While we are on #CRISPR Cas12a, please check out our latest in NAR on the interaction between the Rec2 and Nuc domain of Cas12a that drives target strand cleavage, critical for the Cas12a function. academic.oup.com/nar/article/...

Thanks Vivek, more to come, hopefully soon.

Brief, still a lot of unknown for such small genome. MS2 and in general RNA viruses are really fascinating to observe. Their ability to hijack the host, adapt and thrive is truly incredible.

Final words. Congrats to Ni for being the driving force of this work.

We looked at RNA modification (MS2 must hijack the host to modify its RNA) and the short take is once the RNA modification is there in the full transcript, it doesn't go away. However the short transcripts are amenable to modification. PseudoU are more prone to modification in MS2

We looked at these transcripts. these are 1/ the full length coat protein, require for repression of the replicase and assembly 2/ by products RNA species "parasitic RNA" from the replicase, which is error-prone 3/ We found tones of hybrid reads and de novo mutations. MS2 adapts really quickly !

To replicate, as many viruses, MS2 requires a negative strand template. We looked at the gene expression and read coverage and oh surprise, we found tones of small transcripts (<800 nt). We thought these were not true transcripts, but turned out to be real and accounted over 50% of the read count.

We performed a MS2 phage infection into its natural host (E.coli + F plasmid, critical for MS2 entry). We followed the infection throughout the entire cycle and Nanopore sequenced. 1/ MS2 grows incredibly fast. Formed plaques and 500x fold increase in viral RNA in only 3 hours !

Congrats to the team, especially to Tony and Aakash that have driven the work, Lora for all the mammalian cell work. Pablo from the Palermo lab and of course Giulia on this collaboration.

Conclusion the difference in kinetics in cleavage and the number of contacts with the target strands determines the competition between the DNA strands to access to the catalytic residues, which drives the differences in cleavage behaviour of these Cas12a enzymes.

We next wondered how the NUC/gate are the driver of these differences between the Cas12a orthologues? To answer this question the Palermo lab performed series of molecular dynamics simulations. They showed that the difference in flexibility in this region between the orthologues is the driver.

On the target trajectory, we looked at an important region called the NUC loop that is extended in AsCas12a but not other orthologues. We wondered wether this region is important. Short answer is yes. by mutating this region, it kills all activities.

We first looked at an important part of the protein called the "gate" mutation. This is a critical checkpoint on Cas12a to direct the target DNA to the catalytic site. We found this region is important to drive the competition btw the ≠ DNA strands (unlike Cas9, Cas12a has only one cutting site)

We started with a simple question. Why despite a similar structure, #CRISPR Cas12a vary so much in their function aka LbCas12a more prone to trans-cleavage (molecular detection) whereas FnCas12a doesn't work at all in mammalian cells? We focused on a critical region that interacts with the target.

Thank you mate. Great to see you here again

Will run the analyses and will post here soon