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Benchmarking of TALE- and CRISPR/dCas9-Based Transcriptional Regulators in Mammalian Cells for the Construction of Synthetic Genetic Circuits Tina Lebar†,‡,§ and Roman Jerala*,†,‡ †

Department of Synthetic Biology and Immunology, National Institute of Chemistry, 1000 Ljubljana, Slovenia EN-FIST Centre of Excellence, 1000 Ljubljana, Slovenia § Graduate School of Biomedicine, University of Ljubljana, 1000 Ljubljana, Slovenia Downloaded via KAOHSIUNG MEDICAL UNIV on November 15, 2018 at 13:28:48 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Transcriptional activator-like effector (TALE)- and CRISPR/ Cas9-based designable recognition domains represent a technological breakthrough not only for genome editing but also for building designed genetic circuits. Both platforms are able to target rarely occurring DNA segments, even within complex genomes. TALE and dCas9 domains, genetically fused to transcriptional regulatory domains, can be used for the construction of engineered logic circuits. Here we benchmarked the performance of the two platforms, targeting the same DNA sequences, to compare their advantages for the construction of designed circuits in mammalian cells. Optimal targeting strands for repression and activation of dCas9-based designed transcription factors were identified; both platforms exhibited good orthogonality and were used to construct functionally complete NOR gates. Although the CRISPR/ dCas9 system is clearly easier to construct, TALE-based activators were significantly stronger, and the TALE-based platform performed better, especially for the construction of layered circuits. KEYWORDS: TAL effectors, CRISPR/Cas9, transcriptional regulation, NOR gate, synthetic circuits

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aptamer-binding protein.16−18 TALE and dCas9-based regulators have many similarities, including similar length of the DNA recognition site and monomeric DNA binding. This sets them apart from the mainly oligomeric natural transcription factors19 with characteristic cooperative DNA binding, which is relevant for the construction of genetic switches.8 The designable diversity of TALE-based transcriptional regulators allows not only for the construction of simple repressors or activators but for combinations of several orthogonal designed TFs for construction of complex information processing transcriptional networks. Among these are two- or three-input logic gates or bistable switches, representing epigenetic memory elements, where 1 bit can be stored by a pair of orthogonal designed TALEs.8,11 Design of single-layer, TALE-based NOR gates and wiring of the new TALE-repressor as output means that, in principle, any logic function could be constructed. It has been demonstrated that even logic circuits comprising three layers retained a clear separation between active and inactive states.11

traightforward coding for the recognition of selected DNA targets by CRISPR/Cas9- and TAL effector (TALE)-based systems represents an important technological advancement for synthetic biology. Although zinc finger-based regulators have previously been used for the construction of designed transcription factors (TFs), their drawback was the sensitivity of designed zinc fingers to context,1 requiring a trial-and-error approach. The natural role of Cas9 is the cleavage of foreign DNA, based on targeting by the guide RNA (gRNA);2 the role of TALEs is activation of gene transcription, based on direct modular protein-based DNA recognition.3 Several strategies have been invented for the rapid and massive generation of modular TALEs,4,5 enabling generation of tens of thousands of TALEs. Their modular composition enables generation of transcriptional regulators for virtually any selected gene. TALEbased activators and repressors have been constructed by genetic fusion, using the VP16 domain or its derivatives for designed activators6−8 or the KRAB domain for repressors.8−11 Regulation of endogenous as well as designed gene constructs has been demonstrated. Cas9-based generation of transcriptional regulators has followed a similar strategy, using a catalytically inactivated Cas9 (dCas9) in complex with the targeting gRNA. The transcriptional regulator was either genetically fused to the dCas9 protein12−15 or bound to gRNA by means of fusion to an RNA aptamer, targeted by an © 2016 American Chemical Society

Special Issue: Synthetic Biology in Europe Received: November 30, 2015 Published: June 26, 2016 1050

