Nature Biotechnology导读

锌指核酸酶的特异性锌指核酸酶对选择的目的位点进行特异修饰,但是脱靶对细胞是具有毒害作用的,其程度在基因组范围内尚未得到实验评估。Gabriel等利用慢病毒载体整个标签位点证明了锌指核酸酶的脱靶和中靶活性。

Zinc-finger nucleases （ZFNs） allow gene editing in live cells by inducing a targeted DNA double-strand break （DSB） at a specific genomic locus. However, strategies for characterizing the genome-wide specificity of ZFNs remain limited. We show that nonhomologous endjoining captures integrase-defective lentiviral vectors at DSBs, tagging these transient events. Genome-wide integration site analysis mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or II.2RG. Ranking loci with repeatedly detectable nuclease activity by deep-sequencing allowed us to monitor the degree of ZFN specificity in vivo at these positions. Cleavage required binding of ZFNs in specific spatial arrangements on DNA bearing high homology to the intended target site and only tolerated mismatches at individual positions of the ZFN binding sites. Whereas the consensus binding sequence derived in vivo closely matched that obtained in biochemical experiments, the ranking of in vivo cleavage sites could not be predicted in silico. Comprehensive mapping of ZFN activity in vivo will facilitate the broad application of these reagents in translational research.