Cas9 System for

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RNA Strand Displacement Responsive CRISPR/Cas9 System for mRNA Sensing Yue Li, Xucong Teng, Kaixiang Zhang, Ruijie Deng, and Jinghong Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05238 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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Analytical Chemistry

RNA Strand Displacement Responsive CRISPR/Cas9 System for mRNA Sensing Yue Lia, Xucong Tenga, Kaixiang Zhanga,b, Ruijie Denga, Jinghong Lia* a. Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China. b.

School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China

*to whom corresponding should be addressed. Phone: 86-10-62795290; Fax: 86-10-62771149 Email: [email protected] 1 / 23

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ABSTRACT CRISPR/Cas9 has already become a powerful tool for genomic manipulation, further engineering of the system allows it to be precisely regulated in response to external signals, thus broadening its application possibilities, such as biosensing or bioimaging. However, most stimuli-responsive CRISPR systems are built based on elaborately designed and engineered inducible Cas9 proteins, and external stimuli are still mostly limited as small molecules and light. To construct more precise and easy-to-build responsive CRISPR systems and broaden their responsive species, we seek to engineer conditional guide RNA, rather than Cas9 protein, to mediate conditional CRISPR corresponding to logic operation. Here, we construct mRNA-sensing CRISPR by gRNA reconfiguration and toehold mediated strand displacement, in which each target site could be independently controlled. We show that switches can be embedded into the gRNA and used as RNA sensors, capable of detecting multiple mRNA inputs orthogonally and providing CRISPR/Cas9 response outputs. NOR and NAND logical gates are also constructed, demonstrating its orthogonality and programmability. This strategy promises potential uses in constructing genetic circuits to detect endogenous mRNAs and initiate cellular responses.

Keywords Strand displacement; DNA cleavage; CRISPR; logical gates; mRNA; signal regulation; genetic circuit; RNA biosensor

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Introduction The CRISPR/Cas9 system provides powerful tools for sequence-specific genome editing1-3 and gene regulation4-9 with high efficiency and versatility. The ability to precisely control the cleavage or binding activity of the CRISPR system will enhance the selectivity and spatiotemporal accuracy, thus broadening its application possibilities in biosensing, bioimaging and targeted therapy.10-15 Precisely controlled conditional CRISPR system has been preliminarily explored, such as light-responsive16-18 systems, small molecule inducible systems13,19 and transcriptional regulatory circuits using split-Cas9.20,21 However, most of them rely on protein engineering, which requires sophisticated protein design and optimization processes. Besides, these Cas9 variants can combine with all gRNA indiscriminately, regardless of targeting sequences, which limits their application in the orthogonal systems for controlling different gene targets. Thus, it is necessary to develop a method for easily controlling CRISPR activity. Meanwhile, there are few reported works on gRNA design for constructing conditional CRISPR system. They blocked the 20nt spacer region of gRNA from recognizing the target by a photo-responsive group-modified nucleic acids protector or a ligand-controlled aptamer unit,14 and thus the Cas9 cleavage activity could be controlled by the stimulation of light or small molecules. Although different photo-responsive groups or aptamers could be employed to achieve multiple orthogonal control of gRNA, the available triggers are still quite limited. RNA-based regulatory elements provide potential solution to these limitations, utilizing Watson-Crick base pairing to offer both specificity and orthogonality. The secondary structure of RNA and the interactions between RNA molecules can be predicted by software.22-25 We can also learn from the naturally occurring RNA regulatory components, such as riboswitch, which can bind with a range of RNA sequences or metabolites and regulate transcriptional and post-transcriptional events. The species and expression level of RNA is a biological signature for certain 3 / 23



cell type or the current status of the cell.26-29 Artificial sensors can be designed to sense these signature and modulate downstream activity.30-32 In our case, conditional CRISPR can be regarded as a biosensor that captures stimulus signal and converts it into various CRISPR-based functions. Therefore, RNA is an ideal candidate for input signal of a biosensor. Herein, we have developed an mRNA sensing-CRISPR biosensor, which enables independent control of CRISPR activity with different mRNAs. The structure of gRNA is redesigned with a switchable structure, which can transform from OFF to ON upon the binding of mRNA via strand displacement. The mRNA-sensing guide RNA (msgRNA) is designed to be repressed until the hybridization of target mRNA. After binding with mRNA, configurational changes of the msgRNA are initiated, leading to the formation of activated Cas9/gRNA complex that targets an unrelated silencing gene. Our system could switch on CRISPR by sensing the sequence and level of mRNA with designing flexibility, orthogonality and programmability. Moreover, all of the sensor components are genetically coded, which provides opportunities to install intelligent biomolecule sensors into the genetic circuit.

