#CRISPR interference is great because it lets you use a single dCas9 to turn off multiple loci with different gRNAs. But this has a cost: all those gRNAs compete for the same dCas9 pool. Here, we solve this with a feedback loop! A tweetorial: https://abs.twimg.com/emoji/v2/... draggable="false" alt="👇🏾" title="Rückhand Zeigefinger nach unten (durchschnittlich dunkler Hautton)" aria-label="Emoji: Rückhand Zeigefinger nach unten (durchschnittlich dunkler Hautton)">1/5 #synbio https://www.biorxiv.org/content/10.1101/2020.08.11.246561v1">https://www.biorxiv.org/content/1...

In this work, we show how using multiple gRNAs in a single synthetic biology circuit can result in a decrease in repression efficiency. In this "open loop" system, more targets -> more competition -> less repression for each target -> https://abs.twimg.com/emoji/v2/... draggable="false" alt="☹️" title="Stirnrunzelndes Gesicht" aria-label="Emoji: Stirnrunzelndes Gesicht"> . 2/5

We then show how adding a extra gRNA that represses dCas9 itself can solve this issue. In this "closed loop" system, more targets -> more competition -> less repression -> more dCas9 -> repression back to normal https://abs.twimg.com/emoji/v2/... draggable="false" alt="😃" title="Lächelndes Gesicht mit geöffnetem Mund" aria-label="Emoji: Lächelndes Gesicht mit geöffnetem Mund">! 3/5

This approach allows us to build complex synthetic gene circuits with multiple components based on CRISPRi, while preserving robust, stable dCas9 behavior. Also it& #39;s a fun example of modeling and wet lab feeding off of each other! 4/5

The work was a great collaboration across institutions, spearheaded by the brilliant Hsin-Ho Huang and @yili_qian (Del Vecchio Lab @mitmech) and @Massimo_Bellato ( @BMSlab_unipv). I myself ( @MITdeptofBE) was lucky enough to help problem-solve how the system is put together! 5/5