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If you need more anti-CRISPRs in your life, this is the paper for you! I worked on this study with a wonderful team: @AdairLBorges @jenzyy_97 @allysonparkk and @joeBondyDenomy. We discovered SEVEN Type I-C Acrs and they have some pretty cool properties! https://www.biorxiv.org/content/10.1101/2020.06.15.151498v1
I seem to think the Type I-C CRISPR-Cas system is pretty cool: @BCsorgo and I posted this preprint some months ago, showing how this system can be manipulated for large-scale genome engineering https://www.biorxiv.org/content/10.1101/860999v1
In the latest pre-print, I focus on the biology of Type I-C systems. Where are they encoded? What sorts of elements do they target? And can we identify inhibitors?
At the first JC I went to in the JBD lab in 2015, @AdairLBorges presented this paper: https://mbio.asm.org/content/6/6/e01796-15.abstract
In it, the team identifies Type I-C systems in P. aeruginosa for the first time. They also find that they are encoded on large mobile islands!
In it, the team identifies Type I-C systems in P. aeruginosa for the first time. They also find that they are encoded on large mobile islands!
I dug around for more examples, and found lots more type I-C systems in Pa. We even had a strain in the lab that had a Type I-C system, so we got to work!
These systems had pretty diverse CRISPR arrays. The 47 strains had 300 unique spacers. But I did find that the strains were inter-related. They could be clustered into lineages based on spacer content!
Even our lab strain fit in. It shared most of its spacers with the strains from van Belkum et al, and one spacer that linked it to the group of new strains that I identified!
We found the first new Acr by tracing a self-targeting spacer. The genome it was in had a previously identified anti-CRISPR, AcrIF2. So we tested it against our Type I-C system, and voila! AcrIF2 moonlights as a Type I-C inhibitor!
It turns out that AcrIF2 was hiding a lot more than that though. The Wiedenheft group had published a structure of AcrIF2 bound to the I-F surveillance complex. It seemed to be a straightforward mech: pretend to be DNA, and titrate all those CRISPR-Cas complexes away.
We figured the acidic residues were key. We had determined that AcrIF2 also bound to the I-C complex. So by breaking the acidic residues, surely we could try to disable this anti-CRISPR.
But this was the little protein that could. It refused to falter. It bravely inhibited both systems, no matter the perturbation. I replaced 1, then 2, then 3, then 4 of the acidic residues with alanine, the world's most boring amino acid. And yet it still functioned.
What was going on? It took 8 a.a. swaps to see a change (& this is a tiny 96 a.a. protein): our lab strain actually encodes two CRISPR-Cas systems, I-C, and I-F. The mutant acr, expressed from a phage, suddenly did very poorly against the I-C system.
But are you ready for another twist? When this same mutant phage was tested in strains that encode just one of these systems at a time, it was suddenly quite strong again!
Stacking two systems against one protein was its demise. There appears to be a fitness cost to being a dual inhibitor for the mutant phage. Those extra acidic residues seem to be really important in maintaining the potency of F2 when faced with many complexes in one cell.
This was especially interesting to us because all but one of the new I-C inhibitors were very acidic, just like AcrIF2. Are they all nucleic acid mimics of some kind? And like AcrIF2, our new anti-CRISPRs packed a lot of punch. Three of them were dual I-C / I-E inhibitors.
And two of them even inhibited the Eggethella lenta Type I-C system! Those are some big phylogenetic hoops! If you want to read more about gut bacterial I-C systems, check out this paper by @pa_sope https://www.cell.com/cell-host-microbe/pdfExtended/S1931-3128(19)30417-2
The new Acrs are found in diverse places, like islands, transposons, & of course, phage. On the one hand, you have an easily transferable I-C system, floating along in a conjugative element. & on the other hand, you have other MGEs sneaking in Acrs that disable diverse systems!