EXPERIMENTAL SECTION Reagents and Instruments Cas9 Nuclease, S. pyogenes, EnGen® Spy dCas9, EnGen® Spy Cas9 Nickase, HiScribeTM T7 Quick High Yield RNA Synthesis Kit were purchased from NEB (New England Biolabs, Ipswich, MA, USA). Lipofectamine CRISPRMAX Transfection Reagent and Lipofectamine 3000 were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). PrimeSTAR HS (Premix) used for PCR and RNA marker RL1000 were obtained from TaKaRa Biotechnology Co., Ltd. (Dalian, China). RiboLock RNase inhibitor (40 U mL-1) was purchased from Life Technologies (Carlsbad, CA, USA). Human serum was obtained from Lablead Biotech. Co., Ltd. (Beijing, China). TransStart Green qPCR SuperMix was purchased from TransGen Biotech Co., Ltd. (Beijing, China). All oligonucleotides were purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, 4 / 23


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China). All the PCR products were purified by SanPrep Column DNA Gel Extraction Kit. from Sangon Biotech Co., Ltd. (Shanghai, China). In vitro transcription of msgRNA and mRNA msgRNA and mRNA were transcribed in vitro by HiScribeTM T7 Quick High Yield RNA Synthesis Kit (purchased from NEB) using DNA template. The DNA template contained a T7 promoter sequence and msgRNA or mRNA template sequence. The template DNA was synthesized by PCR reaction using different primers and purified by ethanol precipitation. Briefly, the in vitro transcription reaction was performed in 30 μL volumes at 37°C for 3 h with 1 mg template DNA, 10 μL NTP buffer mix (5 μM of each NTP) and 2 μL T7 RNA polymerase mix. Then to remove the template DNA, 2 μL of DNase I (RNase-free, 2 U μL-1) was added and the mixture was incubated at 37°C for 20 min. The transcribed RNA was purified by Trizol and redissolved in RNase-free water. The purified RNA was quantified based on UV absorbance on a Nanodrop 2000 (Thermo Scientific) and then stored at -80°C for future use. Preparation of the in vitro Cas9 cleavage assay The in vitro Cas9 cleavage assay was performed essentially according to the manufacturer’s instruction. Briefly, 10 μL of the mixture A containing 200 nM of msgRNA, 200 nM (or other concentration according to the ratio) of mRNA, 1 μL 10×Cas9 Nuclease Reaction Buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA), 1 U μL-1 RNase inhibitor (Thermo Fisher) incubated at 37°C for 60 min. Next, 10 μL of a reaction mixture B containing 20 nM purified target dsDNA and 200 nM Cas9 and 1 μL 10×Cas9 Nuclease Reaction Buffer was added into mixture A and incubated at 37°C for 60 min and inactivated by heat at 65°C for 5 min. The reaction mixture was then analyzed by 1% agarose gel electrophoresis. In vitro DNA cleavage activity analysis by quantitative PCR In the quantitative PCR analysis, 1 μL of the product from in vitro Cas9 cleavage assay was diluted to a volume of 30 μL. Then, 1 μL diluent product, 10 μL TransStart Green qPCR SuperMix, 1 μL forward primer and 1 μL reverse primer were diluted to a final volume of 20 μL for qPCR. The qPCR product was analyzed by 1% agarose gel electrophoresis. Each assay was repeated triple times under the same condition. The fluorescence was detected 5 / 23



