Programmable mammalian translational modulators by CRISPR-associated proteins

Translational modulation based on RNA-binding proteins can be used to construct artificial gene circuits, but RNA-binding proteins capable of regulating translation efficiently and orthogonally remain scarce. Here we report CARTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Gene control) to repurpose Cas proteins as translational modulators in mammalian cells. We demonstrate that a set of Cas proteins efficiently and orthogonally repress or activate the translation of designed mRNAs that contain a Cas-binding RNA motif in the 5’-UTR. By linking multiple Cas-mediated translational modulators, we designed and built artificial circuits like logic gates, cascades, and half-subtractor circuits. Moreover, we show that various CRISPR-related technologies like anti-CRISPR and split-Cas9 platforms could be similarly repurposed to control translation. Coupling Cas-mediated translational and transcriptional regulation enhanced the complexity of synthetic circuits built by only introducing a few additional elements. Collectively, CARTRIDGE has enormous potential as a versatile molecular toolkit for mammalian synthetic biology.


Contents
Supplementary Figure Figure 1E. Validated trigger Cas proteins are stated at the top. The bar charts show the calculated reporter expression levels. HEK293FT cells were used in these experiments. Data are represented as the mean ± SD from three independent experiments. ON (trigger -) and OFF (trigger +) states are indicated in red and blue, respectively. Scale bars in cell images represent 200 μm. a.u.: arbitrary unit. Source data are provided as a Source Data file.

Supplementary Figure 4|
The performance of SpCas9-responsive switch in RNA transfection. (A) Schematic diagram of experimental procedures in RNA transfection. Three mRNAs (switch and trigger mRNA (SpCas9 mRNA), and reference mRNA (iRFP670 mRNA)) were transfected into HEK293FT and iPS cells. In the case of HeLa and A549 cells, E3, K3, and B18R mRNAs were transfected in addition to the above three mRNAs to reduce immune response. The cells were analyzed by fluorescence microscopy and flow cytometer. (B) Representative dot plots and histograms of expression levels from switch mRNA with (blue) and without (red) SpCas9 mRNA. Three independent experiments were performed with similar results.

Supplementary Figure 5|
The performance of SpCas9-responsive switch for the iPSC genome integrated Cas9. (A) Representative fluorescence microscopy images of each condition. Scale bar, 200 μm. Three independent experiments were performed with similar results. (B) Representative dot plots of each condition. Red and blue dots indicate Tet-ON dCas9 iPSCs and WT iPSCs, respectively. The x-and y-axes show EGFP and iRFP670, respectively. Efficient EGFP repression was observed when dSpCas9 expression was induced by Dox treatment.

RanCas13b
No gRNA RanCas13b_crRNA Relative intensity [a.u.]  Comparison of translational repression efficiency between wild type AsCas12a and its mutant (H800A). AsCas12a (H800A) lacks RNase activity but maintains other enzymatic activities. Values were normalized by the value in the condition when WT and "No gRNA" reporter plasmids were co-transfected. Data are presented as the mean ± SD from three independent experiments with HEK293FT cells. Error bars represent standard deviations. Statistical analyses were performed by using the unpaired two-tailed Student's t-test. a.u.: arbitrary unit. Source data are provided as a Source Data file. Translational repression occurred only when both protein fragments were present. The reporter assay showed a 33% repression when both input proteins were expressed. Data are presented as the mean ± SD from three independent experiments. HEK293FT cells were used in this experiment. a.u.: arbitrary unit. Source data are provided as a Source Data file. Combinations which are lower than the threshold of 0.7 as OFF switches. Because the set between FnCas12a and LbCas12a-responsive switches, which showed obvious crosstalk, was selected as the best combination, we concluded that there were no 14-Cas proteins combinations that met our criteria for orthogonality. (B) Combinations which were lower than the threshold of 0.7 as ON switches. FnCas12a and LbCas12a-responsive switches were selected as the best combination in the set which selected 12 Cas proteins (right), whereas the set with 11 Cas proteins did not contain them (see Figure 4D). a.u.: arbitrary unit. Source data are provided as a Source Data file. (C) Quantitative data of reporter expression levels were obtained by flow cytometer. OFF switch: a plasmid for expressing TagRFP whose translation is repressed by SaCas9. ON switch: a plasmid for expressing EGFP whose translation is activated by SaCas9. Control OFF and ON switch: plasmids without the sgRNA sequence for SaCas9. HEK293FT cells were used in this experiment. Error bars represent mean ± SD from three independent experiments. Statistical analyses were performed by using the unpaired two-tailed Student's t-test. Source data are provided as a Source Data file.

Supplementary Figure 18|
Cell phenotype control with AND gate circuits related to Figure 6E, F. Representative dot plots of cell viability. The top indicates AND gate consisting of PguCas13b and SaCas9 as inputs and PspCas13b as a mediator. The bottom shows AND gate composed of PspCas13b and SaCas9 as inputs and AkCas12b as a mediator. The x-and y-axes indicate Annexin V and SYTOX Red, respectively. Q1 + Q2 and Q2 + Q3 indicate dead cells and apoptotic cells, respectively. Increases in apoptotic and dead cells were observed only when both inputs were introduced into HEK293FT cells with either AND gate, similar to when a constitutively active hBax plasmid was transfected into the cells.

Supplementary Text 1: Investigating the strategies for improving Cas-responsive switches.
We observed a weak response in our initial design of the NmCas9-responsive switch (NmCas9_gRNA v0), although this Cas protein has genome editing activity in mammalian cells 1 , suggesting that the weak response was not due to inactivity in mammalian cells. We designed seven different types of NmCas9-responsive switches (variants 1-7) by altering the sgRNA sequences and found that variant 7 moderately improved the switch performance (Supplementary Figure 2 and Supplementary Table 2). Similar effects were observed in FnCas9, NcCas9, SpaCas9, AkCas12b, and Cas14a1-responsive switches. Thus, it is possible to improve the repression efficiency of Cas-responsive switches by validating sgRNA sequences.
Switch performance could also be affected by the copy number or insertion position of RNA motifs 2 . We first examined the effects of changing the copy number of sgRNAs and the manner in which they were inserted. Tandem insertion of the sgRNA sequences improved the performance of the PspCas13b and PguCas13bresponsive switches (Supplementary Figure 6A, B), whereas it did not change the efficiency of the CjCas9responsive switch (Supplementary Figure 6C). The use of CjCas9_gRNA in which the spacer sequence was deleted (dSpacer) improved repression efficiency. In contrast, when we connected two sgRNA sequences through an additional 30nt linker to avoid the steric hindrance between two CjCas9 proteins, the repression efficiency was similar to the original construct (Cj_gRNA+30nt-Gluc-Cj_gRNA and Gluc-Cj_gRNA, Supplementary Figure 6C).
Additionally, we assessed the positional effect of the Cas protein binding site (Supplementary Figure 7). Using the dSpacer sequence, in which the spacer sequence was deleted from the sgRNAs used, repression efficiency of the SpCas9-responsive switch varied according to the distance from the 5'-end (Supplementary Figure 7A). However, repression efficiency of the CjCas9-responsive switch became worse (Supplementary Figure 7B). The triple helix in the sgRNA 3 may make the design strategy more complicated in the case of the CjCas9-responsive switches. Further elucidations are required to establish a better strategy to improve translational switches based on Cas protein-sgRNA interactions.  Supplementary Table 3 Components of each 60 AND gate tested in Figure 6. Supplementary Table 4 Statistical analysis related to Figure 6F. Tukey's multiple comparison tests were performed based on the experiments in Fig. 6F Fig 1E, 4, Fig S2,  S6C, S7B, S13