Characterizing the portability of phage-encoded homologous recombination proteins

Nat Chem Biol. 2021 Apr;17(4):394-402. doi: 10.1038/s41589-020-00710-5. Epub 2021 Jan 18.


Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host's single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Bacteriophages / genetics
  • Bacteriophages / metabolism
  • Caulobacter crescentus / metabolism
  • DNA / chemistry
  • DNA / genetics
  • DNA Repair
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • Escherichia coli / metabolism
  • Gene Editing / methods*
  • Homologous Recombination / genetics
  • Homologous Recombination / physiology*
  • Lactococcus / metabolism
  • Mycobacterium smegmatis / metabolism
  • Protein Domains / genetics


  • Bacterial Proteins
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • DNA