Abstract
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.
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Data availability
NGS data for the L. lactis spectinomycin experiment are deposited as a NCBI BioProject under accession no. PRJNA648295. All data and constructs are available from the authors upon reasonable request. For L. rhamnosus plasmids, contact the corresponding authors or anik@tenza.bio. Source data are provided with this paper.
Code availability
The scripts used to analyze doubling times in MATLAB, analyze Illumina sequencing data and generate the adjacency matrix for the force-directed graph are available upon request.
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Acknowledgements
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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Contributions
G.T.F., T.M.W. and G.M.C. conceived the study. X.R., C.J.G. and M.J.L. contributed to project conception. G.T.F. designed the bacterial experiments. T.M.W., F.B.P., I.D.L., J.Z., K.G. and A.D. contributed to bacterial experimental design. G.T.F., F.B.P., I.D.L. and J.Z. carried out the experiments in E. coli, L. lactis, M. smegmatis, C. crescentus and L. rhamnosus and analyzed the data. D.A.S. performed bioinformatic analysis and K.G. performed C. crescentus experiments under the supervision of M.T.L. T.M.W. designed and performed the oligonucleotide annealing experiments. A.D. contributed to experiments in L. rhamnosus. H.K., V.V. and S.W. contributed to the bacterial experiments. G.T.F. wrote the manuscript with input from all other authors. G.T.F. and F.B.P. generated the figures. S.L.S. and J.A. provided supervision. G.M.C. supervised the study.
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G.M.C. is a founder of 64-x, enEvolv and GRO Biosciences. G.T.F., T.M.W. and G.M.C. are named inventors on a patent application related to the technologies described in this Article. Other potentially relevant financial interests are listed at http://arep.med.harvard.edu/gmc/tech.html.
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Extended data
Extended Data Fig. 1 Bicistronic RBS optimization.
In L. lactis, the internal RBS sequence affected recombination efficiency using the bicistronic λ-Red β and EcSSB construct. a, b, RBS 2, which enabled the highest efficiency genome editing in this experiment was selected used in all other constructs unless otherwise indicated. Error bars indicate s.d. from the mean of at least four biologically independent replicates.
Extended Data Fig. 2 Doubling times in E. coli of constructs expressing SSAPs and SSBs reveal that co-expression of SSB can dramatically influence toxicity.
a, Growth curve of cognate SSAP-SSB pairs and SSAPs alone in E. coli under constant induction (7hrs). b, Doubling time measurements for all combinations of the 4 SSAPs and SSBs in E. coli under constant induction (7hrs) with mean and s.d. presented for at least 3 biologically independent replicates. The SSAPs vary in toxicity, with λ-Red β showing considerable toxicity. The co-expression of SSBs reduces SSAP toxicity in a number of cases, especially for PaSSB. There are a number of constructs with low toxicity and high genome editing efficiency (λ-Red β + EcSSB, λ-Red β + PaSSB, PapRecT + PaSSB) showing that there is no direct correlation between toxicity and genome editing efficiency.
Extended Data Fig. 3 Optimization of recombineering efficiency in L. lactis.
a, Optimization of nisin concentration to 10 ng/ml contributed to a significant improvement in genome editing efficiency for PapRecT + PaSSB. a, The optimal oligo amount plateaued at 50 μg of DNA, which corresponds 21.4 μM in 80 μL. b, Expression of the L. lactis MutL variant E33K allowed the efficient introduction of 1 bp mismatches at similar efficiency to 4 bp mismatches which evade MMR. c, d, After optimization from (a, b), PapRecT + PaSSB + LlMutLE33K enabled >20% editing efficiency at the Rif locus (c), and efficient multiplexed editing (d). Error bars indicate s.d. from the mean of at least three biologically independent replicates. b, *: P value < .05; ordinary one-way ANOVA of Log-transformed data, Holm-Sidak multiple comparisons test.
Extended Data Fig. 4 DsDNA recombineering with PapRecT and PaSSB in L. lactis.
Although this work mostly focused on ssDNA recombineering, dsDNA recombineering can be used to integrate larger constructs including genes and resistance markers, and usually requires the presence of a cognate phage exonuclease. These proteins are almost always found within the phage operon containing the SSAP, and can be readily co-expressed to enable dsDNA recombineering. Surprisingly, we find that PapRecT + PaSSB enabled dsDNA recombineering in L. lactis even without including a cognate phage exonuclease suggesting that the co-expressed SSB recruits an endogenous exonuclease, or the SSAP+SSB pair provides the sufficient requirements for dsDNA recombineering. (a) Gene knockins were performed in L. lactis using linear DNA with 500 bp homology arms carrying an Erythromycin resistance cassette. (b) Co-expression of PapRecT + PaSSB enabled the efficient introduction of a 1 kb selectable marker as dsDNA even without the addition of the cognate phage exonuclease. Error bars indicate s.d. from the mean of at least three biologically independent replicates.
Extended Data Fig. 5 Heat maps of RpsE mutagenesis at different mutational depths.
The 5NNK library diversification experiment (Fig. 5) allow us to identify antibiotic resistant single, double, triple, or quintuple mutants when the other codons have WT amino acids. (a) heat maps showing the enrichment of amino acids in the 5NNK library, filtered to separately present those with 1, 2, 3, or 4 mutations vs. WT. The enriched amino acids change at increasing mutational depth. The “5NNK: 1 mutation” library has mutations enriched in the first 2 positions (28 V, and 29 K) similar to the 5x1NNK single-amino acid mutagenesis heat map, while the “5NNK: 4 mutation” library looks similar to the 5NNK library heat map (Fig. 5b), with an enrichment to polar and charged residues at all 5 positions.
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Filsinger, G.T., Wannier, T.M., Pedersen, F.B. et al. Characterizing the portability of phage-encoded homologous recombination proteins. Nat Chem Biol 17, 394–402 (2021). https://doi.org/10.1038/s41589-020-00710-5
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DOI: https://doi.org/10.1038/s41589-020-00710-5
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