lac and thyA fail at 1 MOI but J23119 and thyA succeeds at 1 MOI. Why does thyA help mitigate the toxicity effects with only J23119? I theorize it has something to do with the higher baseline expression levels of J23119 compared to lac. Because lac is inducible, any gene driven by it will take some time after induction to ramp up while any gene driven by J23119 is always highly expressed.

Interesting that the MOI has no impact of the efficacy of the edit with the lac promoter. Additionally, the variability at 10 MOI for lac is extreme, it seems like a large portion of them outright failed. What is it about lac that prevents increasing the MOI from increasing the efficiency? What is it about lac that causes such a high variability for lac 10 MOI?

Cas13a being used its intended way (to destroy a virus) as part of an experimental screening is interesting. I wonder what applications the Cas proteins have while serving their native functions.

An edited population percent of ~0.005% is a pretty low efficiency. That is 1/20,000 cells. Is this sufficient for downstream applications? The specificity is good though. Perhaps increase the efficiency may cause an increase in off target effects.

Contrary to Cas9's moderate tolerance for mismatches in the crRNA, it does not seem to tolerate mismatches in the PAM site very well. This would be beneficial to the bacteria as leniency with PAM sites could open the door for self targeting. For biotechnology applications, this would be a benefit as it would contribute towards the ability to precisely control where Cas9 is cutting.

Cas9 can tolerate a reasonably high degree of mismatch, demonstrating some cleavage with up to 6bp of mismatch (notably, position matters and this is the first 6bp of the guide RNA). This would be useful as it allows the bacteria to potentially overcome minor phage mutations. For biotechnology applications however, this means extra caution must be taken when designing guide RNA. Off-target cleavage is a real concern.

The ability to cleave a supercoiled plasmid would be essential to the current modern day applications of CRISPR. Genomic DNA is often naturally supercoiled, specifically, the majority of the human genome is supercoiled around histones. Bacteria's genomic DNA is also supercoiled. If a CRISPR system is only effective on linear DNA its real world applications would fall short.

tracrRNA is essential for the guide RNA to properly interface with the Cas9 protein complex. Without it, the crRNA is unable to interact with the Cas9 protein and cannot be used to find a specific sequence. This states the critically of both parts of guide RNA to the functioning of a CRISPR system as a whole.