Okay so this answers my earlier question about observed replication after eliminating the lysogeny machinery from the genome. The replication-competent lambda-DART can still generate infectious particles that can systemically spread to other susceptible bacteria, but they deliberately left this as a risk because it provided the tradeoff of enhanced host editing.

So even though they had removed attp and integrase from the genome, lysogenic replication still occurred, if I am interpreting this correctly?

It seems like the paper's own data demonstrates that lambda-DART remains replication-competent and persists within the bacterial population. So they were able to prevent prophage integration but they still don't have complete control over the persistence or propagation of the phage-based editing vector?

I wonder why the authors are omitting the specific details of which constructs they did this step on?

So initially the editing efficiency was 1%?

So they basically removed a 12.3-kb chunk of lambda DNA, which includes attP + integrase (needed for lysogenization), and replaced it with the a 10.8-kb DART genome-editing system to create the new lambda-DART phages?

It's interesting how this DNA was non-essential enough to be removed without completely destroying the genome, but still performs an important function under the normal circumstances. This makes me think about so-called "junk DNA" and how whole chunks of the genome were initially thought to serve no purpose but later were identified as serving critical biological roles. I wonder if someone revisits this experiment in a few decades if we will have learned that this removal had unforeseen consequences that altered the results of the study. There is still so much we do not know about the functions of genetic components.

It's interesting thinking about Cas13a and Cas9, since Cas9 targets dsDNA and permanently edits it, whereas it seems like Cas13a transiently modifies ssRNA without modifications to the genome. Also, I wonder why trans-cleavage specifically? I bet it has to do with steric repulsion and hindrance, since simple peptide molecules favor the trans configuration to reduce steric strain from bulky side chains.

So this is why they modified the phage to prevent it from entering the lysogeny phase, so it wouldn't destroy the host bacteria?

If I'm interpreting this correctly, one of the failsafe measures they used in the lambda phage to control viral reproduction is designing it to be able to deliver and enter the bacterial cell and but it cannot establish a lysogenic state for viral reproduction? The lytic pathway still exists though, right? Because the phage needs to be able to propagate itself to function.

I had to look up what this is because I couldn't remember anything from the slides / lectures or if we had covered this at all, but these can induce single nucleotide polymorphisms (SNPs) in the DNA sequence without having to do a ds or ss break, super interesting!

What are they defining as minimal here? Is there a predetermined number of nucleotides that qualifies as minimal? It would be useful if they clarified their parameters more here.

I haven't gotten all of the way down the paper yet but I wonder if there is some non-engineered bacteriophage that could be used as an alternative method to using phage lambda. Most engineered features on recombinant genetic material come from other organism (example: GFP tag comes from the jellyfish Aequorea victoria), so it would be interesting to see if there was a "natural" (for lack of better word) equivalent that could perform the same or similar functions.