Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.

Introduction

Eukaryotic cells achieve many different states by executing complex transcriptional programs that allow a single genome to be interpreted in numerous, distinct ways. In these programs, specific loci throughout the genome must be regulated independently. For example, during development, it is often critical to activate sets of genes associated with a new cell fate while simultaneously repressing sets of genes associated with a prior or alternative fate. Similarly, environmental conditions often trigger shifts in metabolic state, which requires activating a new set of enzymes and repressing other previously expressed enzymes, leading to new metabolic fluxes. These complex multi-locus, multi-directional expression programs are encoded largely by the pattern of transcriptional activators, repressors, or other regulators that assemble at distinct sites in the genome. Reprogramming these instructions to produce a different cell type or state thus requires precisely targeted changes in gene expression over a broad set of genes.

Gaj et al., 2013 Gaj T.

Gersbach C.A.

Barbas 3rd, C.F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Qi et al., 2013 Qi L.S.

Larson M.H.

Gilbert L.A.

Doudna J.A.

Weissman J.S.

Arkin A.P.

Lim W.A. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Gilbert et al., 2013 Gilbert L.A.

Larson M.H.

Morsut L.

Liu Z.

Brar G.A.

Torres S.E.

Stern-Ginossar N.

Brandman O.

Whitehead E.H.

Doudna J.A.

et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Mali et al., 2013a Mali P.

Aach J.

Stranges P.B.

Esvelt K.M.

Moosburner M.

Kosuri S.

Yang L.

Church G.M. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. How might we engineer novel gene expression programs that match the sophistication of natural programs? Such capabilities would provide powerful tools to probe how changes in gene expression programs lead to diverse cell types. These tools would also provide the ability to engineer more sophisticated designer cell types for therapeutic or biotechnological applications. Although a number of transcriptional engineering platforms have been developed, there are major constraints for constructing complex transcriptional programs. For example, synthetic transcription factors (such as designed zinc fingers or transcription activator-like [TAL] effectors) can target a specific regulatory action to a key genomic locus, but it is challenging to simultaneously target many loci in parallel because each DNA-binding protein must be individually designed and tested (). The bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats) interference system (CRISPRi) provides an alternative suite of tools for genome regulation (). In particular, a catalytically inactive Cas9 (dCas9) protein, which lacks endonuclease activity, can be used to flexibly target many loci in parallel by using Cas9-binding guide RNAs that recognize target DNA sequences based only on predictable Watson-Crick base pairing. CRISPRi regulation can be used to achieve activation or repression by fusing dCas9 to activator or repressor modules (), but these direct protein fusions are constrained to only one direction of regulation. Thus, it remains challenging to engineer regulatory programs in which many loci are targeted simultaneously but with distinct types of regulation at each locus.

Good et al., 2011 Good M.C.

Zalatan J.G.

Lim W.A. Scaffold proteins: hubs for controlling the flow of cellular information. Rinn and Chang, 2012 Rinn J.L.

Chang H.Y. Genome regulation by long noncoding RNAs. Spitale et al., 2011 Spitale R.C.

Tsai M.-C.

Chang H.Y. RNA templating the epigenome: long noncoding RNAs as molecular scaffolds. Delebecque et al., 2011 Delebecque C.J.

Lindner A.B.

Silver P.A.

Aldaye F.A. Organization of intracellular reactions with rationally designed RNA assemblies. Figure 1 Genomic Regulatory Programming Using CRISPR and Multi-Domain Scaffolding RNAs Show full caption (A) lncRNA molecules may act as scaffolds to physically assemble epigenetic modifiers at their genomic targets. Modular RNA structures can encode domains for protein binding and DNA targeting to colocalize proteins to genomic loci. (B) CRISPR RNA scaffold-based recruitment allows simultaneous regulation of independent gene targets. The minimal CRISPRi system silences target genes when dCas9 and an sgRNA assemble to physically block transcription. Fusing dCas9 to transcriptional activators or repressors provides additional functionality. When function is encoded in dCas9 (CRISPRi) or dCas9-effector fusion proteins, the sgRNA recruits the same function to every target site. To encode both target and function in a scaffold RNA, sgRNA molecules are extended with additional domains to recruit RNA-binding proteins that are fused to functional effectors. This approach allows distinct types of regulation to be executed at individual target loci, thus allowing simultaneous activation and repression. To develop a platform for synthetic genome regulation that allows locus-specific action, we took inspiration from natural regulatory systems that encode both target specificity and regulatory function in the same molecule. In cell-signaling pathways, scaffold proteins act to physically assemble interacting components so that functional outcomes can be precisely controlled in time and space (). Similar scaffolding principles apply in genome organization, wherein, for example, long noncoding RNA (lncRNA) molecules are proposed to act as assembly scaffolds that recruit key epigenetic modifiers to specific genomic loci ( Figure 1 A) (). The idea that RNA can be used to coordinate biological assemblies has important implications for engineering. RNA is inherently modular and programmable: DNA targets can be recognized by base pairing, and modular RNA-protein interaction domains can be used to recruit specific proteins ( Figure 1 A). The ability of engineered RNA scaffolds to coordinate functional protein assemblies has already been elegantly demonstrated ().

Mali et al., 2013a Mali P.

Aach J.

Stranges P.B.

Esvelt K.M.

Moosburner M.

Kosuri S.

Yang L.

Church G.M. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. To implement a synthetic, modular RNA-based system for locus-specific transcriptional programming, we can extend the CRISPR single-guide RNA (sgRNA) sequence with modular RNA domains that recruit RNA-binding proteins. This approach converts the sgRNA into a scaffold RNA (scRNA) that physically links DNA binding and protein recruitment activities ( Figure 1 B). Critically, a single scRNA molecule encodes both information about the target locus and instructions about what regulatory function to execute at that locus. This approach allows multidirectional regulation (i.e., simultaneous activation and repression) of different target genes as part of the same regulatory program. Engineering multivalent RNA recruitment sites on each scRNA offers the further possibility of independently tuning the strength of activation or repression at each target site. The potential viability of this approach is supported by a recent report showing that a sgRNA extended with MS2 hairpins can recruit activators to a reporter gene in human cells ().

Here, we demonstrate that CRISPR sgRNAs can be repurposed as scaffolding molecules to recruit transcriptional activators or repressors, thus enabling flexible and parallel programmable locus-specific regulation. We use the budding yeast S. cerevisiae as a testbed to identify three orthogonal RNA-protein binding modules and to optimize scRNA designs for single and multivalent recruitment sites. We show that the system developed in yeast also functions efficiently in human cells to regulate reporter and endogenous target genes, and we extend its scope to include recruitment of chromatin modifiers for gene repression. We then demonstrate the use of CRISPR scaffold RNA molecules to construct synthetic multigene expression programs. Specifically, we are able to regulate multiple genes in a highly branched biosynthetic pathway in yeast to express key enzymes in alternative combinations. These synthetic transcriptional programs, by combinatorially altering metabolic organization, allow us to flexibly redirect the pathway between five distinct possible product output states. Finally, we show that dCas9 can act as a master regulator of these gene expression programs, receiving input signals and acting as a single control point to execute a multigene response encompassing simultaneous activation and repression of downstream target genes.