DOI: 10.1021/acssynbio.5b00259 ACS Synth. Biol. 2016, 5, 1050−1058

Letter

ACS Synthetic Biology

Figure 1. Comparison of TALE- and CRISPR/dCas9-based transcriptional repression. (a) Design of TALE/gRNA target sites. PAMt denotes the PAM sequence for targeting the template strand; PAMnt denotes the PAM sequence for targeting the nontemplate strand. (b) Schematic presentation of dCas9-gRNA complex binding to nontemplate strand target (top) and to template strand target (bottom). (c) Schematic presentation of roadblock transcriptional repression by a TALE-DBD and dCas9:gRNA complexes. When a TALE-DBD or dCas9:gRNA complex is bound to the target site downstream of the CMV promoter, elongation of transcription by RNA polymerase II is inhibited. gRNA(t) denotes gRNA targeting the template strand, and gRNA(nt) denotes gRNA targeting the nontemplate strand. (d) Roadblock repression by binding of a TALE DNA-binding domain or the dCas9:gRNA complex to the exonic region of a reporter gene. (e) Roadblock repression by binding of a TALE DNAbinding domain and dCas9:gRNA complexes to a binding site between the TATA box and the Kozak sequence (statistical significance at levels ***P < 0.001, **P < 0.01 and *P < 0.05). (f) Schematic presentation of KRAB-mediated transcriptional repression by a TALE-KRAB repressor and dCas9-KRAB:gRNA complexes. When a TALE-KRAB repressor or dCas9-KRAB:gRNA complex is bound to the target sites upstream of the CMV promoter, the KRAB domain causes transcriptional repression by chromatin silencing. gRNA(t) denotes gRNA targeting the template strand and gRNA(nt) denotes gRNA targeting the nontemplate strand. (g) KRAB-mediated repression on reporter plasmids with a different number of target sites. Plasmids with 2 or 7 copies of [a_b] target sites were used along with the TALE[B]-KRAB repressor or gRNA[B], targeting the template and the nontemplate strand (statistical significance at levels ***P < 0.001 and **P < 0.01). (h) KRAB-mediated repression of the 3 designed reporter plasmids (statistical significance at levels ***P < 0.001, **P < 0.01 and *P < 0.05).

For a reliable assessment of the advantages of each system, a systematic comparison is therefore warranted between TALEand CRISPR/dCas9-based designed TFs targeting the same DNA sequences. This study investigates the efficiency of dCas9-based transcriptional regulators for the construction of genetic circuits in mammalian cells in a direct comparison with TALE-based genetic circuits. Targeting the same DNA sequence, TALE- and CRISPR/dCas9-based repressors are first compared. Next, we describe the construction of a CRISPR/dCas9-based NOR gate that can be used for the construction of any two-input logic gate. Successful layering of such gates was achieved by KRAB-mediated repression of RNAP III promoter-driven gRNA expression. TALE-based

CRISPR/dCas9-based transcription factors have recently been implemented to perform simple logic functions in bacterial20 and mammalian cells.21−23 The advantage of CRISPR-based multiplexing based on several gRNAs represents an attractive opportunity, as several strategies have been proposed to generate multiple gRNAs in a single transcript.22,24 While TALE- and CRISPR/dCas9-based transcriptional regulators of endogenous genes have been compared by targeting the same enhancer regions,25 a direct comparison by targeting the same DNA sequence has not as yet (to our knowledge) been conducted. Additionally, several frameworks have been proposed for transcriptional activators based on dCas9,17,26 but have not been benchmarked in the same setting. 1051


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Figure 2. Designed transcription factor orthogonality. (a) TALE repressors (left) and gRNAs (right) used in this study were tested against each other’s target sites. Repression of reporter gene expression occurred only when the firefly luciferase reporter plasmid was cotransfected with the appropriate TALE-KRAB repressor or dCas-KRAB and gRNA plasmids. (b) TALE-KRAB repressors (top) and dCas-KRAB:gRNA complexes (bottom) inhibit transcription of the corresponding reporter gene in a single cell.

transcriptional activators induce transcription more strongly than dCas9-based activators. While CRISPR/dCas9-based complex synthetic circuits are feasible and certainly easier to construct, TALEs offer some distinct advantages for the construction of such devices. To perform a direct comparison of TALE- and dCas9-based transcriptional regulation, target sites were first designed for binding of both TALEs and gRNAs. As the gRNA target site spans 21 residues, as compared to a typical 17−18 nucleotides for TALE target sites, the designed target site was a TALE target site, extended by 3−4 nucleotides to allow binding of both TALE and gRNA/dCas9 complex. At both ends of the

target, a PAM sequence (CGG) required for dCas9 recognition was introduced to facilitate investigation of the difference between gRNAs, targeting different strands (Figure 1a,b). For the regulatory transcriptional toolbox elements used in synthetic circuits, the two main requirements are efficiency and orthogonality. We first investigated the efficiency of the roadblock type of repression similar to that used previously.27 We tested repression by targeting the nontemplate strand in the exonic region of a reporter gene, as reported by Qi et al.27 The reporter plasmid was designed as a single gRNA/TALE DNA target site, incorporated into the mCitrine fluorescent protein nucleotide sequence as an N-terminal in-frame fusion. 1052


