Methods

Programmable mammalian translational modulators by CRISPR-associated proteins

Construction of plasmids

Switch plasmids

pAptamerCassette-EGFP (available at Addgene, #140288) was digested by AgeI and BamHI. Single-stranded oligo DNAs were annealed to generate double-stranded DNA and then ligated with the digested plasmid. Links to sequences of all the generated switch plasmids are listed in Supplementary Table 3.

Trigger plasmids

The open reading flame (ORF) of trigger proteins was amplified by PCR using appropriate primers. The amplicons were digested by restriction enzymes and then inserted downstream of the CMV promoter of pcDNA3.1-myc-HisA (invitrogen). Links to sequences of all the generated trigger plasmids are listed in Supplementary Table 3.

Split-Cas9 plasmids

To construct split-Cas9, hCas9 (obtained from Addgene, #41815) was used as the template. N_Cas9 and C_Cas9 were amplified by inverse PCR using appropriate primers. To generate drug-responsive Cas9, DmrA and DmrC were amplified and then fused to each split-Cas9 (N_Cas9 and C_Cas9) using the In-Fusion HD Cloning Kit (Clontech). Links to sequences of all the generated plasmids are listed in Supplementary Table 3.

Plasmid for multiple genetic circuits

The ORF of each Cas protein was amplified using appropriate primers. Using each switch plasmid as a template, the backbone of the plasmids was amplified by inverse PCR. The ORF was inserted in the plasmid backbone using the In-Fusion HD Cloning Kit. Links to sequences of all the generated plasmids were listed in Supplementary Table 3.

All Plasmids were purified using the Midiprep kit (QIAGEN or Promega)

Construction of IVT (in vitro transcription) template

The ORF of SpCas9 (for trigger mRNA) and iRFP670 (for reference mRNA), 5’-UTR fragment, and 3’-UTR fragments were amplified by PCR using appropriate primers and template plasmids or oligo DNAs.

The IVT templates of trigger mRNA and reference mRNA were generated by fusion PCR using the 5’-UTR fragment, the 3’-UTR fragment, the ORF, and appropriate primers.

To make an IVT template of switch mRNA, a fragment including the 5’-UTR and ORF was amplified from SpCas9-responsive switch plasmid by PCR using appropriate primers. The fragment and the 3’-UTR fragment were fused by fusion PCR using appropriate primers.

To remove the template plasmids in the PCR products, 1 µL DpnI was added to each reaction, and the mixture was incubated at 37°C for 30 min. All PCR products were purified using the MinElute PCR purification kit (QIAGEN) or Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s protocols. The set of primers is listed in Supplementary Table 4.

mRNA synthesis and purification

All mRNAs were prepared using a MEGAscript T7 Transcription Kit (Thermo Fisher Scientific). To reduce the immune response, pseudouridine-5′-triphosphate (ΨTP) and 5-methylcytidine-5′-triphosphate (m5CTP) (both from TriLink BioTechnologies) were used instead of natural UTP and CTP, respectively, except for switch mRNA. The IVT reaction included 1 × Enzyme mix, 1 × Reaction buffer, 7.5 mM ΨTP or UTP, 7.5 mM m5CTP or CTP, 7.5 mM ATP, 1.5 mM GTP, 6 mM Anti Reverse Cap Analog (TriLink BioTechnologies), and the template DNA. The mixture was incubated at 37°C for 6 h. To remove the template DNA, TURBO DNase (Thermo Fisher Scientific) was added to the mixture and incubated at 37°C for 30 min. Then the reaction mixtures were purified using a FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech) or Monarch RNA Cleanup kit

(New England Biolabs) and incubated with Antarctic Phosphatase (New England Biolabs) at 37°C for 30 min. The reaction mixtures were purified again using the RNeasy MinElute Cleanup Kit (QIAGEN) or Monarch RNA Cleanup kit according to the manufacturer’s protocols.

All IVT mRNA sequences are shown in the Supplementary Sequences.

Cell culture

HEK293FT cells (Invitrogen) were cultured in DMEM High glucose (nacalai tesque) supplemented with 10% FBS (Biosera), 2 mM L-Glutamine (Invitrogen), 0.1 mM Non-Essential Amino Acids (Invitrogen) and 1 mM Sodium Pyruvate (Sigma) at 37°C with 5% CO2 in a humidified cell culture incubator. In Supplementary Figure 13, HEK293FT cells that constitutively express TagBFP were used. The cells were cultured in the same manner as HEK293FT cells.

Plasmid transfection

HEK293FT cells were seeded in 24-well (1.0×105 cells/well), 96-well (2.0×104 cells/well), or 384-well (2.5×103 cells/well) plates. Appropriate plasmids were transfected into the cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols. Details of the transfection conditions for each experiment are shown in Supplementary Table 5. iRFP670 plasmid was used as a transfection control in all experiments except for those shown in Figure 4, for which TagBFP plasmid was used. For the experiments shown in Figure 2C and D, A/C heterodimerizer-containing media (final concentration 500 nM) were prepared and replaced before the transfection.

mRNA transfection

HEK293FT cells were seeded in 24-well plates (1.0×105 cells/well). Appropriate mRNAs were transfected into the cells using Lipofectamine MessengerMAX (Invitrogen) according to the manufacturer’s protocols.

