Synthetic biology — application-oriented cell engineering

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ScienceDirect Synthetic biology — application-oriented cell engineering Mingqi Xie1, Viktor Haellman1 and Martin Fussenegger1,2 Synthetic biology applies engineering principles to biological systems and reprograms living cells to perform novel and improved functions. In this review, we first provide an update of common tools and design principles that enable userdefined control of mammalian cell activities with spatiotemporal precision. Next, we demonstrate some examples of how engineered mammalian cells can be developed towards biomedical solutions in the context of real-world problems. Addresses 1 Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland 2 University of Basel, Faculty of Science, Mattenstrasse 26, CH-4058 Basel, Switzerland Corresponding author: Fussenegger, Martin ([email protected])

Current Opinion in Biotechnology 2016, 40:139–148 This review comes from a themed issue on Tissue, cell and pathway engineering Edited by April Kloxin and Kyongbum Lee For a complete overview see the Issue and the Editorial

their extension to more diverse applications or specifications [3,4].

Cell engineering tools Genome editing

As the natural ability of cells to control their behaviour is encoded at their genetic level, the capability to engineer complex cell functionalities directly relates to our expertise in manipulating endogenous gene networks. In recent years, the emergent CRISPR/Cas9 technology enabling facile and permanent modification of genomic sequences in mammalian cells and animals has brought the promise of gene editing designer nucleases back into the limelight [3]. Classical designer nucleases, such as zinc-finger nucleases (ZFNs) and transcription-activator like effector nucleases (TALENs), can be programmed to recognize any DNA sequence of interest to trigger specific double strand breaks (DSBs) at defined genomic loci. When provided with an additional donor sequence, precise changes in the targeted genomic sequence can be introduced by manipulating the endogenous DNA repair machinery of the cell [4] (Figure 1a).

Available online 29th April 2016 http://dx.doi.org/10.1016/j.copbio.2016.04.005 0958-1669/Published by Elsevier Ltd.

Introduction Founded at the interface between recombinant DNA technology and biotechnology by the turn of the millennium, synthetic biology is currently perceived as an interdisciplinary field that approaches important problems in biology and global healthcare systems with an engineering mindset. Although there is not a clear definition of its boundaries, the general mission of synthetic biology is to harness the power of biology to achieve rational and diligent control over living cells and animals [1,2]. In doing so, cell engineering in synthetic biology is based on the concept that biological components from different organisms can be reassembled into userdefined genetic networks that operate either in parallel or together with natural biological systems to restore, improve or add essential functions to cells. Therefore, synthetic biologists must first understand all of the phenomena that nature manipulates, and only a clear knowledge of their fundamental mechanisms of action enables www.sciencedirect.com

In contrast to ZFNs and TALENs, the Cas9 protein is by nature a sequence-specific nuclease. Repurposed from the bacterial CRISPR system for cleaving foreign DNA, Cas9 finds its cleavage target through Watson–Crick base pairing interactions between a single stranded guide RNA molecule (gRNA) and an approximately 20 bp target DNA sequence. Thus, the generation of site-specific DSBs can be governed by a simple alteration of a short section within the gRNA and no longer involves laborious reengineering of DNA-binding protein-domains, as is the case of ZFN and TALEN [3] (Figure 1a). However, the site-specificity of Cas9 is minimally constrained by a protospacer-adjacent motif (PAM), a 2–3 bp recognition site on the DNA target (50 -NGG-30 ) required for the gRNA/Cas9-complex to bind and unwind the doublestranded DNA to initiate its cleavage [3,5]. To attenuate this restriction, Bolukbasi et al. engineered a hybrid designer nuclease by fusing a four-finger ZF-array to a Cas9 mutant with attenuated DNA-binding affinity [6]. When guided to a genomic locus with poor PAM-compatibility, the ZF-domain was able to specifically bind a PAM-independent sequence upstream of the gRNA-defined target site and assist Cas9-mediated gene editing to increase on-target specificity. Other practical features that improve user-defined Cas9 performances include (a) varying the gRNA target lengths to increase editing Current Opinion in Biotechnology 2016, 40:139–148

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Regulation of gene expression with designer nucleases. (a) Basic mechanism of gene editing. Designer nucleases trigger DSBs (double strand breaks; red) at specific user-defined target DNA sequences (purple). When provided with a donor sequence (orange) flanked by homologous regions (blue), the donor sequence is incorporated into the target site by endogenous DNA repair systems such as homologous recombination (HR). ZFN (Zinc-Finger nucleases) and TALEN (transcription-activator like effector nucleases) are chimeric fusion proteins consisting of a sequence-specific protein array (ZF or TALE) and a non-specific restriction endonuclease (e.g., FokI), while Cas9 is by nature a sequence-specific nuclease. Sequence specificity is programmed either by reengineering of ZF- or TALE-arrays, or by alteration of the guide RNA’s (gRNA) targeting sequence. (b) Transcriptional and epigenetic regulation. Site-specific transcriptional regulators can be engineered by fusing a target-specific DNAbinding domain (DBD; purple), such as catalytically inactive Cas9*, dCas9, ZF or TALE, to a specific effector domain (ED) to enable (i) repression of target gene transcription with the DBD fused to a repressor domain (KRAB or SID4X) or to DBD only; (ii) activation of target gene transcription with the DBD fused to an activator domain (VP64, VPR or SunTag); (iii) visualization of locus control regions (LCRs; inset) with DBD fused to a fluorescent domain (GFP, HaloTag or SunTag) or (iv) histone modifications of genomic target loci with the DBD fused to methyl- or acetylases/ transferases. (c) Orthogonal regulation of gRNA/dCas9-mediated events. Incorporation of aptamer sequences into editable regions of the gRNA (such as upper stem or 30 hairpins, [5]) enables gRNA-dependent combination of dCas9 with different effector domains: reconstitution of different transcriptional regulators occurs by assembly of different aptamer-binding protein-effector fusions with different aptamer-containing gRNA scaffolds using the same dCas9 orthologue.

