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. [Figure not available: see fulltext.]
Bibliografisk noteFunding Information:
We thank G. Kuznetsov and G. Squyres for reviewing the manuscript and providing helpful feedback. We thank C. Bell for discussions about possible mechanisms of recombineering and J.P. Van Pijkeren along with J.-H. Oh for helping us set up the initial protocols for L. lactis. This work was supported by the National Institute of General Medical Sciences under grant no. 1U01GM110714-01 and the Department of Energy with DE-FG02-02ER63445 to G.M.C. This work was also supported by NIH grant no. R01GM082899 to M.T.L., who is an Investigator of the Howard Hughes Medical Institute. G.T.F. was supported by the National Science Foundation Graduate Research
Fellowship under grant no. DGE1745303. D.A.S. was supported by a Landry Cancer Biology Research Fellowship. K.G. was supported by the National Science Foundation Graduate Research Fellowship under grant no. DGE1745302.
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