Details of the transfection conditions for each experiment are shown in Supplementary Table 5

Cell imaging

Before the flow cytometry measurements, cell images were captured using the Cytell Cell Imaging System (GE Healthcare Life Sciences). To capture each fluorescent image, the following channels were used: Blue channel (Ex 390 nm / Em 430 nm) for TagBFP, Green channel (Ex 473 nm / Em 512.5 nm) for EGFP and hmAG1, Orange channel (Ex 544 nm / Em 588 nm) for TagRFP, and Red channel (Ex 631 nm / Em 702 nm) for iRFP670. The captured images were analyzed using ImageJ (NIH).

Imaging analysis and calculations

The tiff files of the captured fluorescent cell images were analyzed with ImageJ. The background intensities were subtracted using the rolling ball algorithm/method before calculation. The cell area was defined by a reference (iRFP670 or, for experiments shown in Figure 4, TagBFP) positive place. The median value of the reporter/reference within the defined area was calculated and used for the analysis.

In the orthogonality heatmaps, intensities were calculated using the following formulas:

Defined value (DV) = median value of the reporter/reference in each cell.

Relative value (RV) = (DV of trigger +) / (DV of trigger −).

Intensity = (RV) / (RV of No gRNA sample).

Flow cytometry measurements

The cells were washed with PBS, treated with 0.25% Trypsin-EDTA (Thermo Fisher Scientific) and incubated at 37°C with 5% CO2 for 5 min. The cells were analyzed using an Accuri C6 (BD Bioscience) flow cytometer with FL1 (533/30 nm) and FL4 (675/25 nm) filters.

Flow cytometry data analysis

Flow cytometry data sets were analyzed using FlowJo version 10.5.3 (BD Biosciences) and Excel (Microsoft). Gates were generated using mock samples. Data from debris were eliminated when preparing forward-versus side-scatter dot plots (FSC-A versus SSC-A). Then, events on the chart edges in the dot plots of the EGFP intensity versus the iRFP670 intensity were removed. In the histogram where iRFP670-intensity is displayed on the X-axis, the iRFP670-positive (reference-positive) gate was defined (Supplementary Figure 1B). In the following analysis, the median of reporter/reference of each cell was calculated from the reference positive population using FlowJo.

Translational efficiency is defined using the following formulas:

Normalized intensity (NI) = 1000 × median of the ratio (reporter intensity/reference intensity) of each cell.

Relative intensity (RI) = (NI of trigger +) / (NI of trigger −).

Translational efficiency = (RI) / (RI of No gRNA sample).

All values were normalized by the value of the No gRNA sample.

In Supplementary Figure 2, all RI values were normalized by the value of the ON state No gRNA sample.

The fold activations in Figure 1D and Supplementary Figure 7 are defined as RI.

The fold change in Supplementary Figure 14C is defined as RI in ON switch and the reciprocal of RI in OFF switch.

Statistical analysis

All statistical analysis was performed by unpaired two-tailed Student’s t-test using R software or Excel (Microsoft).

Multiple Sequence Alignment for phylogenetic tree

Representative Cas9 gRNA sequences that showed crosstalk were analyzed with Multiple Sequence Comparison by Log-Expectation (MUSCLE: https://www.ebi.ac.uk/Tools/msa/muscle/). The result was exported as a FASTA file, and a phylogenetic tree was drawn by Python3.

Article TitleProgrammable mammalian translational modulators by CRISPR-associated proteins

Abstract

The complexity of synthetic genetic circuits relies on repertories of biological circuitry with high orthogonality. Although post-transcriptional circuitry relying on RNA-binding proteins (RBPs) qualifies as a repertory, the limited pool of regulatory devices hinders network modularity and scalability. Here we propose CaRTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Genomic Engineering) to repurpose CRISPR-associated (Cas) proteins as translational modulators. We demonstrate that a set of Cas proteins are able to repress (OFF) or activate (ON) the translation of mRNAs that contain a Cas-binding RNA motif in the 5’-UTR. We designed 81 different types of translation OFF and ON switches and verified their functional characteristics. Many of them functioned as efficient translational regulators and showed orthogonality in mammalian cells. By interconnecting these switches, we designed and built artificial circuits, including 60 translational AND gates. Moreover, we show that various CRISPR-related technologies, including anti-CRISPR and split-Cas9 platforms, can be repurposed to control translation. Our Cas-mediated translational regulation is compatible with transcriptional regulation by Cas proteins and increases the complexity of synthetic circuits with fewer elements. CaRTRIDGE builds protein-responsive mRNA switches more than ever and leads to the development of both Cas-mediated genome editing and translational regulation technologies.


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