Comprehensive computational design of mCreI homing endonuclease cleavage specificity for genome engineering

被引：27

作者：

Ulge, Umut Y. ^{[2
,3
]}

Baker, David A. ^{[1
,4
,5
]}

Monnat, Raymond J., Jr. ^{[1
,6
]}

机构：

[1] Univ Washington, Dept Genome Sci, Seattle, WA 98195 USA

[2] Univ Washington, Mol & Cellular Biol Grad Program, Seattle, WA 98195 USA

[3] Univ Washington, Med Scientist Training Program, Seattle, WA 98195 USA

[4] Univ Washington, Dept Biochem, Seattle, WA 98195 USA

[5] Univ Washington, Howard Hughes Med Inst, Seattle, WA 98195 USA

[6] Univ Washington, Dept Pathol, Seattle, WA 98195 USA

来源：

NUCLEIC ACIDS RESEARCH | 2011年 / 39卷 / 10期

基金：

美国国家卫生研究院;

关键词：

MAMMALIAN-CELLS; DIRECTED EVOLUTION; DNA-BINDING; I-PPOI; RECOMBINATION; GENE; SITE; MEGANUCLEASES; RECOGNITION; DERIVATIVES;

D O I：

10.1093/nar/gkr022

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Homing endonucleases (HEs) cleave long (similar to 20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements. In order to determine whether comprehensive computational design could be used as a general strategy to engineer new HE target site specificities, we used RosettaDesign (RD) to generate 3200 different variants of the mCreI LAGLIDADG HE towards 16 different base pair positions in the 22 bp mCreI target site. Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved. These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.

引用

页码：4330 / 4339

页数：10

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