President and Fellows of Harvard College;Liu, David R.;Guilinger, John Paul;Pattanayak, Vikram
发明人:
申请号：
EP12845790.0
公开号：
EP2734621B1
申请日:
2012.07.22
申请国别(地区):
EP
年份:
2019
代理人:
摘要：
Engineered nucleases (e.g., zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and others) are promising tools for genome manipulation and determining off-target cleavage sites of these enzymes is of great interest. We developed anin vitroselection method that interrogates 10<；sup>；11<；/sup>；DNA sequences for their ability to be cleaved by active, dimeric nulceases, e.g., ZFNs and TALENs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleavedin vitroby two ZFNs, CCR5-224 and VF2468, which target the endogenous humanCCR5andVEGF-Agenes, respectively. Analysis of the identified sites in cultured human cells revealed CCR5-224-induced mutagenesis at nine off-target loci. 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 nuclease design. It was also pobserved that TALENs can achieve cleavage specificity similar to or higher than that observed in ZFNs.
