Abstract：
: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.
journal_name
Nat Methodsjournal_title
Nature methodsauthors
Guilinger JP,Pattanayak V,Reyon D,Tsai SQ,Sander JD,Joung JK,Liu DRdoi
10.1038/nmeth.2845subject
Has Abstractpub_date
2014-04-01 00:00:00pages
429-35issue
4eissn
1548-7091issn
1548-7105pii
nmeth.2845journal_volume
11pub_type
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