Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds

Abstract

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.

Figure 1. Genomic Regulatory Programming Using CRISPR and Multi-Domain Scaffolding RNAs

(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 co-localize 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.

Figure 2. Multiple Orthogonal RNA Binding Modules Can Be Used to Construct CRISPR Scaffolding RNAs

(A) scRNA constructs with MS2, PP7, or com RNA hairpins recruit their cognate RNA-binding proteins fused to VP64 to activate reporter gene expression in yeast. A yeast strain with an unmodified sgRNA and the dCas9-VP64 fusion protein gives comparatively weaker reporter gene activation. The MS2 and PP7 RNA hairpins bind at a dimer interface on their corresponding MCP and PCP binding partner proteins (Chao et al., 2008), potentially recruiting two VP64 effectors to each RNA hairpin. The structure of the com RNA hairpin in complex with its binding protein has not been reported, but functional data suggest that a single Com monomer protein binds at the base of the com RNA hairpin (Wulczyn and Kahmann, 1991). scRNA constructs and corresponding RNA-binding proteins were expressed in yeast with dCas9 and a 1x tetO-VENUS reporter gene. Representative flow cytometry data are presented in Figure S1. (B) There is no significant crosstalk between mismatched pairs of scRNA sequences and non-cognate binding proteins. scRNA constructs and RNA-binding proteins were expressed in yeast with dCas9, using a 7x tetO-VENUS reporter gene to detect any potential weak crosstalk between mismatched pairs. The y-axis is on a log-scale, and activity with cognate scRNA-binding protein pairs is significantly greater with the 7x tet reporter compared to the 1x reporter. (C) Multivalent recruitment with two RNA hairpins connected by a double-stranded linker produces stronger reporter gene activation compared to single RNA hairpin recruitment domains. The 2x MS2 (wt+f6) construct was designed with an aptamer sequence (f6) selected to bind to the MCP protein (Hirao et al., 1998). This construct has two distinct sequences to recruit the same protein, which may help to prevent misfolding between hairpin domains that can occur when two identical hairpins are linked on the same RNA. (D) A mixed MS2-PP7 scRNA construct constructed using the 2x double-stranded linker architecture recruits both MCP and PCP. Fold-change values in (A)–(D) are fluorescence levels relative to parent yeast strains lacking scRNA. Values are median ± SD for at least three measurements. RNA sequences are reported in Table S1.

Figure 3. CRISPR RNA Scaffold Recruitment Can Activate or Repress Gene Expression in Human Cells

(A) scRNA constructs with MS2, PP7, or com RNA hairpins recruit corresponding RNA-binding proteins fused to VP64 to activate reporter gene expression in HEK293T cells. scRNA and RNA binding proteins were expressed in a cell line with dCas9 and a TRE3G-EGFP reporter containing a 7x repeat of a tet operator site. For comparison, an unmodified sgRNA targeting the same reporter gene was expressed in a cell line with the dCas9-VP64 fusion protein. Representative flow cytometry data are presented in Figure S3. (B) The 2x MS2 (wt+f6) scRNA construct recruits MCP-VP64 to activate expression of endogenous CXCR4 in HEK293T cells expressing dCas9. Comparatively weak activation is observed in cells with dCas9-VP64 and unmodified sgRNA. There is no significant activation of CXCR4 in cells with dCas9 and unmodified sgRNA. Similar effects were observed at each of three individual target sites located within ~200 bases of the transcriptional start site (TSS). Cell surface expression of CXCR4 was measured by antibody staining. (C) The com scRNA construct recruits Com-KRAB to silence a SV40-driven EGFP reporter gene in HEK293T cells expressing dCas9. At the P1 site, upstream of the TSS, recruitment of dCas9 (i.e. CRISPRi) does not silence EGFP, but scRNA-mediated KRAB recruitment does. At the NT1 site, overlapping the TSS, CRISPRi partially silences EGFP, and scRNA-mediated KRAB recruitment further enhances silencing. The P1 and NT1 target sites were selected from a panel of sites examined in a prior study (Gilbert et al., 2013). Fold-change values in (A)–(C) are fluorescence levels relative to a parent cell line lacking scRNA. Values are median ± SD for at least three measurements. (D) scRNA constructs mediate simultaneous activation and repression at endogenous human genes in HEK293T cells, measured by RT-qPCR. A 2x MS2 (wt+f6) scRNA construct recruits MCP-VP64 to activate CXCR4, and a 1x com scRNA construct recruits COM-KRAB to silence B4GALNT1. Fold-change values are gene expression levels (mean ± SD) from two RT-qPCR measurements, relative to negative control cell lines. The observed change in CXCR4 mRNA level measured by RT-qPCR corresponds to an increased protein level (Figure S3D).