using Biorad CFX96. Preparation of in vitro transcription inhibition assay The target mRNA was transcribed in vitro as mentioned above. 1 μM T7-target-spinach plasmid was conducted the in vitro Cas9 cleavage assay using 10 μM target mRNA X, 10 μM msgRNA X and 10 μM Cas9 nuclease in a 20 μL of reaction mixture. The in vitro transcription assay was conducted at 37C for 1 h in a 30 μL of a reaction mixture containing 10 μL Cas9 DNA cleavage product, 25 mM DFHBI, 10 μL NTP buffer mix and 2 μL T7 RNA polymerase mix. After the reaction, 30 μL of the solution was transferred to 384-well plate and the fluorescence spectra were measured on Microplate Reader. Fluorescence emission was detected at an excitation wavelength of 468 nm and emission range of 503-600 nm with a slit width of 5 nm. Results were the average of three consecutive independent measurements and each measurement was performed in triplicate. The data points represented the average of these triplicate measurements. The error bars in all data indicated the standard deviation from triplicate replicates. All measurements were done at 37C. Data analysis of the gel electrophoresis To characterize the activation and inhibition performance for msgRNA, gels were run for quantification of ON and OFF states. Gel imaging used champgel 5000 gel imaging system. Image J software was used to calculate the band intensity. The background intensity of the lane near the band was subtracted from the intensity of each band to correct the inhomogeneity of the gel imaging or the UV shadow of the loading buffer. The percentage of the uncleaved DNA was calculated by using the formula: % Uncleaved DNA= % (a)/(a + b), where a was integrated intensity of uncleaved DNA while b was the sum of integrated intensity of all cleavage products after background correction. The calculated values for ON and OFF states were then normalized to the negative control assay without Cas9. The error bars in all data indicated the standard deviation from at least two independent replicates. CRISPR activated in complex biological samples Human serum sample was centrifuged at 15000 rpm for 10 min and supernatants were obtained. Genomic DNA and total RNA were extracted from cultured MCF-7 cells using standard methods. The test for the activation of CRISPR in the human serum sample was performed under 6 / 23


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similar experimental conditions of in vitro DNA cleavage assay, excepting the addition of 2 μL of human serum sample supernatants or 5 ng/μL genomic DNA or 1 ng/μL total RNA. Cell culture and transfections Hela cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37oC in a humidified atmosphere with 5% CO2. For transfection experiments, cells were seeded in a 24-well plate and transfected at ~50% confluency. The cells were transfected with 300 ng pmaxGFP plasmid using 1.5 μL Lipofectamine 3000 in 500 μL Opti-Mem media at 37oC for 6 h. After 6 h, the Opti-Mem transfection mixtures were removed from the cells and replaced with DMEM growth media for 24 h incubation. Then, 3 μmol of Cas9 protein, 3 μmol of mRNA and 3 μmol of either gRNA or msgRNA were co-transfected into cells using Lipofectamine CRISPRMAX Transfection Reagent in 500 μL Opti-Mem media at 37oC for 6 h. After 6 h, the Opti-Mem transfection mixtures were removed from the cells and replaced with DMEM growth media for 48 h incubation. Cells from 24-well plates were collected 48 h post transfection and diluted into PBS buffer. Flow cytometry was performed using a BD FACSCalibur. At least 5000 individual cells per condition were analyzed. Flow cytometry analysis was performed using FlowJo software (Ashland, OR, USA).

RESULTS AND DISCUSSION Design of gRNA with blocked CRISPR activity In the original gRNA design, the natural dual trans-activating CRISPR RNA (tracrRNA)–CRISPR RNA (crRNA) structure33,34 is simplified by a synthetic sgRNA. sgRNA is composed of two main parts: a 20 bp spacer which is complementary with the target DNA cleavage site, and a conserved scaffold that could be recognized and combined with Cas9 protein.35,36 Cas9 protein interacts with the repeat-antirepeat duplex scaffold of sgRNA in a sequence-dependent manner via main chain of its residues.37,38 Correct secondary structure is crucial for sgRNA to recognize the Cas9 protein. It can be speculated that if the secondary structure changes, sgRNA 7 / 23