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Figure 3. Construction of functional NOR gates using designed TALE- and CRISPR/dCas9-based transcriptional repressors. (a) The TALE-KRAB repressor-based NOR gate. (b) The dCas9-KRAB repressor-based NOR gate. (c, d) TALE- (c) and dCas9-based (d) NOR gates were demonstrated with luciferase activity measurements (left), flow cytometry (middle) and confocal microscopy (right). The scale bar represents 250 μm. Amounts of transfected plasmids are listed in Supplementary Table S4.

means of chromatin silencing, we hypothesized that it might not be sensitive to strand selection. However, for all tested gRNAs, more efficient repression was observed when targeting the nontemplate strand and was comparable to TALE-KRAB mediated transcriptional repression (Figure 1f,g). While the mechanism responsible for the difference in KRAB-mediated repression between the template and nontemplate strand remains unclear, it seems likely that the orientation of the dCas9-KRAB protein bound to the DNA with respect to the promoter may play some role, although the effect remains for multiple copies of the target site. For optimal benchmarking of CRISPR/dCas9-based transcriptional repressors, we therefore used nontemplate strand targeting gRNAs in all of the following experiments. In addition to the HEK293T cell line, repression experiments were performed in different commonly used mammalian cell lines, such as HeLa, Caco-2, NIH3T3 and CHO (Supplementary Figure S2). The performance of both platforms was efficient in all cell types, however lower than in the originally tested HEK293T cell line, which was used in the following experiments. Because complex synthetic circuits require transcription factor orthogonality in order to function independently from the cells̀ endogenous regulatory logic, TALE repressor and gRNA cross-reactivity were tested. Inhibition of reporter gene expression occurred only when the reporter plasmid was cotransfected with the appropriate repressor, but not with other repressors (Figure 2a). When HEK293T cells were cotransfected with two fluorescent reporter plasmids, each containing different target sites, only the corresponding reporter̀s expression was inhibited when a TALE/gRNA repressor was present (Figure 2b). Our next goal was to design a NOR gate using CRISPR/ dCas9-based transcriptional repressors. As NOR gates are

Cotransfection of either dCas9 and gRNA either TALE encoding plasmids did not result in any decrease in reporter activity (Figure 1c,d). Next we tried a different strategy by relocating the target site 15 nucleotides downstream of the CMV promoter TATA box and directly upstream of the Kozak sequence. We examined potential differences in targeting each of the two DNA strands. Repression by direct binding of both TALE and dCas9 was observed (Figure 1c,e), but dCas9 exhibited only 3- to 7-fold repression as compared to substantially stronger repression by the corresponding TALE roadblock. The roadblock-type repression demonstrated better efficiency by targeting the nontemplate strand; this can be attributed to the differential efficiency in blocking progression of the RNA polymerase, as reported previously for bacterial cells.27 To confirm that the differences in TALE and dCas9 repression efficiency are not an attribute of expression levels, we performed a Western blot analysis, which confirmed similar expression levels of both proteins (Supplementary Figure S1). In mammalian cells, KRAB-based repression can be used, targeted to regions upstream from the promoter to exclude the effect on constitutive transcription.11 Additionally, introduction of multiple copies of the operator increases the efficiency of upor down-regulation and decreases potential off-target effects. To test this strategy, reporter plasmids were designed with multiple target sites upstream of the CMV promoter for binding of either TALE-KRAB repressors or dCas9-KRAB:gRNA complexes (Figure 1f−h). Both TALE- and dCas9-based KRAB repressors exhibited high levels of repression for two target sites. Increasing the number of target sites also significantly improved repression for dCas9-KRAB, which reached 69-fold for 7 copies of the operator, making it comparable to repression by TALE-KRAB (Figure 1f). As KRAB-based repression is generally believed to function by 1053


