Structural and Functional Characterization of the Integrase HK022

Abstract

There is great demand for sophisticated molecular tools for precise genome editing and engineering. The current state of the art for this purpose is to use site‐specific nucleases to generate targeted double‐stranded breaks (DSBs) that are repaired by the cell. Though effective, nucleases often display off‐target cleavage and generate heterogeneous repair products via operations such as non‐homologous end joining. In contrast, tyrosine site‐specific recombinases (SSRs), like Cre and FLP which function biologically to drive integration of phage/viral genomes, generate precise repair outcomes. These outcomes include genetic insertions, deletions or inversions and are performed with considerably lower off‐target activity. HK022 is a relatively under‐studied tyrosine SSR with considerable sequence similarity to the well‐studied lambda phage integrase. In contrast to that enzyme, HK022 displays a recognition profile that is appropriate for targeting disease‐causing mutations, is highly site‐specific, and does not require auxiliary proteins for its function. Additionally, HK022 has the potential to recognize and recombine non‐identical DNA sites which provides the ability to selectively promote recombination in one direction, instead of an equilibrium between non‐recombined and recombined forms. The flexibility and small size of the DNA recognition site, combined with its ability to utilize large DNA cassette inserts make HK022 a promising candidate for optimization for genome engineering. Three forms of HK022 (optimized for synaptic, post‐strand exchange, and Holliday junction structures with DNA) have successfully been expressed and purified for co‐crystallization with unique DNA of interest. Given the success with the lambda integrase, we suspect that HK022 will be equally amenable to crystallization. Solving the structure of HK022 in its many forms provides the ability to compare its recognition pattern to lambda integrase to further understand the diversity of this enzyme class. In addition to generating the first crystal structure of HK022, we wish to enhance the overall stability and activity of the enzyme and optimize it for therapeutic and biotechnological purposes.Support or Funding InformationNational Science Foundation Graduate Research Fellowship Program (NSF GRFP) DGE‐1762114. National Institute of Health (NIH) R01 GM105691.