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specificity [7,8]; (b) increasing the homologous recombination efficiencies either by the rational design of donor DNA structures [9] or by manipulating cell cycle stages [10]; and (c) generating functional gRNA molecules from Pol II promoters to render the editing event compatible with endogenous, synthetic and tuneable promoters [11,12,13]. Currently, the toolbox of CRISPR/Cas9-based designer nucleases is expanding at such a dramatic pace that the prospect of ‘genome surgery’ becoming an eligible therapy to treat genetic disorders appears to be achievable [4]. For example, the recent discovery of another RNAguided endonuclease Cpf1 might further increase CRISPR-based editing capacities within single cells [14], and the ability to repurpose Cas9 to cleave single stranded RNA molecules by the simple addition of short nucleic acid structures called PAMmers [15] could expand this editing technology towards post-transcriptional target events. Transcriptional control Basic architecture of synthetic transcriptional regulators

In contrast to genome editing, gene regulation at the transcriptional level is reversible in most cases and poses an only temporary manipulation of expression levels. Recently, it was shown that shortening the gRNA targeting sequences to 16 nt in length could fully attenuate the nuclease activity of Cas9 while retaining the DNA-binding capability of the gRNA/Cas9 complex [16,17]. Therefore, similar to the dCas9 mutant in which the nuclease’s catalytically active sites were permanently removed, the fusion of different effector domains to catalytically inactive Cas9, dCas9, or ZF/TALE-arrays yields a variety of site-specific transcriptional and epigenetic regulators (Figure 1b). For example, when targeted to a promoter or enhancer region within the mammalian nucleus, sequence-specific DNA-binding proteins [17,18] or their chimeric fusions with a repressor domain (such as the Kru¨ppel-associated box (KRAB) domain of the kox1 gene [19] or four concatenated copies of the mSin3 interaction domain (SID4X) [20]) could tightly repress the transcription of target genes (Figure 1b-i). Likewise, fusing the same DNA-binding domains (DBDs) to an activator domain such as VP64 [21] or VPR (i.e., a tripartite effector composed of the activators VP64, p65, and Rta in tandem, [22]) triggers strong transcriptional activation of target genes (Figure 1b-ii). However, as genomic DNA in eukaryotes is often packed around histone proteins to yield dense chromatin structures called nucleosomes, particular locus control regions (LCRs) might have complex tertiary structures so that target gene sequences that are theoretically distal to enhancer regions might unpredictably fold into the vicinity of the transcription initiation complex (Figure 1b-iii) [23]. To elucidate the LCR structure at transcription initiation complexes, a variety www.sciencedirect.com

of visualization tools were developed by fusing sequence-specific DBDs to fluorescent proteins [24], ligand-activated fluorescent tags such as HaloTag [25], or a versatile protein-scaffold, SunTag, which consists of a tandem array of scFv recognition peptides that could recruit up to 24 copies of VP64-GFP-scFv fusions [26]. When targeting SunTag-fusion proteins to different genomic loci, the authors demonstrated unprecedented levels of transcriptional activation and cellular imaging (Figure 1b-iii) [26]. KRAB- and SID4X-mediated transcriptional repression at the genomic level often triggers histone modifications, such as promoter hypermethylation or deacetylation [19,20]. In most cases, the reversibility of target gene expression is greatly compromised by the high repressive efficiency that strongly delays transcriptional reactivation [18]. To address this problem, Maeder et al. targeted a TET1 hydroxylase domain to silenced genomic regions, which efficiently induced histone demethylation at sitespecific CpG motifs (Figure 1b-iv) [27]. Conversely, histone methyltransferase domains such as G9a-SET reinforce histone methylation and therefore further silence local gene expression (Figure 1b-iv) [28]. Regulation of multiple parallel transcriptional events

Classical RNA-guided DNA-binding is often limited by the fact that one dCas9 orthologue can only either promote gene activation or repression, but not both [29]. Providing a valuable resource to the CRISPR/Cas9-technology, Briner et al. carefully analysed the structure, function and design principles of the gRNA and eventually identified regions in the molecule that are either dispensable to or tolerant of sequence variations (Figure 1c) [5]. Later, Zalatan et al. capitalized on this achievement and introduced a powerful design in which effector domains must no longer be fused to the dCas9protein to reconstitute the transcriptional regulator [30]. In particular, the authors incorporated aptamer-motifs into the editable regions of the gRNA, and after coexpressing individual fusion constructs between different aptamer-binding proteins (ABP) and different effector domains, different gRNA-aptamer/ABP-effector complexes could use the same dCas9 protein to regulate different types of transcriptional activities when recruited to different gRNA-specific DNA target sites (Figure 1c) [30]. Trigger-inducible transcriptional regulation

Although designable DNA-binding proteins can bind any sequence and provide limitless DNA targets, the lack of an inducible component greatly constrains the level of complexity and tunability that can be achieved when integrating these DNA-binding proteins into gene networks [31]. Indeed, as the sensing of environmental signals is an essential feature of living cells, synthetic gene circuits should also allow their host cells to respond Current Opinion in Biotechnology 2016, 40:139–148