will have problems in binding with Cas9, which will abolish the DNA cleavage ability. To block the function of gRNA, the complementary sequences were inserted in the tetraloop or at the 3‘ end respectively, forming a hairpin structure (Figure 1A). The inserted sequences formed a blocking region, separating the spacer region from the rest of gRNA and misfolding the scaffold region, which significantly hindered the function of gRNA based on the structure of the Cas9/gRNA complex. To test the feasibility of designed blocking strategy, gRNAs with different inserted sequences were transcribed in vitro and incubated with Cas9 protein to cleave the dsDNA in the Cas9 nuclease reaction buffer. The cleavage products were analysed by 1% agarose gel electrophoresis.39,40 As illustrated in Figure 1B, when sequence was inserted into the tetraloop and/or at the 3‘ end of the gRNA in Lane 2-4, there presented two bands, indicating that the cleavage activity was almost undiminished in comparison to native gRNA (Lane 1). Only when the inserted sequences hybridized to each other, Cas9 would lose its cleavage ability, thus one band for uncleaved DNA was observed in the gel (Lane 5). These findings demonstrate that distorting gRNA structure by inserting complementary sequences could efficiently inhibit the CRISPR ability. And msgRNA could be designed on the basis of these results.

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Figure 1. Blocking the activity of gRNA by inserting complementary sequences. (A) Schematic representation of the blocked region of msgRNA forbids the Cas9 from binding. (B) Gel electrophoresis analysis of in vitro cleavage products with gRNA variants. Target dsDNA was incubated with the following gRNA for 1 h at 37oC under the condition of in vitro Cas9 cleavage assay: Native gRNA (lane 1); gRNA with sequence inserted in the tetraloop (Lane 2) or at the 3’ end (Lane 3); gRNA with sequences inserted in the tetraloop and at the 3’ end, not complementary (Lane 4) or complementary to each other (Lane 5). The last lane served as control without gRNA.

Design and optimization of mRNA-sensing gRNA Next, we developed strategies to regenerate the ability of the msgRNA by toehold mediated strand displacement. The sequence integrated into the tetraloop or the 3’ end was the same or complementary to the partial sequence of trigger mRNA. In the absence of the mRNA, the msgRNA remained in the off state, because the gRNA scaffold was positioned in the loop region of the hairpin-like RNA switch, which hindered Cas9 binding and dsDNA cleavage. In the presence of the mRNA, the mRNA hybridized to the toehold region and unzipped the hairpin into a linear state, thereby turning the msgRNA on (Figure 2A). mRNA was tagged along at the 3’ end, thus resulting in negligible sabotage of the function of msgRNA.41 9 / 23



To investigate whether the developed approach could effectively turn on msgRNA with trigger mRNA, the DNA cleavage assay was done to determine the efficiency of blocked and reactivated msgRNAs. A short sequence from mCherry mRNA was selected as trigger mRNA. As shown in Figure 2C, in the absence of trigger mRNA, the msgRNA retained the complete blocking efficiency and eliminated virtually all Cas9-mediated cleavage. After interacting with mRNA for one hour, the target eGFP gene containing dsDNA was cut into two halves and two bands were observed, indicating significant DNA cleavage (Figure 2C, Lane 2). These findings demonstrate that msgRNA could be reactivated under the control of mRNA. The Gibbs free energy of the RNA secondary structure was calculated to further explain the process of strand displacement. When the blocking region with complementary sequence was inserted into the native gRNA, the resulting hairpin like structure had a lower free energy than native gRNA (Table S1, supporting information, SI), indicating that the structure of msgRNA was more stable. When the mRNA interacted with msgRNA, the free energy further decreased. Intermolecular binding affinity between mRNA and msgRNA was larger than the intramolecular binding affinity in the msgRNA molecule, thus strand displacement process could proceed (Table S2 and S3, SI). We then set to optimize to maximize the restoration of msgRNA ability. We varied the length of the blocking sequence (Figure S1A and S1C, Table S2, SI). msgRNAs with the blocking sequence of 10-30 bp were constructed and incubated with Cas9 protein and target dsDNA under the same condition of in vitro DNA cleavage assay and later detected under gel electrophoresis. As expected, the activity of msgRNA decreased with the blocking sequence length increasing. We chose a 30 nt blocking length msgRNA for further research because it had substantially decreased or undetectable activities in the off state comparing to a negative control lacking gRNA, and had the potential to restore the ability when mRNA removed the blockage via strand displacement. Then msgRNAs with different lengths of toehold sequence (0, 5, 10, 20, 40 nt) were 10 / 23


