Directed evolution of recombinase specificity by split gene reassembly

被引:39
作者
Gersbach, Charles A.
Gaj, Thomas
Gordley, Russell M.
Barbas, Carlos F., III [1 ]
机构
[1] Scripps Res Inst, Skaggs Inst Chem Biol, La Jolla, CA 92037 USA
基金
美国国家卫生研究院;
关键词
SITE-SPECIFIC RECOMBINASE; ZINC-FINGER NUCLEASES; BETA-LACTAMASE; FLP RECOMBINASE; MUTATIONS; VARIANTS;
D O I
10.1093/nar/gkq125
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
The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding p-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications.
引用
收藏
页码:4198 / 4206
页数:9
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