Abstract
Site-specific recombinases (SSRs) are valuable tools for genetic engineering due to their ability to manipulate DNA in a highly specific manner. Engineered zinc-finger and TAL effector recombinases, in particular, are two classes of SSRs composed of custom-designed DNA-binding domains fused to a catalytic domain derived from the resolvase/invertase family of serine recombinases. While TAL effector and zinc-finger proteins can be assembled to recognize a wide range of possible DNA sequences, recombinase catalytic specificity has been constrained by inherent base requirements present within each enzyme. In order to further expand the targeted recombinase repertoire, we used a genetic screen to isolate enhanced mutants of the Bin and Tn21 recombinases that recognize target sites outside the scope of other engineered recombinases. We determined the specific base requirements for recombination by these enzymes and demonstrate their potential for genome engineering by selecting for variants capable of specifically recombining target sites present in the human CCR5 gene and the AAVS1 safe harbor locus. Taken together, these findings demonstrate that complementing functional characterization with protein engineering is a potentially powerful approach for generating recombinases with expanded targeting capabilities.
Highlights
Genome engineering has emerged as a powerful approach for introducing custom alterations within biological systems [1]
Zinc-finger nucleases (ZFNs) [2,3,4,5], TAL effector nucleases (TALENs) [6,7,8] and CRISPR/Cas9 [9,10,11,12] have surfaced as tools capable of modifying both human cells and model organisms
Redesigning Recombinase Specificity with high efficiency and flexibility. These enzymes induce targeted DNA double-strand breaks (DSBs), which stimulate the DNA damage response machinery and lead to the introduction of small insertions or deletions via non-homologous end joining (NHEJ) [13] or integration/correction by homology-directed repair (HDR) [3,4,5, 14]. Despite their broad success, the utility of nuclease-based technologies is hampered by the formation of DSBs, which can be toxic to cells and lead to unknown and deleterious mutations at off-target sites [15,16,17,18]
Summary
Genome engineering has emerged as a powerful approach for introducing custom alterations within biological systems [1]. Redesigning Recombinase Specificity with high efficiency and flexibility These enzymes induce targeted DNA double-strand breaks (DSBs), which stimulate the DNA damage response machinery and lead to the introduction of small insertions or deletions via non-homologous end joining (NHEJ) [13] or integration/correction by homology-directed repair (HDR) [3,4,5, 14]. High rates of modification via HDR can be difficult to achieve in post-mitotic cell types Together, these limitations underscore the need for the development of new technologies capable of inducing robust and safe genomic modifications
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