High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides

TLDR

Homologous recombination is a fundamental but rare process that requires complex reactions and extensive homology. The study demonstrates that the Beta protein of phage λ can generate chromosomal recombinants using synthetic single‑stranded DNAs as short as 30 bases. Beta protein, a Rad52‑like protein, binds and anneals the ssDNA donor to a complementary single strand near the replication fork to create the recombinant. The ssDNA recombination achieves up to 6 % efficiency, enabling mutagenesis and repair of chromosomes and providing a broadly applicable tool for genome editing across bacteria and eukaryotes.

Abstract

Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage λ generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.