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to changing conditions by translating external signals into a decision that changes the cell state. To achieve this feature, prokaryotic transcription factors (pTFs) of the TetR, GntR and MarR families, which are naturally evolved biosensors, have been extensively used in the past decade to remote-control gene expression in mammalian cells with a multitude of cell permeable trigger compounds [32]. The presence of a pTF-specific trigger compound renders the transcription factor incapable of binding a target promoter region engineered to contain its specific operator sequence, and depending on the effector domain fused to pTF, this change of promoter occupancy either results in activation or repression of gene transcription (Figure 2a-i). This basic gene switch system was implemented in non-limiting mammalian cell designs to enable transcriptional regulation with different types of input signals, including chronic disease markers [33], food additives [34] and cosmetics [35]. To regulate gene expression with cell impermeable trigger compounds, a semi-synthetic engineering strategy is commonly used to rewire extracellular input signals to the mammalian host cell’s signal transduction machinery. Because many intracellular signalling cascades are mediated by second messengers (SMs), engineering target genes under the control of SM-specific promoters enables regulation of mammalian cell activities with trigger signals that activate gene transcription by occupying the cell’s natural pathways (Figure 2a-ii). In this metabolically economic way, these semi-synthetic signalling cascades were able to synchronize mammalian transcriptional activities using signal molecules with low bioavailability and minimal residence times at the cell surface, such as acidic microenvironments [36], pro-inflammatory cytokines [37], neurotransmitters [38] or blue light [39]. Lately, the engineering of optogenetic systems that incorporate light-responsive SM-producing enzymes into mammalian cells has gained increased attention. In contrast to molecular trigger compounds, light is a non-toxic stimulus with an unlimited supply that can regulate target events with maximal spatiotemporal precision [40]. Inspired by a pioneering study by Folcher et al. [41], Kim and colleagues engineered an optogenetic device by coupling cGMP-production by a blue light-activated guanylate cyclase (bGC) to a synthetic cGMP-regulated promoter in a variety of mammalian cell types (Figure 2aii). As the molecular mechanism of many drugs that promote sexual arousal — such as Viagra1 — is through inhibition of cGMP-specific phosphodiesterases, bGC could be applied to mice to provide photostimulated penile erection as a potential therapeutic strategy for treating sexual dysfunctions in humans [42]. Another classical design principle for optogenetic systems is the use of light-inducible dimerization protein partners. Similar to constitutive dimerization modules such as the Current Opinion in Biotechnology 2016, 40:139–148

DocS/Coh2 interaction-domains or intein-domains [31,43] and chemically induced dimerization modules such as the rapamycin-inducible FKBP-FRB protein pair, light also triggers the formation of a variety of dimers, such as Cry2/CIB1, UVR8/UVR8, and PIF6/PhyB (Figure 2b), which have all been elegantly repurposed in mammalian cells to reconstitute a variety of effector proteins, including transcriptional regulators [20,44–46], translational regulators [47], ion channel modulators [48] and protein scavengers [49,50]. Translational control

The regulation of gene expression at the translational level is relatively fast acting and therefore useful for finetuning expression dynamics [43]. For example, aptamer motifs can be engineered into the 50 -UTR of mammalian mRNA sequences so that the binding of an aptamerbinding protein to this chimeric transcript would prevent the mRNA from ribosomal recognition and thereby inhibit translational initiation (Figure 2c-i). The most commonly used aptamer-binding proteins in mammalian cell engineering include the C/D-box-binding L7Ae [51], the MS2-box-binding MCP [30,51] and the boxB-binding N-peptide [47,52]. Recently, Cao et al. coupled the boxB/N-peptide system with the blue light-triggered Cry2/CIB1 dimerization module to create a synthetic optogenetic device that enabled trigger-inducible 50 cap-independent translational initiation in human cells (Figure 2c-ii) [47]. Another elegant way to engineer inducible translational control elements is the use of noncoding RNA structures such as ribozymes [52,53]. As the hammerhead ribozyme (HHR) is a RNA structure capable of efficiently cleaving any nucleic acid molecule containing the HHR structure, S. Ausla¨nder and colleagues engineered the boxB aptamer-motif into the HHR catalytic active sites so that self-cleavage and degradation of a target mRNA containing this chimeric HHR was regulated in an N-peptide-dependent manner (Figure 2ciii) [52]. Finally, RNA interference (RNAi) is another powerful tool that uses non-coding RNAs to control translational activities [12,54]. Short regulatory RNA molecules such as shRNAs, siRNAs or miRNAs consisting of approximately 20 nucleotides can be incorporated into mammalian cells to bind and knock down any target mRNA molecule that contains a complementary nucleotide sequence. Depending on the degree of sequence complementarity with the short RNAs, the targeted mRNA can undergo either degradation or temporary silencing (Figure 2c-iv) [55]. Protein localization control

Control over intracellular protein trafficking is a key control parameter in the compartmentalized mammalian cell, especially in contrast to prokaryotic cell types [56– 58]. The Avena sativa phototropin 1 derived LOV2-Ja protein is a versatile tool for the engineering of optogenetic regulatory elements in the cytoplasm. In the dark, www.sciencedirect.com