Figure 4. Reprogramming the Output of a Branched Metabolic Pathway with a 3-Gene scRNA CRISPR ON/OFF Switch

(A) Heterologous expression of bacterial violacein biosynthesis pathway in yeast produces violacein from L-Trp with five enzymatic steps and one non-enzymatic step. Branch points at the last two enzymatic transformations catalyzed by VioD and VioC produce four possible pathway outputs. (B) An scRNA program regulates three genes simultaneously to control flux into the pathway and to direct the choice of product. The yML025 yeast strain (Table S4) has VioBED genes strongly expressed (ON), and VioAC genes weakly expressed (OFF). A 2x PP7 scRNA targets VioA and a 1x MS2 scRNA targets VioC for activation. An unmodified sgRNA targets VioD for repression by CRISPRi. (C) scRNA programs flexibly redirect the output of the violacein pathway. The yML025 yeast strain expressing dCas9, MCP-VP64, and PCP-VP64 was transformed with an empty parent vector (pRS316) or with a plasmid containing one, two, or three scRNA constructs to route the pathway to all four product output states (Table S6). Yeast strains were grown on SD –Ura agar plates. Product distribution was analyzed by HPLC. Stars on the chromatograms indicate the expected product of the engineered pathway. Quantitative values for changes in gene expression (by RT-qPCR) and product distributions are reported in Figure S4B.

Figure 5. The dCas9 Master Regulator Inducibly Executes scRNA-Encoded Programs

(A) dCas9 can act as a synthetic master regulator of scRNA-encoded circuits. We placed dCas9 under the control of an inducible Gal10 promoter. The yML017 yeast strain (Table S4) has VioABED genes strongly expressed (ON), and VioC weakly expressed (OFF). A 1x MS2 scRNA targets VioC for activation. An unmodified sgRNA targets VioD for repression by CRISPRi. (B) The presence of the master regulator dCas9 controls execution of the scRNA program. Yeast expressing a two-component scRNA program and MCP-VP64 were grown on agar plates in the presence or absence of galactose to control dCas9 expression. When the dCas9 master regulator is not present (−Gal), Vio pathway gene expression remains in the basal state and pathway flux proceeds to the PV product. When dCas9 is present (+Gal), VioC switches ON, VioD switches OFF, and pathway flux diverts to the DV product.

Figure 6. Encoding Complex dCas9/scRNA Regulatory Programs

scRNAs can be combined with dCas9 to construct designer transcriptional programs in which distinct target genes can be simultaneously activated or repressed, or subject to other types of regulation. Temporal control of the synthetic program can be achieved by inducing the dCas9 protein as a master regulator. Alternative scRNA gene expression programs could be achieved in the same cell by harnessing orthogonal dCas9 proteins that recognize their guide RNAs through distinct sequences (Esvelt et al., 2013). Each orthogonal dCas9 protein could control a distinct set of scRNAs, allowing independent control over distinct gene expression programs. Each scRNA, in turn, allows independent control at the level of an individual gene. Distinct dCas9 proteins could be placed under the control of different extracellular signals or inducible promoters.