Delighted to share our latest on #CRISPR In this paper we tried to determine the mechanisms of trans-cleavage activity of #CRISPR Cas12a enzymes, property used for molecular detection. Bottom line -> electrostatic interactions near the RuvC active site are critical.

www.biorxiv.org/content/10.1...

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 !

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.

Happy to share our latest paper in collaboration with A/Prof Giulia Palermo's lab at UC Riverside on #CRISPR Cas12a cleavage. Here we describe 1/ Why CRISPR Cas12 orthologues are so different despite similar structure 2/ We focused on a specific part of the protein

www.biorxiv.org/content/10.1...

Great to see this work from Gavin Knott's group out on the De novo design of #CRISPR Cas13 inhibitors aka (AIcrs) using RF-Diffusion. Great potency in inhibiting Cas13 nuclease.

www.biorxiv.org/content/10.1...