RNA-based engineering of inducible CRISPR-Cas9 transcription factors for de novo assembly of eukaryotic gene circuits

Synthetic biology in mammalian cells holds great promise for reverse engineering biological processes and rewiring cellular behaviors for therapeutic purpose. An essential aspect in our ability to reprogram the cellular code is the availability of highly orthogonal, inducible transcriptional regulators. CRISPR-based strategies employing effector-domain tethering to the single guide RNA (sgRNA)-dCas9 complex have greatly advanced this field by allowing for precise activation or repression of any gene via simple sgRNA reprograming. However, the implementation of inducible CRISPR-based transcriptional regulators (CRISPR-TRs) has so far been restricted to dCas9 protein engineering and conditional effector tethering. Although elegant, these approaches are limited by dCas9 promiscuous loading of sgRNAs, which hinders their use for the creation of independent multi-gene transcriptional programs. To address this limitation, I have developed a modular framework for the rational design of inducible CRISPR-TR, based on simple and reversible modifications of the sgRNA sequence. At the core of this conceptual framework lies the ability to inactivate native sgRNAs by appending on their 5’-end a short RNA segment, which folds to form a spacer-blocking hairpin (SBH). Base-pairing between the extension and the sgRNA spacer prevents docking of the CRISPR-TR on-target, fully abrogating its activity. Subsequently, I have created inducible SBH variants (iSBH) by replacing the hairpin loop with conditional RNA cleaving units. Using a variety of sensing-loops, I was able to engineer a panel of switchable iSBH-sgRNAs, designed to activate specifically in the presence of protein, oligonucleotide, and small molecule inducers. Leveraging the versatility of this method, I demonstrate that iSBH-sgRNAs expression can be multiplexed to assemble synthetic gene circuits implementing parallel and orthogonal regulation of multiple endogenous gene targets. Finally, I have distilled the design principles derived throughout this project to develop a web tool that automates the creation of iSBH- sgRNAs. Already a valuable addition to the synthetic biology toolkit, iSBH-based inducibility should in theory also be applicate to all CRISPR-Cas9 derivatives (genome editing, epigenetic alteration, DNA labelling, etc.) as well as other newly characterized RNA-guide nucleases from the CRISPR family.