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Figure 4. KRAB domain-mediated repression of the RNAPIII promoter. (a) Schematic presentation of layered repression with the CRISPR/dCas9 system. In the absence of gRNA[B] (left), gRNA[D] is expressed and can mediate transcriptional repression of the reporter gene in complex with dCas9-KRAB. When gRNA[B] is present (right), the dCas9-KRAB:gRNA[B] complex inhibits activity of the mU6 promoter, driving expression of gRNA[D], and the reporter gene is expressed. (b) KRAB-mediated repression of the mU6 promoter. HEK293T cells were transfected with 50 ng of 7[d]_pCMV-fLuc reporter plasmid, 100 ng of pCMV_dCas9-KRAB plasmid, either 0 or 50 ng mU6_gRNA[B] plasmid and increasing amounts of the 7[b]_mU6_gRNA[D] plasmid. (c, e) Schematic presentation of TALE- (c) and CRISPR/dCas9-based (e) AND gates. (d, f) The TALE- (d) and CRISPR/dCas9-based (f) AND gates were demonstrated with luciferase activity measurements (left) and flow cytometry (right). Amounts of transfected plasmids are listed in Supplementary Table S4.

transcribes mRNA. Recently, Kiani et al.21 demonstrated a strategy for layered transcriptional regulation with CRISPR/ dCas9 by creating synthetic RNAPII and RNAPIII promoters, which can be regulated by CRISPR/dCas9 roadblock. However, such an approach can influence the constitutive expression of a promoter by inserting different DNA sequences in close proximity to the TATA box and other regulatory elements. Although there are reports of successful downregulation of RNAPIII promoters (including the U6 promoter, used for gRNA expression in this study) by the KRAB chromatin silencing domain,28−30 such an approach has not yet been used in the context of the CRISPR/Cas9 system. To test whether the KRAB domain can inhibit transcription of gRNA, we constructed a plasmid comprising the U6 promoter, driving the expression of gRNA[D], and 7 copies of target operator sites [b] upstream of the promoter. In the absence of gRNA[B], gRNA[D] should be expressed, inhibiting transcription of the firefly luciferase reporter in combination with dCas9-KRAB. However, as the dCas9-KRAB:gRNA[B] complex should inhibit transcription of gRNA[D], activity of the firefly luciferase reporter should be observed (Figure 4a). We demonstrated a 5-fold difference between the low and high states at lower amounts of the 7[b]_mU6_gRNA[D] plasmid,

functionally complete logic gates and can therefore be combined to construct any logic gate, such a gate should facilitate straightforward construction of more complex circuits using the CRISPR/dCas9 system. As demonstrated in our previous work,11 a single-layer, TALE-based NOR gate employs KRAB domain-based repressors as inputs and two operators positioned upstream of a constitutive promoter, driving the expression of a reporter gene as output (Figure 3a). We sought to construct a NOR gate with the same topology as that implemented for TALEs using the CRISPR/dCas9 system (Figure 3b) and to compare it to the TALE-based NOR gate. As expected from the conceptual similarity of the repressor in both systems (Figure 1), both NOR gates exhibited similarly efficient performance (Figures 3c,d), suggesting that the CRISPR/dCas9 system could be exploited for construction of more complex circuits, following an approach similar to that for TALE-based circuits. The main potential weakness of CRISPR/dCas9-based circuits is the regulation of gRNA expression, which is normally expressed from RNAP III-driven promoters in mammalian cells. Complex circuits must comprise multiple layers, suggesting that gRNA expression would have to be either repressed or activated. However, RNAP III-driven promoters differ in transcription regulation from RNAP II, which 1054