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Trigger-inducible gene expression in mammalian cells. (a) Transcriptional control. (i) Cell-permeable trigger compounds. Binding of a pTF (prokaryotic transcription factor)-specific ligand to pTF abolishes pTF’s capacity to bind its cognate DNA sequence (OTF, purple). Depending on the effector domain (ED) fused to pTF (AD, activation domain [green]; RD, repression domain [red]), the device provides ligand-triggered OFF or ON transcription switches of target genes in mammalian cells. (ii) Regulation via the cell surface. Changes in intracellular second messenger (SM) levels triggered by plasma membrane receptors or ion channels are rewired to SM-specific promoters to regulate target gene transcription in response to a variety of external signals including light. (b) Dimerization technologies. Conditional or trigger-inducible reconstitution of a transcriptional regulator DBD-ED can be programmed by independently expressing DBD and ED fused to (i) constitutive protein dimerization domains such as Coh2/DocS or intein-domains; (ii) small molecule-triggered protein dimerization domains such as the rapamycin-inducible FKBPFRB; or (iii) light-triggered protein dimerization domains such as the blue light-triggered Cry2/CIB1 (heterodimerization at 450–488 nm), the ultraviolet B triggered UVR8/UVR8 (homodimerization at 280–315 nm) or the phycocyanobilin-dependent PIF6/PhyB (red light-triggered heterodimerization at 650 nm; far red light-triggered dissociation at 750 nm). (c) Translational control. (i) Inhibition of translational initiation. Incorporation of aptamer sequences into the 50 -UTR of the target mRNA enables aptamer-binding proteins (ABPs) to bind the transcript and prevent its ribosomal recognition, thereby resulting in ABP-mediated translational inhibition. (ii) Activation of translational initiation. Incorporation of aptamer sequences into non-coding regions upstream of 50 -cap independent open reading frames (ORFs) creates RNA-tethers for recruiting ABP domains. Co-expression with ABP-CIB1 and Cry2-eIF4E (eIF4E, eukaryotic translation initiation factor 4E) enables blue light-triggered activation of translational inhibition. (iii) Trigger-inducible control of transcript stability. Incorporation of aptamer sequences into catalytically active HHR regions of the target mRNA’s 30 -UTR enables ABPs to bind and prevent the transcript’s self-cleavage and degradation, thereby resulting in ABP-mediated translational activation. (iv) RNA interference. Short regulatory RNA molecules (shRNA, siRNA or miRNA) bind and knock down target mRNA molecules containing a complementary nucleotide sequence after formation of a catalytic complex with host endonucleases (RISC). (d) Optogenetic control of cytosolic events. (i) Protein sequestration. By fusing CIB1 to a multimeric protein and Cry2 to a target-specific nanobody, target proteins can be trapped upon formation of a blue light-triggered oligomerization network. (ii) Protein localization. By fusing target proteins to the LOV2-domain and different signal peptides to the Ja-domain of the Avena sativa phototropin 1-derived LOV2-Ja, blue light (440–473 nm) can be used to target proteins to different intracellular locations.

the Ja-domain is tightly bound to the LOV2 core. However, blue light induces covalent bond formation between the LOV2 and a flavin mononucleotide chromophore to yield a conformational change that triggers the dissociation and unwinding of the Ja-helix. Therefore, following www.sciencedirect.com

a design scheme called photocaging, cargo proteins such as localization signals can be appended to the C-terminus of the Ja-helix such that the accessibility to the embedded signal peptide is only provided upon photo-illumination. In this manner, a panoply of control elements have Current Opinion in Biotechnology 2016, 40:139–148

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been engineered to enable optogenetic regulation of localization events, including nuclear-cytoplasmic trafficking [57], peroxisome targeting [58] and protein degradation [59] (Figure 2d). Proteins can also be targeted to different cellular compartments using classical dimerization modules (Figure 2a). By anchoring one split component to the destination and fusing its dimerization partner to a protein of interest, the blue light-triggered Cry2/CIB1 heterodimerization module, for example, was able to recruit apoptotic effector proteins to the mitochondria to increase the control capacity over human cancer cells [60]. Interestingly, Cry2/CIB1 was also used to engineer a triggerinducible protein oligomerization system that could be implemented as a cytosolic protein scavenger reminiscent of a targeted ‘fishing net’ [49,50] (Figure 2d). The Wnt/ b-catenin signalling pathway is an important protein network in stem cells that regulates a variety of key processes, such as self-renewal and pluripotency. At some stages, the transcriptional activator b-catenin is clustered and degraded by a variety of cytoplasmic proteins. To enable the nuclear translocation of b-catenin, Bugaj et al. used the blue light-triggered protein scavenger to specifically trap the degradation complex and unblock b-catenin for transcriptional regulation [50].

Synthetic biology applications in the real world Drug discovery

The founding goal of synthetic biology was to make biology more efficient and to develop biological systems for industrial, agricultural, environmental and medical applications ranging from drug discovery and drug production to gene-based and cell-based therapies [61–63]. For example, gene switches (Figure 2a–c) that can instantly monitor precise levels of external stimuli to give customizable phenotypic outputs could be implemented as high-throughput screening platforms for pharmaceutical targets [62,64]. In recent years, non-coding RNAs (ncRNAs) have emerged as valuable therapeutic targets affecting and regulating a variety of key cellular processes [65]. Capitalizing on an elegant cloning strategy and the ability to express multiple miRNAs from a tandem precursor-array [55], Wong et al. developed a high-

throughput RNAi-screening system capable of identifying cancer-specific genomic signatures during chemotherapies (Figure 3a) [66]. Likewise, Shechner et al. incorporated a library of functionally unknown RNA structures into the flexible regions of gRNA (Figure 1c) [13] and used dCas9 as a robust screening machine (CRISP-Disp) to identify the biological roles of the ncRNA candidates (Figure 3b). Molecular diagnostics

In vitro diagnostic tests using living cells are gaining importance in the global health arena due to their non-invasive nature and their ease of use and scale [67]. For example, Courbet et al. showed that bacterial biosensors encapsulated into polyvinyl acetate (PVA)/ alginate hydrogel beads were able to detect pathological glycosuria in urine from diabetic patients (Figure 3c) [67]. In contrast to bacterial sensors, however, human cells sense a much wider variety of disease metabolites, such as serum-specific markers of the immune system. To this end, D. Ausla¨nder and colleagues engineered a synthetic allergy profiler in human cells by coupling a histamine-responsive GPCR to an endogenous SM-specific gene promoter (Figure 3c) [68]. The engineered cells were not only able to score the allergen-triggered release of histamine in human whole-blood samples with comparable scores to standard skin-prick tests, but the non-invasive design of the system did also not necessitate a patient’s direct exposure to potential allergens [68]. To further develop human cellbased biosensors towards clinical applications, Schukur and colleagues recently developed a culture technique by encapsulating diagnostic human cells into clinically validated alginate-poly-lysine-alginate capsules to guarantee optimal microenvironments between the sensor cells and substrates (Figure 3c) [69]. Notably, because the microcapsules’ pore size can be tuned to achieve selective permeability for a user-defined set of target molecules, this system is also used as a standard implant architecture for delivering medically relevant gene circuits into living animals to simultaneously sense, treat and prevent specific diseases [70]. One major concern of using cell-based diagnostics outside of the laboratory might be their limited degrees

