I wanna use Cas9/sgRNA complex to edit E. coli genome.
Is it workable?
Cas9/sgRNA complex is in solution.
Transformation method could be chemical (Ca+) or electrophoration.
if you have prefect Cas9/sgRNA complex, you can easily do transformation with that either by using Chemical/ Electroporation method.
but the result will depends on your Cas9/sgRNA complex designing and the specific region of your gene of interest in Ecoli which you want to edit.
Is there a specific reason you'd use this instead of something with a high success rate like Lambda Red or allelic exchange?
This sounds like a complicated and expensive way to make mutations compared to pretty much any other way. Also the actual rate of mutation will probably be low because the typical result will probably either be a dead cell or homologous recombination that restores the wild-type sequence.
Firstly, I make a 95% purity of Cas9 protein solution, then synthesise sgRNA (one or more sgRNA targeting one or more genes) in advance. Secondly, I am planning to mix Cas9 protein and sgRNA, enable them to integrate together in vitro in tube. When I wanna target any gene, I could just synthesize targeting sgRNA in advance, then delivering them to E. coli. Finally, I just use PCR and corresponding primers to validate target gene.
As for whether adding template (dsDNA), this depends on whether all indel mutants are lethal. If few colonies can survive on plates. I could pick them all to run PCR.
An obvious cost could be in synthesis of ~20 bp sgRNA. And if targeting multiple sites could be required to synthesize more sgRNA.
And main operation is to take ready-made Cas9 protein to mix sgRNA, then transform complex to competent E. coli cell.
If we can use high concentration of competent E. coli cells and relatively high concentration of Cas9/sgRNA complex. Even many cells are lethal. few can survive and may be the desired mutant. Multiple targeting could be relatively low success rate, but single target sites or few targeting could be feasible.
How are you going to select for mutants vs. the wild type though? No matter how efficient your competent cells are, I think very few will actually be transformed with the Cas9/sgRNA complex. With how you've described it I think you would literally have to screen hundreds of thousands of colonies to find positives. People in my lab have electroporated Tn5 transposome complexes into various species of bacteria, and gotten hundreds of colonies per reaction with antibiotic selection. But that's hundreds of colonies out of billions of cells.
All mutagenesis studies I've seen using CRISPR-Cas in bacteria have the guide RNA and Cas9 gene on plasmid(s) that can be selected for.
Selection method is to use T7E1(T7 endonuclease 1) which can cut bulge of dsDNA between wild type DNA chain and mutant DNA chain.
How mmany is the molecular weight of Tn5 transposome complex?
Cas9 protein is 158.4 KDa
Here is someone who make a λ red cassette and Cas9 in a plasmid to knockout bacterial gene.
https://www.addgene.org/browse/sequence/198231/
I don't think you really understand what I'm saying. What you are describing has no way to kill off wild-type cells that have not been transformed with the complex. This will be almost ALL of your cells. T7E1 isn't going to kill wild-type cells and allow the mutant to grow. You're completely lacking a method to narrow down colonies that might contain the desired mutations. There's no selective advantage for them.