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constructed (Figure S1B and S1D, Table S3, SI), of which the msgRNA with toehold length 20 nt was the most active. The incubation time of msgRNA interacting with mRNA was optimized to 2 h (Figure S2, SI). In addition, the effect of mRNA length on activation ability has been explored. The result showed that the shorter mRNA tended to have better performance because of less complex secondary structure (Figure S3, Table S5, SI). As an important control, we also confirmed the formation of the mRNA/msgRNA complex by verifying the position and length of mRNA X/msgRNA X complex using agarose gel electrophoresis. We designed and tested the interactions of msgRNA with varied concentrations of target mRNA and random mRNA (Figure S4, SI).

Figure 2. Restoring blocked msgRNA activity with mRNA. (A) Schematic representation of the structure of msgRNA with mRNA sensing region embedded and expected structural change of msgRNA in the presence of trigger mRNA. Trigger mRNA interacted with the toehold region of msgRNA, and anti-mRNA sensing region was displaced by mRNA. msgRNA structure refolds with mRNA tagging along at the 3’ end. (B) msgRNA is comprised of four main regions: Spacer (Region I) that targets dsDNA with a specific sequence; mRNA sensing region (Region III) and toehold sequence (Region IV) at the 3’ end hybridize to a partial sequence of mRNA. Anti-mRNA sensing 11 / 23



region (Region II) located in the tetraloop completely hybridizes to the sequence of mRNA sensing region. The cleavage occurs after mRNA sensing region hybridizes to the mRNA. (C) Gel electrophoresis analysis of the activity of blocked and reactivated msgRNAs. The msgRNA activity regained when interacting with the corresponding mRNA (Lane 2) and did not respond to random mRNA (Lane 3); Lane 1 served as a positive control; Lane 5 served as a negative control without gRNA.

Validation of the ability to control CRISPR activity To characterize the ability of mRNA to control CRISPR activity, an in vitro DNA cleavage assay was done to determine the efficiency. When increased concentration of mRNA added into the system, the cleavage efficiency of target dsDNA also increased (Figure 3A, 3B). The quantity of the uncleaved DNA was then analyzed by qPCR. According to the real-time fluorescence intensity of qPCR (Figure 3C) and the positive correlation between the ΔCt and mRNA concentration (Figure 3D), a strong dose-dependent signal was observed, suggesting that the target mRNA activated DNA cleavage. We could take advantage of this dose dependent relationship to monitor the mRNA expression. The msgRNA was engineered to integrate mRNA sensing region, thus linking output signal to mRNA levels. High-throughput method for measuring CRISPR activity would help us to screen and optimize the most efficient msgRNA sequence, such as the cleavage of fluorophore/quencher-labeled DNA substrates assay.42 At present, we only prepare 1-3 msgRNA, which can be optimized by agarose gel electrophoresis method. However, when constructing large numbers of RNA responsive systems in the future, highthroughput screening methods will be relied on.

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Figure 3. mRNA-responsive Cas9-mediated cleavage enabled by msgRNA. (A) DNA cleavage in vitro with different concentrations of mRNA. The 3 kb target eGFP-containing DNA (10 nM) was cut by the Cas9/gRNA complex upon mRNA X activation to yield 2 kb and 1 kb bands. Assays contained Cas9 (100 nM), msgRNA X (100 nM) and increasing amounts of mRNA (20, 40, 60, 80, 100 and 120 nM, respectively), with the assay containing no gRNA serving as the negative control. (B) Quantification of replicates from (A), where data are expressed as the fraction of uncleaved DNA, as calculated based on band intensities. Mean values with standard deviation are plotted (n=3). (C) qPCR reaction for analysis of cleavage efficiency with different mRNA concentration. (D) Ct values from (C).

