Abstract
CRISPR-based transcription regulators (CRISPR-TRs) have transformed the current synthetic biology landscape by allowing specific activation or repression of any target gene. Here we report a modular and versatile framework enabling rapid implementation of inducible CRISPR-TRs in mammalian cells. This strategy relies on the design of a spacer-blocking hairpin (SBH) structure at the 5′ end of the single guide RNA (sgRNA), which abrogates the function of CRISPR-transcriptional activators. By replacing the SBH loop with ligand-controlled RNA-cleaving units, we demonstrate conditional activation of quiescent sgRNAs programmed to respond to genetically encoded or externally delivered triggers. We use this system to couple multiple synthetic and endogenous target genes with specific inducers, and assemble gene regulatory modules demonstrating parallel and orthogonal transcriptional programs. We anticipate that this ‘plug and play’ approach will be a valuable addition to the synthetic biology toolkit, facilitating the understanding of natural gene circuits and the design of cell-based therapeutic strategies.
Highlights
clustered regularly interspaced short palindromic repeats (CRISPR)-based transcription regulators (CRISPR-TRs) have transformed the current synthetic biology landscape by allowing specific activation or repression of any target gene
Binding to specific DNA targets occurs when the single guide RNA (sgRNA)–Cas[9] complex encounters a spacer-matching sequence upstream of an NGG protospacer adjacent motif[11]. Since this process is dependent on Watson– Crick base pairing, we reasoned that appending a spacercomplementary ‘back-fold’ extension at the 50 end of the sgRNA would generate a ‘spacer blocking hairpin’, effectively silencing CRISPR-TR activity (Fig. 1b)
The advantage of using sgRNA-based inducible systems for synthetic biology applications has been recently showcased in a study demonstrating the ability to rewire cellular pathways by CRISPR-TR with modified sgRNAs containing ligand-responsive riboswitches[29]
Summary
CRISPR-based transcription regulators (CRISPR-TRs) have transformed the current synthetic biology landscape by allowing specific activation or repression of any target gene. We report a modular and versatile framework enabling rapid implementation of inducible CRISPR-TRs in mammalian cells This strategy relies on the design of a spacer-blocking hairpin (SBH) structure at the 50 end of the single guide RNA (sgRNA), which abrogates the function of CRISPR-transcriptional activators. By replacing the SBH loop with ligandcontrolled RNA-cleaving units, we demonstrate conditional activation of quiescent sgRNAs programmed to respond to genetically encoded or externally delivered triggers We use this system to couple multiple synthetic and endogenous target genes with specific inducers, and assemble gene regulatory modules demonstrating parallel and orthogonal transcriptional programs. While Cas[9] variants with divergent protospacer adjacent motif specificities can provide an orthogonal framework for CRISPR-TRs8, their utility in the design of inducible systems is mitigated by the necessity of extensive protein engineering and the metabolic costs associated with protein delivery To address these limitations, we have developed a versatile inducible-CRISPR-TR platform based on minimal engineering of the sgRNA. Using this platform, we demonstrate highly specific parallel activation of multiple genes using a single inducer and independent control with orthogonal inducer/gene target pairs
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