Characterizing the portability of phage-encoded homologous recombination proteins

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.

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 SD 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 standard deviation 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 10ng/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 1bp mismatches at similar efficiency to 4bp 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 SD 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 500bp homology arms carrying an Erythromycin resistance cassette. (b) Co-expression of PapRecT + PaSSB enabled the efficient introduction of a 1kb selectable marker as dsDNA even without the addition of the cognate phage exonuclease. Error bars indicate SD 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 (28V, and 29K) similar to the 5×1NNK 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.

Fig. 1:. SSBs are key mediators of SSAP functionality

(a) Model of ssDNA annealing inhibition by EcSSB or LlSSB, and ability of λ-Red β to overcome annealing inhibition by EcSSB. (b) In-vitro ssDNA annealing without SSB, pre-coated with EcSSB, or pre-coated with LlSSB. Shaded area represents the SEM of at least 2 replicates (c) In-vitro ssDNA annealing in the presence of λ-Red β when pre-coated with EcSSB or LlSSB. Shaded area represents the SEM of at least 2 replicates (d) Model for SSAP-mediated editing at the replication fork. An interaction between SSAP and the host SSB enables oligo annealing to the lagging strand. **Co-expressing an exogenous SSB that is compatible with the SSAP can in some species enable functionality even without host compatibility. (e),(f), The efficiency of editing in E. coli (e) and L. lactis (f) is compared using either SSAPs, SSBs, or “cognate pairs” (as described in the text). Error bars indicate SD from the mean of at least 3 biologically independent experiments. *: P value < .05; Welch’s two tailed t-test of Log-transformed data.

Fig. 2:. The C-terminal tail of SSB affects SSAP compatibility.

(a), A crystal structure of homotetrameric E. coli SSB bound to ssDNA (PDB-ID 1EYG). The amino acid sequence of the flexible C-terminal tail is diagramed in the right panel, along with the design of a 9AA C-terminal truncation to SSB. (b), The L. lactis SSB C-terminal tail is diagramed, along with an example of an SSB C-terminal tail replacement. In this case, the 9 C-terminal amino acids of the L. lactis SSB are replaced with the corresponding residues from E. coli SSB. The notation “LlSSB C9:EcSSB” is used as shorthand. (c), Editing efficiency in L. lactis of λ-Red β co-expressed with a 9AA C-terminally truncated EcSSB mutant. (d), Editing efficiency in L. lactis of λ-Red β co-expressed with LlSSB, or mutants of LlSSB with C3, C7, C8, or C9 terminal residues replaced with the corresponding residues from EcSSB. (e, f) Editing efficiency in L. lactis of PapRecT (e) or MspRecT (f) co-expressed with LlSSB, or mutants of LlSSB with the C7 or C8 terminal residues replaced with the corresponding residues from the cognate SSB. All experiments have at least 3 biologically independent replicates, error bars indicate SD from the mean. (c, d, e, f) *: P value < .05; ordinary one-way ANOVA of Log-transformed data, Holm-Sidak multiple comparisons test.

Fig. 3.. SSAP-SSB interactions match SSAP portability across bacterial species

(a, b) Heat map showing the fold improvement in editing efficiency due to SSB co-expression in (a) L. lactis or (b) E. coli of SSAP-SSB pairs as compared to the SSAP alone. (c), C-terminal sequences of SSBs as well as SSAP compatibility given by (a, b). (d), Editing efficiency in L. lactis of PapRecT co-expressed with LrSSB, MsSSB, or mutants of LrSSB which had the C7 or C8 terminal residues replaced with the corresponding residues from the MsSSB. (e), Editing efficiency in M. smegmatis of λ-Red β, PapRecT, MspRecT, and LrpRecT. (f), Editing efficiency in L. rhamnosus of λ-Red β, PapRecT, MspRecT, and LrpRecT. All experiments have at least 3 biologically independent replicates, error bars indicate SD from the mean. (d, e, f) *: P value < .05; ordinary one-way ANOVA of Log-transformed data, Holm-Sidak multiple comparisons test.

Fig. 4. SSAP-SSB pairs function across a broader host range than SSAPs alone.

(a), Editing efficiency in C. crescentus of two SSAP-SSB protein pairs, λ-Red β + PaSSB and PapRecT + PaSSB which had high genome editing efficiency in both E. coli and L. lactis. (b), Editing efficiency in C. crescentus of λ-Red β + PaSSB with ribosomal binding sites optimized for translation rate and using an oligo designed to evade mismatch repair. All experiments have at least 3 biologically independent replicates, error bars indicate SD from the men. (a, b) *: P value < .05; welch’s two tailed t-test of Log-transformed data.

Fig. 5:. Using SSAP-SSB pairs to interrogate complex phenotypic landscapes.

(a), Oligonucleotide design strategy. A sequence alignment of RpsE between E. coli, N. gonorrhoeae (a pathogen targeted by spectinomycin), and L. lactis shows a conserved 5AA region around the spectinomycin binding pocket (E. coli Lys26). 5 oligos can be pooled to introduce single degenerate codons at each amino acid position (5×1NNK), and a single oligo containing a fully degenerate sequence (5NNK) can be used to diversify the entire region. A crystal structure of E. coli RpsE bound to spectinomycin shows the approximate location of the antibiotic relative to the 5 targeted residues (Supplementary Fig. 12). (b), Normalized heat maps after selection and enrichment for the 5×1NNK single-amino acid saturation mutagenesis experiment vs. 5NNK combination mutagenesis experiment. (c), Force directed graph of all spectinomycin resistant combination mutants with at least 10 reads, lines connect variants that could be accessed through a single-nucleotide mutation, and the size of dots reflects the relative enrichment (d), Shortest paths to highly enriched double mutant FNGGR, as well as the nucleotide conversions required (e), Fitness of selected mutants in the presence and absence of antibiotic. Error bars represent the standard deviation from the mean of four biologically independent replicates.