To further test the controlling ability of msgRNA in transcription process, we constructed a plasmid that constitutively expressing the spinach aptamer with T7 promoter. Spinach RNA aptamer transcribed in vitro could bind and switch on the fluorescence of 3.5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). When activated by the strand displacement of mRNA X, msgRNA refolded and combined with Cas9. Then, Cas9 was guided to cut the target sequence between T7 promoter and spinach RNA aptamer, which hindered the transcription process (Figure 4A). We 13 / 23



measured the fluorescence output 1 h after transcription. As expected, the sensor transfer function demonstrated a steady decrease in fluorescence as mRNA/msgRNA ratio increased to 1.2, beyond which levels plateaued (Figure 4B, 4C). The results show that increased mRNA concentration would turn on more msgRNA and increased the cleavage efficiency of the target dsDNA in a concentration dependent manner. The msgRNA strategy has also been verified in complex biological environments, such as whole RNA, genomic DNA and human serum (Figure S5, SI). msgRNA was turned on by transcribed mRNA X and controlled the transcription of spinach aptamer via Cas9 cleavage. Two independent transcription processes were successfully linked by msgRNA. Under the same experiment condition, the mRNA sensing mechanism was also tested and proved to be applicable to other Cas9 variants including catalytically dead Cas9 and Cas9 nickase (Figure S6, SI). We also tried to validate the msgRNA approach in living cells. Cas9 protein and msgRNA were co-transfected into copGFPexpressing Hela cells along with either matched mRNA or random mRNA. The percentage of copGFP positive cells decreased in the presence of matched mRNA (Figure S7, SI), indicating that the activated msgRNA could be applied in genome editing in living cells. Considerable challenge remains to realize robust msgRNA signal transduction within living cells. The design ability of msgRNA demonstrates that the current work will facilitate flexible adjustments in engineering.

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Figure 4. mRNA-controlled Spinach aptamer transcription inhibition. (A) msgRNA was activated by mRNA X and recognized and cleaved the target sequence, thereby preventing the transcription processes of Spinach aptamer. (B) Spinach aptamer/DFHBI fluorescence loss was quantified by comparing the fluorescence in assays from none to varied concentrations of mRNA (0.2, 0.4, 0.6, 0.8, 1.0, 1.2. and 1.4 μM, respectively). (C) Fluorescence value at excitation wavelength 512 nm of (B).

Evaluation of msgRNA multiplexed regulation To demonstrate the full multiplexing capabilities of msgRNAs, we performed a cleavage assay with three msgRNAs in the same reaction system. mCherry and eGFP genes containing dsDNA were targeted at one and two cleavage sites respectively (Figure 5A and 5B, Table S4 and S5, SI). Figure 5C presents the outcome of the multiplexing experiments with the cleavage ability presented in terms of the percentage of DNA cleaved. The three msgRNAs were independently confirmed their activity. Lanes 2-4 of Figure 5A display that all three msgRNAs were activated separately by their corresponding mRNA triggers with significant cleavage. The msgRNA was basically not activated by other mRNAs, indicating low crosstalk among mRNA triggers. All two or three combinations of mRNAs were also tested and observed all the expected cleavage combinations in Lanes 15 / 23



5-8. The mRNA responsive target DNA cleavage was specific to the presence of the target mRNA even in the system of mixing msgRNAs and different cleavage sites. We systematically modulated all inputs and measured the outputs for all combinations of mRNA input subset combinations. The results agree with expectation in all eight cases. msgRNA has proven to be capable of processing multiple targets with highly specific editing ability.

Figure 5. msgRNA enabled the simultaneous targeting of multiple genes. (A, B) Gel electrophoresis detection of DNA cleavage with three different msgRNAs activated by corresponding mRNA. Three msgRNAs coexisted in the system. In the absence of any trigger mRNA, virtually none DNA target was uncleaved. eGFP gene containing DNA (3 kb) was cut into two shorter fragment (1.5kb, 1.5 kb) activated by mRNA X or two shorter fragments (1 kb, 2 kb) activated by mRNA Y. mCherry gene containing DNA (1.2 kb) was cut into two shorter fragments (0.8 kb, 0.4 kb) activated by mRNA Z. (C) Percentage of DNA cleaved by a set of eight different trigger mRNA combinations. Gray and coloured circles were used to identify the particular trigger mRNA being used.