(Figure 3 Legend) Biomedical applications of engineered mammalian cells. (a) RNAi-based screen for cancer-specific genomic signatures (CombiGEM, [66]). Human cells transduced with high-coverage libraries of barcoded miRNAs can be used to screen for typical genomic profiles that are acquired under specific environmental conditions. (b) CRISPR/Cas9-based screen from non-coding RNA function (CRISP-Disp, [13]). Targeting of gRNA molecules which harbour functionally unknown non-coding RNAs to gRNA-specific promoters of known transcriptional output enables Cas9-mediated screening for effector function: increase or decrease in target gene transcription implies activator- or repressor-functions of the respective RNA structure. (c) in vitro diagnostic tests with living cells. (left) Diagnostic bacteria. Bacteria can be engineered to carry synthetic gene circuits that specifically detect particular disease markers. When encapsulated into polyvinyl acetate/alginate hydrogel beads, these bacterial biosensors can diagnose pathologic levels of particular metabolites from body fluids such as human urine (adapted from [67]). (right) Diagnostic human cells. Human cells can also be engineered with disease-specific gene circuits to detect pathologic metabolite levels in body fluids such as human blood, either by direct exposure of diagnostic cells to diluted serum (adapted from [68]) or by microencapsulation into alginate-poly-L-lysine beads compatible with whole blood cultures (adapted from [69]). (d, e) Synthetic gene circuits in cell-free systems. Transcriptional or translational gene switches can be incorporated on (d) paper or (e) ZnO/Al2O3 nanowall surfaces to detect target metabolites using cell-free systems. (d) Paper-based synthetic gene networks (adapted from [2]). (e) Nanowall-based biosensor (adapted from [64]). www.sciencedirect.com

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of biosafety or their cell-type dependent restrictions on target sensitivities [2]. To this end, Pardee et al. were able to freeze-dry bacterial or mammalian cell-free transcription/translation systems onto conventional paper to create inexpensive testing-strips that were stable for long-term storage and compatible with easy usage under common environmental conditions [2]. By incorporating different gene circuits such as transcriptional networks, toehold switches [1] and FRETbiosensors into the testing platform, a variety of biomedically important trigger signals such as antibiotics, glucose and viral markers could be sensed in a manner that was previously unachievable with cell-based systems (Figure 3d). Similarly, Menzel et al. engineered a novel biohybrid nanosensor enabling the electrical realtime detection and quantification of food contaminants [64]. By incorporating complexes of pTF-bound pTFspecific DNA sequences (Figure 2a) onto a ZnO/Al2O3 nanowall surface, the release of DNA triggered by the target compound rapidly reduced the local net surface charge, which was precisely manifested in the device’s conductance time-course (Figure 3e).

Conclusion and perspectives Today, whereas synthetic biology-inspired metabolic engineering approaches are already established processes in industrial biotechnology [63,71], growing expertise in engineering living cells could assist the pharmaceutical industry in developing high-throughput approaches for drug discovery [2]. Additionally, whereas cell-based and cell-free diagnostics might advance the success of pointof-care technologies [72], one important next step in mammalian synthetic biology research will be the synchronization of this precise diagnostic capacity with automated therapeutic responses. Indeed, with a panoply of such closed-loop prosthetic networks [73] already being successfully validated in different human compatible cell types and animal models, and as human pluripotent cells can be converted into a variety of autologous cell types using synthetic gene switches [74] and circuits [75], future technologies that could enable the long-term and safe operation of gene-based and cell-based therapies in human patients may best integrate the promises of personalized and regenerative medicine.

Acknowledgements The authors thank Pratik Saxena and Hui Wang for their generous advice. Work in the M.F. lab is supported by a European Research Council (ERC) advanced grant (ProNet – No. 321381) and, in part, by the National Centre of Competence in Research (NCCR) Molecular Systems Engineering.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. 

Green AA, Silver PA, Collins JJ, Yin P: Toehold switches: denovo-designed regulators of gene expression. Cell 2014, 159:925-939.

Current Opinion in Biotechnology 2016, 40:139–148

This elegant switch-design is capable of sensing a variety of RNA signals. However, although this system operates perfectly in bacteria and outside the cell, its functionality in mammalian cells might remain elusive. 2. 

Pardee K, Green AA, Ferrante T, Cameron DE, DaleyKeyser A, Yin P, Collins JJ: Paper-based synthetic gene networks. Cell 2014, 159:940-954. This is probably one of the breakthrough works, which demonstrates the efficient translation of synthetic biology-based gene circuits into the real world. Commercially available cell-free systems were freeze-dried onto paper (discs or self-made assay-layouts), enabling the inexpensive, sterile, and abiotic distribution of biosensing technologies for use in the clinic, global health, industry, research, and education. 3.

Sternberg SH, Doudna JA: Expanding the biologist’s toolkit with CRISPR-Cas9. Mol Cell 2015, 58:568-574.

4.

Doudna JA: Genomic engineering and the future of medicine. JAMA 2015, 313:791-792.

5. 

Briner AE, Donohoue PD, Gomaa AA, Selle K, Slorach EM, Nye CH, Haurwitz RE, Beisel CL, May AP, Barrangou R: Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell 2014, 56:333-339. This paper describes an exceptional advance of the CRISPR/Cas9 technology. The authors identified and characterised six conserved modules within the gRNA structure and concluded that while the bulge and nexus modules are necessary for DNA cleavage, the upper stem and hairpin I are dispensable without affecting Cas9-interactions. This work paved the way for a variety of follow-up CRISPR/Cas9 breakthroughs [13,30].

6. 

