Controlling the Activity of CRISPR Transcriptional Regulators with Inducible sgRNAs.

The type-II CRISPR-Cas9 system has been repurposed to create synthetic programmable transcriptional regulators (CRISPR-TRs). Subsequent modifications of the system now allow for spatiotemporal control of CRISPR-mediated gene activation and repression. Among these solutions, the development of inducible spacer-blocking hairpin guide RNAs (iSBH-sgRNAs) provide an easy to implement and versatile way to condition the activation of most CRISPR-TRs on the presence of a user defined inducer.

Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression.

Spatial/temporal control of Cas9 guide RNA expression could considerably expand the utility of CRISPR-based technologies. Current approaches based on tRNA processing offer a promising strategy but suffer from high background. Here, to address this limitation, we present a screening platform which allows simultaneous measurements of the promoter strength, 5', and 3' processing efficiencies across a library of tRNA variants.

In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering.

RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs.

Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs.

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.