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Construction of NOR or NAND logic gates The components of the mRNA-sensing CRISPR switches, including input and output signals, are all biological macromolecules, which can be used to build complex genetic programs. We constructed 2-input NOR or NAND logic gates consisting of two mRNA/msgRNA pairs. Spinach aptamer expressing plasmids contained Cas9 cleavage sites adjacent to T7 promoter. We employed mRNA X and mRNA Y as inputs and defined the presence and absence of mRNA X and mRNA Y as ‘‘1’’ and ‘‘0’’, respectively. In a typical assay using 10 μM mRNA X or mRNA Y as the inputs, a threshold value of 2000 a.u. was defined to separate the OFF and ON logic states of the output fluorescence signal at 468 nm. As shown in Figure 6, after the activation of Cas9/msgRNA in the presence of either mRNA X or mRNA Y, the expression of spinach decreased. The fluorescence was only significantly high (output = 1) when the input was in the (0/0) state, and was 0 when the inputs were (1/0), (0/1), and (1/1) indicating the successful construction of the NOR gate that is “ON” only when both inputs are “OFF” (Figure 6B). Similar conditions happened in the construction of the NAND gate (Figure 6D). The expressing of the spinach aptamer could be totally declined only in the presence of both of mRNA X and mRNA Y. The fluorescence was only significantly low (output = 0) when the input was in the (1/1) state, and was 1 when the inputs were (1/0), (0/1), and (0/0) indicating the successful construction of the NAND gate that is “OFF” only when both inputs are “ON”. These results presented the successful construction of the NOR or NAND logic gate on the msgRNA strategy. The biological signal responsive CRISPR facilitates the construction of biomolecular logic gates that conditionally control gene expression.

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Figure 6. NOR or NAND logic gates using the fluorescence of spinach aptamer as the signal output. (A, C) Schematic showing the principle for NOR or NAND logic gate. Spinach aptamer cannot be transcribed from the T7 promoter after the activation of CRISPR in the presence of mRNA. (B, D) The fluorescence signal of the spinach aptamer for the NOR or NAND logic functions operating with mRNA X and mRNA Y. Groups 1–4 in the fluorescent results correspond to the four input combinations in truth tables of the NOR or NAND logic gate. A threshold value of 2000 a.u. was defined to separate the OFF and ON logic states of the output spinach signal.

CONCLUSIONS In conclusions, we have developed a simple and effective mRNA-sensing CRISPR system in which genomic manipulation could be orthogonally activated by untagged, endogenous sequence of RNA triggers. This strategy embeds the mRNA sensor into the gRNA and employs a strand-displacement mechanism to switch the CRISPR system from OFF to ON state. msgRNA has low designing restriction, which can be rationally designed de novo by placing mRNA sequence and its complement in the tetraloop and the 3’ region of the msgRNA. Compared to protein engineering, the engineering of 18 / 23


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gRNA is much faster and needs less effort in terms of labor and consumables. In addition, the dose-dependent manner enables the regulation of Cas9 cleavage activity more subtly. With the advantages above, our msgRNA approach possesses the ability to sense the sequences and levels of multiple mRNAs and also offers good flexibility in terms of signal outputs using the various functions of CRISPR/Cas9 system. The msgRNA strategy could be further explored in the future to detect disease-associated RNAs and construct artificial genetic circuits in living cells, highlighting the potential toward intracellular RNA expression level monitoring and cell-based therapeutic strategy design.

Acknowledgement This work was financially supported by National Natural Science Foundation of China (No. 21621003, No. 21235004, No. 21327806), National Key Research and Development Program of China (No. 2016YFA0203101) and Tsinghua University Initiative Scientific Research Program.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at Sequences of oligonucleotides; optimization of the length of the blocked region and the toehold region; the incubation time of msgRNA interacting with mRNA; msgRNA incubated with different length of mRNA; conditional formation of functional msgRNA/mRNA complex and CRISPR activated in complex biological samples.

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