Bolukbasi MF, Gupta A, Oikemus S, Derr AG, Garber M, Brodsky MH, Zhu LJ, Wolfe SA: DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat Methods 2015, 12:1150-1156. This is a typical synthetic biology-inspired approach to fine-tuning Cas9 specificity. The authors fused ZF-arrays specific for 12bp target sequences to a Cas9 mutant with attenuated DNA-binding affinity. Thus, user-defined ZF-specific DNA-binding was provided instead of PAMdependence to achieve optimal specificity for genome editing. 7.

Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK: Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 2014, 32:279-284.

8.

Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim JS: Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 2014, 24:132-141.

9.

Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE: Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 2016, 34:339-344.

10. Lin S, Staahl BT, Alla RK, Doudna JA: Enhanced homologydirected human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 2014, 3:e04766. 11. Gao Y, Zhao Y: Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol 2014, 56:343-349. 12. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK: Multiplexed  and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell 2014, 54:698-710. This impressive work contains design principles that might greatly contribute to the synthetic biology community. The authors used the RNA endonuclease Csy4 (i) to enable programmable gRNA and miRNA expression and (ii) to stabilise immature RNA transcripts for polycistronic and intronic expression. The authors also used cis-acting ribozymes to generate mature gRNA structures from Pol II promoters. 13. Shechner DM, Hacisuleyman E, Younger ST, Rinn JL:  Multiplexable, locus-specific targeting of long RNAs with CRISPR-display. Nat Methods 2015, 12:664-670. Capitalizing on editable and dispensable regions in the gRNA such as those shown previously [5], the authors applied dCas9 as a highthroughput screening device for non-coding RNA functions. 14. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM,  Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A et al.: Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 2015, 163:759-771. www.sciencedirect.com

Application-oriented cell engineering Xie, Haellman and Fussenegger 147

This paper describes the discovery of another mammalian cell-compatible RNA-guided endonuclease, Cpf1, that mediates robust DNA targeting with features distinct from Cas9. Cpf1 uses a 50 T-rich PAM and a shorter gRNA analogue to cleave double stranded DNA into products with sticky ends. 15. O’Connell MR, Oakes BL, Sternberg SH, East-Seletsky A,  Kaplan M, Doudna JA: Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 2014, 516:263-266. Introducing single-stranded nucleic acid oligomers called PAMmers to the gRNA/Cas9-complex manipulates Cas9 to bind and cleave RNA targets by mimicking a local double stranded structure that is essential for PAM recognition. 16. Dahlman JE, Abudayyeh OO, Joung J, Gootenberg JS, Zhang F, Konermann S: Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat Biotechnol 2015, 33:1159-1161. 17. Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian J, Pruitt BW, Beal J, Vora S et al.: Cas9 gRNA engineering for genome editing, activation and repression. Nat Methods 2015, 12:1051-1054. 18. Li Y, Jiang Y, Chen H, Liao W, Li Z, Weiss R, Xie Z: Modular  construction of mammalian gene circuits using TALE transcriptional repressors. Nat Chem Biol 2015, 11:207-213. The authors used TALE-arrays as transcriptional repressors that showed good compatibility with complex synthetic gene circuits. This well-written paper contains highly instructive design principles for TALE-construction. 19. Thakore PI, D’Ippolito AM, Song L, Safi A, Shivakumar NK, Kabadi AM, Reddy TE, Crawford GE, Gersbach CA: Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods 2015, 12:1143-1149. 20. Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M, Cong L, Platt RJ, Scott DA, Church GM, Zhang F: Optical control of mammalian endogenous transcription and epigenetic states. Nature 2013, 500:472-476. 21. Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H et al.: Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 2015, 517:583-588. 22. Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle MEPRI, Lin S, Kiani S, Guzman CD, Wiegand DJ et al.: Highly efficient Cas9mediated transcriptional programming. Nat Methods 2015, 12:326-328. 23. Keung AJ, Joung JK, Khalil AS, Collins JJ: Chromatin regulation at the frontier of synthetic biology. Nat Rev Genet 2015, 16:159-171. 24. Chen B, Hu J, Almeida R, Liu H, Balakrishnan S, Covill-Cooke C, Lim WA, Huang B: Expanding the CRISPR imaging toolset with Staphylococcus aureus Cas9 for simultaneous imaging of multiple genomic loci. Nucleic Acids Res 2016. 25. Knight SC, Xie L, Deng W, Guglielmi B, Witkowsky LB, Bosanac L, Zhang ET, El Beheiry M, Masson JB, Dahan M et al.: Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science 2015, 350:823-826. 26. Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD: A  protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 2014, 159:635-646. This is a very interesting and, to some extent, unusual approach of creating a versatile tagging system (SunTag) capable of appending a variety of effector functions to target proteins. SunTag is an array of repetitive epitope structures capable of recruiting up to 24 copies of antibody-linked effector proteins.

