Nat Methods

Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have activity against related off-target sequences. To better understand TALEN specificity, we profiled 30 unique TALENs with different target sites, array length and domain sequences for their abilities to cleave any of 10(12) potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant that exhibits equal on-target cleavage activity but tenfold lower average off-target activity in human cells.DP1 GM105378/DP/NCCDPHP CDC HHS/United StatesDP1 GM105378/GM/NIGMS NIH HHS/United StatesR01 GM065865/GM/NIGMS NIH HHS/United StatesR01 GM095501/GM/NIGMS NIH HHS/United StatesR01 GM095501/GM/NIGMS NIH HHS/United StatesT32 GM007753/GM/NIGMS NIH HHS/United StatesT32GM007753/GM/NIGMS NIH HHS/United StatesHoward Hughes Medical Institute/United StatesHoward Hughes Medical Institute/United States2014-10-01T00:00:00Z24531420PMC401012