Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Renewed Utility of Tyrosine Integrases

Authors:

Kelly P. Williams1*, Jesse Cahill1, Lauren G. Clark2, Michael C. Jewett2,3, Joseph S. Schoeniger1

Institutions:

1Sandia National Laboratories; 2Northwestern University; 3Stanford University

Abstract

The Intrinsic Control for Genome and Transcriptome Editing in Communities (InCoGenTEC) Science Focus Area aims to develop strategies for biocontainment, enable safe transformation of non-model prokaryotes using phage vectors, and understand gene mobility in microbial communities. The overall project goals are to: (1) mechanistically understand gene mobility events through comprehensive computational mapping of integrase- and transposon-driven mobility; (2) perform functional genomics studies to identify genes and pathways responsible for mobility and identify novel genes for use in biocontainment mechanisms; and (3) utilize prophages from a genomic island database to transform non-model microbes toward the goal of safe microbial community transformation.

DNA integrases catalyze recombination between specific attachment (att) sites on two circular DNAs: the attB site on a bacterial chromosome and the attP site on a pre-island circle. This results in a single circle with the genomic island precisely integrated into the chromosome. Integrases retain all DNA strands until after recombination is complete, forming covalent intermediates at either a catalytic tyrosine or serine residue depending on the protein family. If an att site is available for use in a locus, this reaction mechanism can provide an inherently more efficient and safer approach to genome editing than CRISPR methods which first introduce a double-stranded break in the chromosome. Additional safety and control come from the property of directionality: integrases require an additional partner protein (e.g., excisionase or recombination directionality factor) to catalyze the reverse excision reaction but not the forward integration reaction.

Serine integrases have been favored for genome editing applications because tyrosine integrases are perceived to require additional protein factors from their bacterial hosts. This dependence on host factors, particularly the integration host factor (IHF), occurs for certain classical tyrosine integrases (e.g., phage lambda) but may not apply to integrases from the many bacterial phyla that do not harbor IHF genes, nor to all integrases from IHF+ species. Using project software to precisely map hundreds of thousands of tyrosine integrase att sites, the research team assembled a panel of diverse tyrosine integrases which were assayed using E. coli-based in vivo and cell-free assays. Many integrases from phyla not known to bear IHF genes were functional, even in IHF-deficient genetic backgrounds, and in cell-free assays where IHF was diluted ~10-fold.

This work demonstrates that bias against tyrosine integrases has resulted from a misperception; most are not dependent on host factors. Tyrosine integrases are ~8-fold more abundant than serine integrases, offering far more site-specificity. Vetting numerous tyrosine integrases by assay, with diverse site-specifities, is expected to expand safe gene editing biotechnology.

Funding Information

Sandia National Laboratories is managed and operated by National Technology & Engineering Solutions of Sandia, LLC, under the DOE National Nuclear Security Administration contract DE-NA0003525.