RESUMO
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. In this chapter, I cover the know-how relating to the design and synthesis of iSBH-sgRNAs, as well as the implementation in mammalian cells of inducible CRISPR-TR strategies based on this technology.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Ativação Transcricional/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
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. This analysis reveals that the sequence determinants underlying these activities, while overlapping, are dissociable. Rational design based on the ensuing principles allowed us to engineer an improved tRNA scaffold that enables highly specific guide RNA production from a Pol-II promoter. When benchmarked against other reported systems this tRNA scaffold is superior to most alternatives, and is equivalent in function to an optimized version of the Csy4-based guide RNA release system. The results and methods described in this manuscript enable avenues of research both in genome engineering and basic tRNA biology.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , RNA Polimerase II/genética , RNA Guia de Cinetoplastídeos/genética , RNA de Transferência/genética , Edição de Genes , Regulação da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismoRESUMO
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. We use GenERA to survey the entire regulatory landscape of a 3'UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Regiões 3' não Traduzidas/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma/genética , Humanos , MicroRNAs/genética , Elementos de Resposta/genéticaRESUMO
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.