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Figure 5. Comparison of TALE- and CRISPR/Cas9-based transcriptional activation. (a) Schematic presentation of transcriptional activation by TALE- and dCas9-based activators. Upon binding of a transcriptional activator to the target sites upstream of a minimal promoter (pMIN), the reporter gene is expressed. gRNA(t) denotes gRNA targeting the template strand, and gRNA(nt) denotes gRNA targeting the nontemplate strand. (b) Transcriptional activation by the VP16 activation domain (statistical significance at levels ***P < 0.001 and **P < 0.01). Reporters contained either one copy of target site [d] and one copy of target site [bT] (TALE[B] target site) or 7 copies of target sites [b] upstream of a minimal promoter, driving the expression of firefly luciferase. (c) Transcriptional activation by the VPR activation domain (statistical significance at levels ***P < 0.001 and **P < 0.01). Reporter plasmids contained either one copy of target site [d] and one copy of target site [bT] (TALE[B] target site) or 7 copies of target sites [b] upstream of a minimal promoter, driving the expression of firefly luciferase. (d) Schematic presentation of RNA aptamer-mediated transcriptional activation. Upon binding of the MS2 RNA aptamer-binding protein, fused to an activation domain to its binding site on the gRNA, the reporter gene is expressed. (e) Transcriptional activation by the MS2 aptamer binding protein, fused to the VPR activation domain (statistical significance at level *P < 0.05). The reporter plasmid contained 7 copies of target sites [b] upstream of a minimal promoter, driving the expression of firefly luciferase.

performed a similar comparison for VP16-based activators, constructing reporter plasmids with one or multiple copies of target sites upstream of a minimal promoter (Figure 5a). In contrast to TALE-based activators, the dCas9-based VP16 activator was unable to activate transcription of a reporter gene on a plasmid with a single target site at a significant level when targeting either the template or the nontemplate strand (Figure 5b). This aligns with results relating to dCas9-based activation of endogenous genes, which often required gRNAs, targeting several positions to achieve significant gene activation.32,33 While increasing the number of target sites upstream of the promoter resulted in sufficient activation by all tested VP16 activators, use of the TALE-VP16 activator resulted in considerably higher expression levels (over 1000-fold) than activation with dCas9-based VP16 activators (less than 100fold). The results of one recent study comparing both systems for regulation of endogenous genomic loci for reprogramming somatic cells into iPS cells25 suggest that both systems show similar trends in transcriptional repression. However, they also indicated that TALE transcriptional activators exhibited more potent activity for the regulation of endogenous genes. While

suggesting successful repression of the U6 promoter by a dCas9-KRAB:gRNA complex (Figure 4b). We next attempted to construct a two-layered AND-gate with the same topology as for TALE repressors before,11 which demonstrated significantly decreased performance of dCas9based circuits (Figure 4c−f). The efficiency of this gate is limited to two layers, while up to four layers have been demonstrated for TALE-based circuits.11 A CRISPR/dCas9based AND gate with a similar topology was recently constructed in combination with the bacterial transcriptional repressor LacI,23 in which two RNAPII inducible promoters were used to drive the expression of either dCas9 or gRNA, flanked by two ribozymes at each end. The same strategy for RNAPII promoter-driven gRNA expression, comparable in strength to expression from RNAPIII-driven promoters, has previously been demonstrated by other groups.22,31 Although activators are not absolutely required for the construction of logic gates, they are required for the introduction of positive feedback loops and therefore represent an important regulatory element in the synthetic biology toolbox. As well as benchmarking KRAB-based repressors, we 1055


