Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity

被引：161

作者：

Guilinger, John P. ^{[1
,2
]}

Pattanayak, Vikram ^{[1
,2
]}

Reyon, Deepak ^{[3
,4
,5
]}

Tsai, Shengdar Q. ^{[3
,4
,5
]}

Sander, Jeffry D. ^{[3
,4
,5
]}

Joung, J. Keith ^{[3
,4
,5
]}

Liu, David R. ^{[1
,2
]}

机构：

[1] Harvard Univ, Dept Chem & Biol Chem, Cambridge, MA 02138 USA

[2] Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USA

[3] Massachusetts Gen Hosp, Ctr Canc Res, Mol Pathol Unit, Charlestown, MA USA

[4] Massachusetts Gen Hosp, Ctr Computat & Integrat Biol, Charlestown, MA USA

[5] Harvard Univ, Sch Med, Dept Pathol, Boston, MA 02115 USA

来源：

NATURE METHODS | 2014年 / 11卷 / 04期

基金：

美国国家卫生研究院;

关键词：

ZINC-FINGER NUCLEASES; EFFECTOR NUCLEASES; GENOME; KNOCKOUT; REVEALS; DISEASE; SYSTEM; ZFNS;

D O I：

10.1038/nmeth.2845

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：429 / +

页数：9

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