Thread by @reworkman0

Excited to share my first first-author paper from my PhD in the @jwmodell lab, out in @CellCellPress today!! Read about how we discovered bacteria evolved their very own CRISPRi sgRNAs to control their native CRISPR-Cas systems, story thread below (authors.elsevier.com/c/1cNb~L7PXcMJ7)

Our story starts with a twist! When we performed a transposon screen to search for bacterial regulators of CRISPR immunity, instead of identifying host factors we actually found that disruption of the long isoform of tracrRNA (tracr-L) increased immunity against phage infection!

Before now, tracrRNA was considered only an essential scaffold for crRNA, which directs Cas9 to its targets. In genome engineering, tracrRNA and crRNA are fused to form the sgRNAs we all know and love. But why tracrRNA is made by two promoters has been a mystery.

So we hypothesized that tracr-L could have a unique role to play in CRISPR-Cas immunity. We found that tracr-L deletion impacts CRISPR memory formation and usage, and can be rescued by tracr-L complementation. So tracr-L is a repressor of CRISPR-Cas immunity.

But how is tracr-L working? Dtracr-L cells have dramatically increased Cas9 protein levels, Cas operon mRNA levels, tracr-S and crRNA levels, which explains why immunity is so much more active. But again, how is this repressor working?

We discovered the 5’ end of tracr-L contains a short sequence with a perfect match in the Cas operon, adjacent to a PAM, which Cas9 uses to find its targets.This made us wonder if perhaps bacteria had figured out about sgRNAs before us…

Long story short they have! Tracr-L forms a natural sgRNA, sans crRNA, that binds to Cas9 to form a repressive RNP, and then binds to the Cas operon promoter to repress transcription of the Cas operon. But you may be wondering at this point, why would bacteria want this?

We think that the heightened immunity associated with CRISPR-Cas overexpression also leads to heightened lethal autoimmunity. When we competed WT and Dtracr-L cells against each other, WT handily won in a matter of days!

So we think tracr-L acts as a buffer to maintain Cas expression and activity within acceptable levels to mitigate autoimmunity, and allow CRISPR-Cas to be induced when needed. But when is it needed and what de-represses tracr-L?

These are big questions going forward that I hope to study during the remainder of my PhD, so stay tuned! Thanks for reading, and thank you so much to all my co-authors, collaborators, and CRISPR scientists who came before who made this work happen!