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plasmid 32279, Addgene) and from TALEN1295 (TALE[D]; plasmid 32283, Addgene). The KRAB domain was obtained from pLVPT-rtTR-KRAB-2SM2 (plasmid 11652, Addgene) and the VP16 domain from the vector pSGVP, provided by Prof. Mark Ptashne (Memorial Sloan−Kettering Cancer Center, New York). The VPR activator sequence was adapted from the Addgene plasmid 63801 and synthesized by Genewiz. All gRNA/TALE target sites were designed as 7 repeats, separated by hypervariable linkers and synthesized by Genewiz. One copy of the gRNA/TALE target site was introduced into reporter plasmids with PCR. Reporter genes were PCRamplified from commercial plasmids: firefly luciferase from pGL4.16 (Promega), TagBFP from pTagBFP-N (Evrogen) and mCitrine was obtained as a gift from Dr. Oliver Griesbeck (the Max Planck Institute of Neurobiology, Munchen, Germany). Renilla luciferase (phRL-TK, Promega), iRFP670 (synthesized by LifeTechnologies) and mCherry (pmCherry-C1, Clontech) were used as transfection controls. The SV40 large T-antigen nuclear localization sequence (NLS), the hexa-histidine tag and the minimal promoter were introduced into the constructs with PCR. Cell Culture and Transfection. The human embryonic kidney (HEK) 293T cell line (ATCC), the HeLa cell line (ATCC) and the Caco-2 cell line (ATCC) were cultured in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (BioWhittaker) at 37 °C in a 5% CO 2 environment. The CHO cell line (ATCC) and the NIH-3T3 cell line (ATCC) were cultured in DMEM F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum (BioWhittaker) at 37 °C in a 5% CO2 environment. For luciferase and fluorescence spectroscopy experiments 2 × 104 HEK293T cells per well were seeded in CoStar White 96-well plates (Corning). For Western blot analysis 5 × 105 HEK293T cells per well were seeded in 6-well plates (TPP). For flow cytometry experiments 2 × 105 HEK293T cells per well were seeded in 12-well plates (TPP). For confocal microscopy experiments 5 × 104 HEK293T cells per well were seeded in 8well tissue culture chambers (Ibidi). At 30−90% confluence, they were transfected with a mixture of DNA and PEI (6 μL/ 500 ng DNA, stock concentration 0.324 mg/mL, pH 7.5). Unless otherwise stated, the ratio of reporter plasmids and TALE transcriptional regulators was 1:1, and the ratio of reporter plasmids, dCas9 transcriptional regulators and gRNAs was 1:1:1. Fluorescence Spectroscopy. Three days after transfection, the cells were harvested and lysed with 30 μL of 1× passive lysis buffer (Promega). Fluorescence was measured using a Synergy Mx automated microplate reader (BioTec, Winooski, VT, USA). Fluorescence emission was detected at 520−540 nm (mCitrine) and 600−620 nm (mCherry transfection control). The relative fluorescence was calculated by normalizing the mCitrine fluorescence of each sample by the constitutive mCherry fluorescence measured within the same sample. Graphs represent the mean and standard deviation of three measurements within an experiment. The data are representative of at least two independent experiments. Luciferase Assays. Three days after transfection, the cells were harvested and lysed with 30 μL of 1× passive lysis buffer (Promega). Firefly luciferase and Renilla luciferase expression were measured using a dual luciferase assay (Promega) and an Orion II microplate reader (Berthold Technologies). Relative luciferase activity was calculated by normalizing each sample’s firefly luciferase activity to the constitutive Renilla luciferase

transcriptional activation by the hybrid VPR activation domain26 resulted in significantly higher levels of expression for all tested activators, activation with dCas9-based VPR activators was substantially lower than for the TALE-VPR activator (Figure 5c). In this case, targeting the template strand yielded a stronger effect than targeting the nontemplate strand, regardless of the number of target sites. Granted that transcriptional activation and repression domains recruit very different protein complexes, it was nevertheless surprising that activation and repression clearly favored targeting different strands. TALEs allow independent combination of activators and repressors, which can be accomplished by dCas9 systems through combination of two orthogonal Cas proteins or through implementation of RNA aptamers to gRNAs. Different aptamer-binding proteins could then prompt the activation or repression of selected target genes.17,18 We tested transcriptional activation by gRNAs, extended with 2 copies of the MS2 RNA aptamer, finding that the dCas9 protein only mediates DNA binding while the MS2 aptamer-binding protein fused with the VPR activation domain activates transcription (Figure 5d). Coupling of the VPR to the RNA-aptamer binding domain resulted in strong activation (over 900-fold), which is comparable to activation with dCas9-VPR activator (Figure 5e). This result implies that such an approach could be used for circuits with both repressors and activators. In conclusion, benchmarking of TALE and dCas9-based designed TFs revealed that, in general, TALEs cause a stronger regulatory effect; this is particularly important for layered circuits, where the efficiency of repression must be as high as possible. We show that gRNA expression from a RNAPIIIdriven promoter can be controlled by the KRAB domain; however, the efficiency of repression was not sufficient for construction of layered CRISPR/dCas9-based logic. Nevertheless, for less complex logic functionsespecially those that can be performed in a single layerdCas9 renders sufficiently strong transcriptional modulation and confers the advantage of significantly easier construction. The optimal strategy for the design of efficient genetic circuits may be to use the CRISPR/ dCas9 system for rapid prototyping and screening and to implement the circuit intended for durable application using TALEs (unless the number of proteins to be introduced is not excessive).

