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Inducible Spacer Blocking Hairpins (iSBH)


CRISPR-based transcription regulators (CRISPR-TR) 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.
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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.
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iSBH design using iSBHfold


iSBHfold is a webtool allowing users to design their own iSBH-sgRNAs. The algorithm takes as input a set of user defined spacer sequences (also know as sgRNA guide sequence). After choosing which inducer (endoribonucleases and/or ASOs) is to control which sgRNA, iSBHfold will compute the DNA sequences of the corresponding iSBHs. Additionally RNA secondary structure predictions are given along with primer sequences to be used to clone the iSBHs in a give sgRNA scaffold expressing plasmid.
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Created, designed and implemented by Quentin RV FERRY (2017), Hosted by CBRG