Structure-guided reprogramming of serine recombinase DNA sequence specificity

Abstract

Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences >10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with >80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.gene targeting | protein engineering | site-specific recombination | zinc-finger recombinase S ite-specific recombinases are essential for a variety of diverse biological processes, including the integration and excision of viral genomes, the transposition of mobile genetic elements, and the regulation of gene expression (1). Recently, site-specific recombinases have emerged as powerful tools for advanced genome engineering (2, 3). The exquisite sequence specificities of recombination systems such as Cre/lox, FLP/FRT, and φC31/ att allow researchers to accurately modify genetic information for a variety of applications (4-6). However, DNA sequence constraints imposed by site-specific recombinases make routine modification of cellular genomes contingent on the presence of artificially introduced recognition sequences. As a result, a number of attempts have been made to circumvent or reprogram the strict DNA sequence specificities observed in these systems (7-9). 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...

Reengineering Cro Protein Functional Specificity with an Evolutionary Code

“…In addition, we have recently determined the structure of N15 Cro bound to consensus cognate DNA (B. M. Hall, M. S. Dubrava, S. A. Roberts and M. H. Cordes, unpublished results), which will allow comparison of docking geometries and contacts for Cro proteins of different fold. Finally, incorporation of in vitro or in vivo evolution (see, e.g., Gaj et al 43 ), including randomization of the coding residues and/or other sites in Cro proteins, might yield improved models of direct and/or contextual effects on specificity and could yield insights not accessible to pure comparative methods or to structure-based design.…”

Rewritable digital data storage in live cells via engineered control of recombination directionality

“…Additional biochemically independent RAD modules could likely be identified from the increasing set of known natural recombinases, (37)(38)(39) or perhaps by engineering synthetic integrase excisionase pairs with altered DNA recognition specificity (40)(41)(42). Such work, along with puzzles of integrating dozens of competing biochemical functions, suggest that engineering increased capacity in vivo data storage systems will help define and challenge the limits of synthetic biology.…”

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

“…Moreover, although our optimization was conducted with the native gix core sequence (41–43), several studies have shown that zinc finger-Gin or TALE-Gin fusions are active, and in some cases more active, on slightly altered core sites (28,30,31,34,45–47). Thus, we next sought to target sequences found within the human genome to test if unmodified human genomic sequences were capable of being targeted by recCas9 and to test if varying the guide RNA and core sequences would increase recCas9 activity.…”