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METHODS Plasmid Construction. All plasmids used were constructed using classic molecular cloning methods or the Gibson assembly method, as listed in Supplementary Table S1. Nucleotide sequences are set out in Supplementary Table S2, followed by amino acid sequences in Supplementary Table S3. All gRNA guide sequences were introduced with PCR into the plasmid pgRNA-humanized (plasmid 44248, Addgene). The gRNAs and TALE repressors for layered repression were PCRamplified along with the U6 or CMV promoter and transferred into pcDNA3 plasmids containing gRNA/TALE target sites. The nuclease-deficient Cas9 was obtained from pHR-SFFVdCas9-BFP-KRAB (dCas9; plasmid 46911, Addgene). The gRNA containing MS2 aptamer sequences (gRNA_2 ms2) was synthesized by LifeTechnologies, amplified with PCR and inserted into the plasmid pgRNA-humanized. The MS2 protein sequence was adapted from the Addgene plasmid 27121 and synthesized by LifeTechnologies. The TALE DNA-binding domains were PCR amplified from TALEN1257 (TALE[A]; plasmid 32280, Addgene), from TALEN1297 (TALE[B]; 1056


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ACS Synthetic Biology Author Contributions

activity determined within the same sample. All graphs represent the mean and standard deviation of three measurements within an experiment. The data are representative of at least three independent experiments. SDS-PAGE and Western Blotting. Two days after transfection, the cells were harvested with 500 mL of PBS, centrifuged for 10 min at 2000 rpm and lysed in 100 μL of 1xPassive lysis buffer (Promega). The lysates were kept on ice for 15 min and centrifuged for 30 min at 14500 rpm Protein concentration was determined with the BCA assay. Samples (50 μg of total protein) were mixed with reducing SDS-PAGE loading buffer and incubated for 10 min at 95 °C. Proteins were separated at 200 V on an 8% polyacrylamide gel following transfer to a nitrocellulose membrane at 350 mA. The membrane was blocked in 0.2% I-Block (Applied Biosystems) for 30 min at room temperature and incubated in primary antibodies (mouse anti-tetraHis IgG; Qiagen) for 90 min at room temperature. After three washing steps in PBST (PBS containing 0.1% Tween-20), the membrane was incubated in secondary antibodies (goat antimouse IgG conjugated with horseradish peroxidase (HRP); Santa Cruz Biotechnology) for 30 min at room temperature. Following two washing steps in PBST, chemiluminescence was detected with the Supersignal West Femto Maximum sensitivity substrate (Thermo Scientific). Flow Cytometry. Three days after transfection, the cells were washed with PBS and harvested with 500 mL of FACS buffer (3% fetal bovine serum in PBS). Flow cytometry analysis was performed using a CyFlow space flow cytometer (Partec). A 488 nm diode laser was used to detect mCitrine, a 405 nm diode laser was used to detect TagBFP, and a 633 nm diode laser was used to detect the iRFP670 transfection control. In each sample, at least 30 000 cells were analyzed and gated to iRFP670-positive cells. The collected data were processed using FlowJo software (TreeStar). The data are representative of at least three independent experiments. Confocal Microscopy. Microscopic images were acquired 3 days after transfection, using the Leica TCS SP5 inverted laser-scanning microscope on a Leica DMI 6000 CS module equipped with a HCX PL Fluotar L 20×, numerical aperture 0.4 (Leica Microsystems). A 514 nm laser line of a 100 mW argon laser with 25% laser power was used for mCitrine excitation, and the emitted light was detected between 510 and 550 nm. A 1 mW 543 nm HeNe laser was used for mCherry transfection control excitation, and the emitted light was detected between 590 and 630 nm. Leica LAS AF software was used for acquisition, and ImageJ software was used for image processing. The data are representative of at least three independent experiments and five separate observations within each experiment.

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RJ designed the study and wrote the manuscript. TL designed the study, designed and cloned the plasmid constructs, performed all experiments and wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grants from the Slovenian Research Agency and in part by EU structural funds assigned to the EN-FIST Centre of Excellence. We thank Dr. Mark Ptashne for the plasmid containing the VP16 domain and Dr. Oliver Griesbeck for the plasmid with the mCitrine fluorescent protein. We thank Ana Halužan-Vasle and Ana Milovanović for help in performing initial experiments on comparison of transcriptional repressors. We are grateful to Rok Gaber, Anže Smole and Luka Smole for help with binding site design.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.5b00259. Supporting tables and figures (PDF)

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