Synthesis of programmable integrases

Abstract

Accurate modification of the 3 billion-base-pair human genome requires tools with exceptional sequence specificity. Here, we describe a general strategy for the design of enzymes that target a single site within the genome. We generated chimeric zinc finger recombinases with cooperative DNA-binding and catalytic specificities that integrate transgenes with >98% accuracy into the human genome. These modular recombinases can be reprogrammed: New combinations of zinc finger domains and serine recombinase catalytic domains generate novel enzymes with distinct substrate sequence specificities. Because of their accuracy and versatility, the recombinases/integrases reported in this work are suitable for a wide variety of applications in biological research, medicine, and biotechnology where accurate delivery of DNA is desired.recombinases ͉ zinc finger ͉ gene delivery ͉ gene targeting ͉ protein engineering T he postgenomic era of medicine will be defined by our ability to achieve biological control through genetic reprogramming. New tools are needed to accurately rewrite the genomic script and specifically alter genes, gene expression, and epigenetic state at any desired loci. To date, no enzyme-natural or synthetic-has been able to accurately modify only a single targeted site within the human genome (1). Scientists in biology, biotechnology, stem cell research, and gene therapy currently rely on naturally occurring enzymes to perform functions like DNA integration and excision. However, these enzymes recognize multiple sites within the human genome, often resulting in off-target DNA integration and chromosomal translocation (2-6). Our recent work with serine resolvases and invertases led us to hypothesize that we could use a modular approach that capitalizes on cooperative specificity to design synthetic enzymes that would uniquely recognize a single site within the 3 billion-base-pair human genome and allow us to deliver DNA specifically to this site (Fig. 1A) (7).In their native contexts, serine resolvases and invertases selectively recombine target sites within the same DNA molecule. This intramolecular specificity is assured by obligate assembly of large protein complexes, wherein accessory factors bound at neighboring sites impose topological and spatial constraints on the recombination reaction (8). Hyperactive mutants of several serine resolvases and invertases have been discovered that efficiently catalyze unrestricted recombination between minimal dimer-binding sites (Table S1) (9, 10). Furthermore, unlike other site-specific recombinases, serine resolvases and invertases are well suited to synthetic reengineering. These enzymes are modular in both structure and function, each comprised of a distinct catalytic domain flexibly tethered to a small helix-turnhelix DNA-binding domain (DBD). However, these DBDs are poorly suited for accurate genomic recombination (11) because the recognition motifs are short (4-6 bp) and degenerate (12, 13).In contrast, zinc finger DNA-binding proteins recognize target sites of v...

Structure-guided reprogramming of serine recombinase DNA sequence specificity

“…A ZFR target site consists of two zinc-finger protein binding-sites (ZFBS) flanking a 20-bp core sequence recognized by the serine resolvase or invertase catalytic domain. Unlike zinc-finger nucleases, which are built from the sequence-independent catalytic domain of the FokI restriction endonuclease (22), ZFR sequence specificity is the cooperative product of modular site-specific DNA recognition (23-28) and sequence-dependent catalysis (20). By taking advantage of cooperative sequence specificity, ZFRs are able to mediate the excision of transgenetic elements from the human genome and to catalyze plasmid integration into a genomic locus with exceptional efficiency and specificity (19,20).…”

“…These successes, however, have been dependent on the presence of ZFR target sites that contain 20-bp core sequences derived from the native serine resolvase or invertase recombination sites (19,20). To address this limitation, we previously used enzyme libraries consisting of random amino acid substitutions to evolve recombinase variants to react with nonnative DNA sequences (13,19).…”

“…Unlike zinc-finger nucleases, which are built from the sequence-independent catalytic domain of the FokI restriction endonuclease (22), ZFR sequence specificity is the cooperative product of modular site-specific DNA recognition (23-28) and sequence-dependent catalysis (20). By taking advantage of cooperative sequence specificity, ZFRs are able to mediate the excision of transgenetic elements from the human genome and to catalyze plasmid integration into a genomic locus with exceptional efficiency and specificity (19,20).These successes, however, have been dependent on the presence of ZFR target sites that contain 20-bp core sequences derived from the native serine resolvase or invertase recombination sites (19,20). To address this limitation, we previously used enzyme libraries consisting of random amino acid substitutions to evolve recombinase variants to react with nonnative DNA sequences (13,19).…”

“…Despite these efforts, engineered site-specific recombinase variants often exhibit considerably relaxed DNA sequence specificities (8, 10-14), a detrimental byproduct that often results in adverse off-target chromosomal modification (15-17). Thus, there is significant interest in the development of generalized protein engineering strategies capable of comprehensively redesigning DNA sequence specificity for genome engineering and therapeutic applications.In recent years, our group and others have demonstrated that engineered zinc-finger recombinase (ZFR) fusion proteins are versatile alternatives to existing site-specific recombinase technologies (13,(18)(19)(20). ZFRs are artificial DNA-modifying enzymes generated by fusing site-specific DNA-binding zincfinger proteins (ZFPs) to highly selective serine resolvase or invertase catalytic domains (21).…”

“…In recent years, our group and others have demonstrated that engineered zinc-finger recombinase (ZFR) fusion proteins are versatile alternatives to existing site-specific recombinase technologies (13,(18)(19)(20). ZFRs are artificial DNA-modifying enzymes generated by fusing site-specific DNA-binding zincfinger proteins (ZFPs) to highly selective serine resolvase or invertase catalytic domains (21).…”