• Replicable Genetic Element:Consider using a plasmid as the genetic element. Plasmids are replicable in E. coli and can harbor specific DNA sequences. Design a plasmid with a region that forms the desired ssDNA bubble.
• Bubble Stability:To avoid proteins or toxic chemicals, focus on intrinsic DNA properties. Explore sequences with low stability (e.g., high AT content) to promote bubble formation. Ensure the bubble remains accessible for protein interactions.
• Bioorthogonal Conditions:Opt for sequences that are stable under physiological conditions (pH, salt, temperature). Test the bubble’s stability at 37 °C in E. coli

Since the E.coli contains specific DNA sequences try to use plasmid which contains the desire ssDNA bubble in it sequence.

Any "overexpression" trancscription bubble is open all of the time. T7 produces RNA like hell, which is why it's used for overexpression with the assertion "much RNA means much protein".

I wonder how you'd want to get a transciption bubble without proteins "supporting" it, after all it is created by proteins. You'd have to be more specific on your requirements.

Andre Marschall

The results of a colleague suggest that Cas9 proteins bind to transcription bubbles next to their respective PAM (https://doi.org/10.1038/s41594-019-0188-z). This binding can lead to cleavage or other off-target events via base/prime editors. We can perform high throughput screenings/directed evolution within E. coli. Each cell contains one different Cas9 variant. The big clue would be to have an open "trancsription bubble" next to the PAM on the same plasmid carrying Cas9. It would therefore cleave itself and only non-off-target binding Cas9s remain (there is a second step reassuring active cleavage and editing efficiency as readout).

Or could you think of any other way, utilizing this off-target ssDNA bubble binding by Cas9 as screening readout within a HTS in E. coli?

Well, creating a semi-permeable transcription bubble can be challenging in the context of the structural stability of DNA as it tends to reanneal to its double-stranded form. Next is the concern of replication, which involves the fact that semi-permanent unwinding can potentially hinder the replication machinery from proceeding with DNA replication. Lastly, to maintain the transcription bubble to its semi-permanent unwound state, RNA polymerase is required to be halted in its activity at the transcription site, which could, in turn, lead to instability and interference in the replication of the plasmid. Considering these aspects, I believe three probable yet theoretical strategies can be adopted in this regard. First is genetically engineering a modified RNA polymerase, which can maintain the plasmid DNA at its single-stranded state by getting associated at a precise plasmid location without hindering the transcription process. Second is implementing genetically engineered single-strand binding (SSB) proteins, which can keep the plasmid DNA at its unwound state without interfering with RNA synthesis. Lastly, chemical molecules such as intercalating agents are introduced, which can develop proximal unwinding by being inserted at the nitrogenous base pairs of plasmid DNA; Molecules that are enhancers or activators of helicases; Hydrogen bond destabilizers like Di-Methyl Sulfoxide (DMSO), Urea or Formamide which can perform denaturation of double-stranded DNA; Cross-linking agents like Psoralens which forms covalent cross-linkages between single-stranded DNA molecules and DNA or RNA polymerases; Ligands which associate with single-stranded DNA such as Peptide Nucleic Acids (PNAs) and nucleic analogs which stabilizes the single-stranded structures of DNA; Alkylating agents such as Nitrogen and Sulfur derivatives of Mustard gas, Ethyl Methanesulfonate (EMS), Methyl Methanesulfonate (MMS), N-Nitrosoureas and Temozolomide. Nevertheless, besides being hypothetical, all these strategies have cons of their own.