29. Kleinstiver BP, Prew MS, Tsai SQ, Nguyen NT, Topkar VV, Zheng Z, Joung JK: Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat Biotechnol 2015, 33:1293-1298. 30. Zalatan JG, Lee ME, Almeida R, Gilbert LA, Whitehead EH, La  Russa M, Tsai JC, Weissman JS, Dueber JE, Qi LS et al.: Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 2015, 160:339-350. Capitalizing on editable and dispensable regions in the gRNA similar to those shown previously [5], the authors incorporated protein-binding aptamer motifs into the gRNA molecule so that effector domains must no longer be fused to dCas9 to constitute a transcriptional regulator. In this way, an identical dCas9 orthologue could perform many parallel effector functions in the same cell. 31. Slomovic S, Collins JJ: DNA sense-and-respond protein modules  for mammalian cells. Nat Methods 2015, 12:1085-1090. This excellent paper shows medically useful applications of the protein splicing technology. The authors used intein domains and ZF-proteins to design DNA-sensitive transcriptional activators in mammalian cells that could be applied as endogenous virus detectors. 32. Auslander S, Fussenegger M: From gene switches to mammalian designer cells: present and future prospects. Trends Biotechnol 2013, 31:155-168. 33. Rossger K, Charpin-El-Hamri G, Fussenegger M: A closed-loop synthetic gene circuit for the treatment of diet-induced obesity in mice. Nat Commun 2013, 4:2825. 34. Xie M, Ye H, Charpin-El-Hamri G, Fussenegger M: Antagonistic  control of a dual-input mammalian gene switch by food additives. Nucleic Acids Res 2014, 42:e116. As the title implies, the authors engineered the Comamonas testosteroni transcriptional repressor CbaR into a unique gene switch in mammalian cells that accepted antagonistic inputs of benzoate and vanillate. This simple, non-binary gene switch might become useful in a variety of biomedical settings such as gene delivery, bioproduction and biocomputation. 35. Wang H, Ye H, Xie M, Daoud El-Baba M, Fussenegger M: Cosmetics-triggered percutaneous remote control of transgene expression in mice. Nucleic Acids Res 2015, 43:e91. 36. Auslander D, Auslander S, Charpin-El Hamri G, Sedlmayer F,  Muller M, Frey O, Hierlemann A, Stelling J, Fussenegger M: A synthetic multifunctional mammalian pH sensor and CO2 transgene-control device. Mol Cell 2014, 55:397-408. The authors engineered a pH- and CO2-regulated gene expression system in mammalian cells by rewiring a proton-responsive GPCR to a cAMP-responsive promoter. When implanted into type 1 diabetic mice, this device detected diabetic ketoacidosis and coordinated an automated therapeutic response by producing insulin to restore glucose and pH homeostasis. 37. Schukur L, Geering B, Charpin-El Hamri G, Fussenegger M:  Implantable synthetic cytokine converter cells with AND-gate logic treat experimental psoriasis. Sci Transl Med 2015, 7 318ra201. The authors repurposed the NFkB- and JAK/STAT3 pathways in human cells to engineer a therapeutic gene circuit capable of processing multiple disease-specific inputs. When implanted into mice developing the inflammatory skin disorder psoriasis, this device serially detected pathologic TNFa- and IL22-levels and rapidly initiated a self-sufficient therapeutic response by expressing the anti-inflammatory cytokines IL4 and IL10. 38. Rossger K, Charpin-El Hamri G, Fussenegger M: Reward-based hypertension control by a synthetic brain-dopamine interface. Proc Natl Acad Sci U S A 2013, 110:18150-18155. 39. He L, Zhang Y, Ma G, Tan P, Li Z, Zang S, Wu X, Jing J, Fang S, Zhou L et al.: Near-infrared photoactivatable control of Ca(2+) signaling and optogenetic immunomodulation. eLife 2015, 4.

27. Maeder ML, Angstman JF, Richardson ME, Linder SJ, Cascio VM, Tsai SQ, Ho QH, Sander JD, Reyon D, Bernstein BE et al.: Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat Biotechnol 2013, 31:1137-1142.

40. Muller K, Naumann S, Weber W, Zurbriggen MD: Optogenetics for gene expression in mammalian cells. Biol Chem 2015, 396:145-152.

28. Agne M, Blank I, Emhardt AJ, Gabelein CG, Gawlas F, Gillich N, Gonschorek P, Juretschke TJ, Kramer SD, Louis N et al.: Modularized CRISPR/dCas9 effector toolkit for targetspecific gene regulation. ACS Synth Biol 2014, 3:986-989.

41. Folcher M, Oesterle S, Zwicky K, Thekkottil T, Heymoz J,  Hohmann M, Christen M, Daoud El-Baba M, Buchmann P, Fussenegger M: Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant. Nat Commun 2014, 5:5392.

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Current Opinion in Biotechnology 2016, 40:139–148

148 Tissue, cell and pathway engineering

The authors synchronised the output of a near infrared light (NIR)-activated c-di-GMP-producing enzyme with a human c-di-GMP responsive interferon promoter. To facilitate remote control in biomedical applications, the authors also engineered a brain-computer interface device translating human brainwaves into different NIR-signals to eventually enable mind-controlled activation of target genes in mammalian cells and mice. 42. Kim T, Folcher M, Doaud-El Baba M, Fussenegger M: A synthetic  erectile optogenetic stimulator enabling blue-light-inducible penile erection. Angew Chem Int Ed Engl 2015, 54:5933-5938. When applying an engineered blue light-activated guanylate cyclase to the corpus cavernosum of male rats, local photostimulated cGMP production simulated the pharmacological activities of Viagra1, leading to the remote control of penile erection.

56. Beyer HM, Juillot S, Herbst K, Samodelov SL, Muller K, Schamel WW, Romer W, Schafer E, Nagy F, Strahle U et al.: Red light-regulated reversible nuclear localization of proteins in mammalian cells and zebrafish. ACS Synth Biol 2015, 4:951-958. 57. Niopek D, Benzinger D, Roensch J, Draebing T, Wehler P, Eils R, Di Ventura B: Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells. Nat Commun 2014, 5:4404. 58. Spiltoir JI, Strickland D, Glotzer M, Tucker CL: Optical control of peroxisomal trafficking. ACS Synth Biol 2015. 59. Renicke C, Schuster D, Usherenko S, Essen LO, Taxis C: A LOV2 domain-based optogenetic tool to control protein degradation and cellular function. Chem Biol 2013, 20:619-626.

43. Lienert F, Torella JP, Chen JH, Norsworthy M, Richardson RR, Silver PA: Two- and three-input TALE-based AND logic computation in embryonic stem cells. Nucleic Acids Res 2013, 41:9967-9975.

60. Hughes RM, Freeman DJ, Lamb KN, Pollet RM, Smith WJ, Lawrence DS: Optogenetic apoptosis: light-triggered cell death. Angew Chem Int Ed Engl 2015, 54:12064-12068.

44. Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M: CRISPRCas9-based photoactivatable transcription system. Chem Biol 2015, 22:169-174.

61. Lienert F, Lohmueller JJ, Garg A, Silver PA: Synthetic biology in mammalian cells: next generation research tools and therapeutics. Nat Rev Mol Cell Biol 2014, 15:95-107.

45. Polstein LR, Gersbach CA: A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat Chem Biol 2015, 11:198-200.

62. Zhang J, Jensen MK, Keasling JD: Development of biosensors and their application in metabolic engineering. Curr Opin Chem Biol 2015, 28:1-8.

46. Muller K, Zurbriggen MD, Weber W: Control of gene expression using a red- and far-red light-responsive bi-stable toggle switch. Nat Protoc 2014, 9:622-632.

63. Paddon CJ, Keasling JD: Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development. Nat Rev Microbiol 2014, 12:355-367.

47. Cao J, Arha M, Sudrik C, Bugaj LJ, Schaffer DV, Kane RS: Light inducible activation of target mRNA translation in mammalian cells. Chem Commun (Camb) 2013, 49:8338-8340. In this first optogenetic device regulating translational target events, the authors used the boxB-aptamer as an RNA tether to recruit N-peptide tagged CIB1 and Cry2-fused eIF4E, eventually achieving blue lightdependent translational initiation.

64. Menzel A, Gubeli RJ, Guder F, Weber W, Zacharias M: Detection of real-time dynamics of drug–target interactions by ultralong nanowalls. Lab Chip 2013, 13:4173-4179.

48. Kyung T, Lee S, Kim JE, Cho T, Park H, Jeong YM, Kim D, Shin A, Kim S, Baek J et al.: Optogenetic control of endogenous Ca(2+) channels in vivo. Nat Biotechnol 2015, 33:1092-1096.

65. Singh MS, Peer D: RNA nanomedicines: the next generation drugs? Curr Opin Biotechnol 2016, 39:28-34. 66. Wong AS, Choi GC, Cheng AA, Purcell O, Lu TK: Massively parallel high-order combinatorial genetics in human cells. Nat Biotechnol 2015, 33:952-961. 67. Courbet A, Endy D, Renard E, Molina F, Bonnet J: Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci Transl Med 2015, 7 289ra283.

49. Lee S, Park H, Kyung T, Kim NY, Kim S, Kim J, Heo WD:  Reversible protein inactivation by optogenetic trapping in cells. Nat Methods 2014, 11:633-636. In a highly attractive design, the authors fused CIB1 to a multimeric protein to engineer a protein scavenger that uses antibody-conjugated Cry2 to trap target proteins in a blue light-triggered manner.

68. Auslander D, Eggerschwiler B, Kemmer C, Geering B, Auslander S, Fussenegger M: A designer cell-based histamine-specific human allergy profiler. Nat Commun 2014, 5:4408.

50. Bugaj LJ, Choksi AT, Mesuda CK, Kane RS, Schaffer DV: Optogenetic protein clustering and signaling activation in mammalian cells. Nat Methods 2013, 10:249-252.

69. Schukur L, Geering B, Fussenegger M: Human whole-blood culture system for ex vivo characterization of designer-cell function. Biotechnol Bioeng 2016, 113:588-597.

51. Wroblewska L, Kitada T, Endo K, Siciliano V, Stillo B, Saito H, Weiss R: Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery. Nat Biotechnol 2015, 33:839-841.

70. Yang HK, Yoon KH: Current status of encapsulated islet transplantation. J Diabetes Complicat 2015, 29:737-743.

52. Auslander S, Stucheli P, Rehm C, Auslander D, Hartig JS,  Fussenegger M: A general design strategy for proteinresponsive riboswitches in mammalian cells. Nat Methods 2014, 11:1154-1160. This impressive work shows the general design principles for engineering protein-responsive ribozymes from constitutive cis-acting ribozymes. The authors incorporated aptamer structures, such as boxB, into the HHR catalytic domain without disrupting the self-cleavage ability, thereby permitting the programmability of translational initiation of the target genes. 53. Kennedy AB, Vowles JV, d’Espaux L, Smolke CD: Proteinresponsive ribozyme switches in eukaryotic cells. Nucleic Acids Res 2014, 42:12306-12321. 54. Bloom RJ, Winkler SM, Smolke CD: A quantitative framework for the forward design of synthetic miRNA circuits. Nat Methods 2014, 11:1147-1153. 55. Wang T, Xie Y, Tan A, Li S, Xie Z: Construction and characterization of a synthetic microRNA cluster for multiplex RNA interference in mammalian cells. ACS Synth Biol 2015.

Current Opinion in Biotechnology 2016, 40:139–148

71. Jullesson D, David F, Pfleger B, Nielsen J: Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals. Biotechnol Adv 2015, 33:1395-1402. 72. Vashist SK, Luppa PB, Yeo LY, Ozcan A, Luong JH: Emerging technologies for next-generation point-of-care testing. Trends Biotechnol 2015, 33:692-705. 73. Heng BC, Aubel D, Fussenegger M: Prosthetic gene networks as an alternative to standard pharmacotherapies for metabolic disorders. Curr Opin Biotechnol 2015, 35:37-45. 74. Guye P, Ebrahimkhani MR, Kipniss N, Velazquez JJ, Schoenfeld E, Kiani S, Griffith LG, Weiss R: Genetically engineering selforganization of human pluripotent stem cells into a liver budlike tissue using Gata6. Nat Commun 2016, 7:10243. 75. Saxena P, Heng BC, Bai P, Folcher M, Zulewski H, Fussenegger M: A programmable synthetic lineage-control network that differentiates human iPSCs into glucose-sensitive insulinsecreting beta-like cells. Nat Commun 2016, 7:11247 PMID: 27063289.

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