in one cultured human cell line revealed CCR5-224–induced changes at nine off-target loci, though this remains to be tested in other relevant cell types. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future ZFN design.
Subfields: Molecular Biology, Plant Science
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Citations in the corpus
- Delivering CRISPR: a review of the challenges and approaches (2018, unknown): “Another significant challenge is off-target cutting (Gabriel et al., 2011; Pattanayak et al., 2011).”
- Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases (2014, background): “In addition, in vitro selection of cleavage sites (Pattanayak et al. 2011) or the IDLV capture approach (Gabriel et al.”