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#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: 
1/5 #synbio https://www.biorxiv.org/content/10.1101/2020.08.11.246561v1
1/5 #synbio https://www.biorxiv.org/content/10.1101/2020.08.11.246561v1
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 -> 
. 2/5
. 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 
! 3/5
! 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'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
