Results

In our effort to develop an RNA-based molecular recording system, we aimed to construct recording tapes, assess fluorescent proteins for visualization, and evaluate the precision of RNA editing using the REPAIR v2 system. A proof-of-concept experiment was designed to test the tapes’ ability to switch between distinct translational states. Using CRISPR/Cas-mediated RNA editing and fluorescent protein markers, we tracked these state transitions in living cells. The following sections detail the construction of the recording tapes, the experimental setup for sequential RNA editing, and the visualization of different states using fluorescent readouts.

Cloning of ProgRAM-recording-tapes Golden Gate assembly

Cloning of our final constructs, pRAM_ProgRAM-recording-tapes were proceeded by many cloning steps. First, it included the creation of a minimal vector for SynBio applications, pRAM (BBa_K5102000). The construction of pRAM began with a pcDNA3.4-TOPO vector available to us in the lab and the vector was obtained by several Gibson assembly and KLD reactions. In the end, the backbone includes a CMV enhancer, promoter and 5’UTR, T7 promoter and terminator, Woodchuck posttranscriptional regulatory element (WPRE), SV40 polyA element, plasmid ori, as well as AmpR promoter and CDS for selection. In the end, two BsmBI-v2 recognition sites have been introduced to allow for Golden Gate assembly.

Due to the high sequence similarity of the P2A-eUnaG-T2A sequences, cloning of the pRAM_ProgRAM-recording-tapes vectors were assembled together in a single tube Golden-Gate reaction using BsmBI-v2 enzyme. The reaction included pRAM backbone, ProgRAM recording tape, and three gBlocks: T2A-miRFP670nano3-P2A-eUnaG, T2A-mScarlet3-P2A-eUnaG, T2A-mTagBFP2-P2A-eUnaG.

Following E. coli transformation, eight colonies per construct were picked and screened by colony PCR, using a forward primer binding to the plasmid backbone and a reverse primer complementary to the insert. The results were verified via DNA electrophoresis, and colonies with the expected band size were used to inoculate overnight E. coli cultures. The next day, plasmid DNA was miniprepped and verified by Whole Plasmid Sequencing.

Preliminary XFPs analysis

Each recorded state of the ProgRAM recording tape is visualized by the expression of one of three fluorescent proteins. We selected three fluorescent proteins — miRFP670nano3, mScarlet-3, and mTagBFP2 — based on criteria such as emission and excitation wavelengths, maturation time, and fluorescence intensity. However, for preliminary analysis, we decided to test imaging with some XFPs with similar excitation/emission wavelengths that were already available to us in the lab: miRFP670nano (BBa_K5102063), mScarlet-I (BBa_K5102062), as well as mTagBFP2 (BBa_K5102061). Our positive control for this measurement was transfection with the plasmid encoding for Lyn-mCherry-FLAG fusion (BBa_K5102127), which has an emission wavelength of 610 nm.

XFP	Excitation [nm]	Emission [nm]
miRFP670nano	645	670
miRFP670nano3	645	670
mScarlet-I	569	593
mScarlet3	569	592
mCherry	587	610
mTagBFP2	399	454

The selected fluorescent proteins were cloned individually into pcDNA_Zeo Mammalian Expression Vector. Successful cloning was confirmed via whole plasmid sequencing. To evaluate the cross-excitation of the selected XFPs and assess imaging quality, we visualized the transfected HEK293T cells after 24 hours using a CX7 imager.

The results showed that the proteins with similar excitation and emission wavelength to our XFPs can be imaged without cross-excitation on a CX7 imager. Based on this, we infer that our chosen fluorescent proteins are likely to perform effectively, thus confirming our decision to move forward with them.

REPAIR Confirmation - NLuc Restoration

To evaluate the functionality of the REPAIR v2 system, we developed a NanoLuciferase (NLuc) restoration assay. This assay relied on introducing a G35A point mutation via KLD reaction, which created a STOP codon in the open reading frame. We then designed a guide RNA (gRNA), which, bound to dPspCas13b, directs ADAR2DD to the target. The guide RNA design also included an A to C mismatch for precise targeting and increased deamination efficiency.

This system would restore NanoLuciferase activity only if the gRNA correctly directed the REPAIR editor (the dPspCas13b/ADAR2DD fusion) to modify the target adenosine, eliminating the TAG STOP codon in the CDS.

The assay involved transfecting HEK293T cells with three DNA constructs:

1. a plasmid encoding a NanoLuciferase with a STOP codon at the N-terminus, which renders the protein non-functional;
2. a plasmid encoding dPspCas13b/ADAR2DD (REPAIR v2);
3. a plasmid encoding the gRNA to guide dPspCas13b/ADAR2DD to the mutation site.

Transfections were performed at different transfection ratios of plasmid encoding the non-functional NLuc, gRNA, and the dPspCas13b/ADAR2DD editor were varied in the following proportions, respectively: 1:1:1, 1:2:2, 1:3:3, 1:4:4, and 1:5:5 (since REPAIR and gRNA were always delivered in equal molar ratios, results are shown in a 1:X ratio on the figure below).

Control groups were included by omitting either the gRNA or the editor to account for off-target activity. Additionally, a positive control involved the transfection of a plasmid encoding a functional NanoLuciferase.

Experiment	NLuc-STOP	NLuc-STOP-PP7	dPspCas13b/ADAR2DD	dPspCas13b/ADAR2DD-tdPCP	gRNA	NLuc (+ ctrl)
Restoration Experiment	+	-	+	-	+	-
Restoration Experiment with addition of PP7/PCP system	-	+	-	+	+	-
Off-target (no Editor)	+	-	-	-	+	-
Off-target (no gRNA)	+	-	+	-	-	-
Positive control	-	-	-	-	-	+

After 72h, luminescence readings were taken to monitor restoration levels. The bar graph shows these readings, indicating NanoLuciferase activity levels, normalized to our positive control. The experimental group (REPAIR v2 + gRNA) demonstrates significantly higher luminescence compared to control groups (No gRNA and No Editor), confirming successful REPAIR v2-mediated adenosine modification and subsequent NanoLuciferase restoration.

Conclusions: The restoration of NanoLuciferase activity in our assays confirmed that the REPAIR v2 system functions as intended, allowing us to proceed with further experiments. Additionally, results determined that the 1:5:5 molar ratio of target:editor:gRNA plasmids was chosen for future experiments.

Tape-Switching - NLuc Assay

After obtaining tape sequences, we tested REPAIR’s ability to switch from state zero to one. State zero allows protein translation in the first reading frame, while state one shifts the frame by deaminating the first adenosine in the START codon. To validate this, we used a NanoLuciferase assay with NanoLuc CDS in the second reading frame. Since in state 0, NanoLuc is out of frame with the first START codon, successful REPAIR conversion from state 0 to 1 should increase luminescence, indicating restored NanoLuciferase activity.

To design this experiment, we cloned our tapes into the desired plasmid containing GSG_T2A (BBa_K5102113) and the nano-luciferase (BBa_K5102056). For each of our five tapes, we designed and synthesized one in state 0 and one in state 1 that would serve as positive control. Tape switching was tested with the Nano-Glo® Luciferase Assay System (Promega, N110), which uses a reporter system that quantitatively detects the presence of NanoLuciferase, allowing us to assess whether the state switching has occurred as intended.

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Due to inconclusive results with the NLuc Assay, we recognized its limitations for testing tape-switching efficiency in our system. Due to time and availability constraints, we opted to shift to testing with XFPs to explore whether their inherent readout output correlates better with successful tape-switching events in future attempts.

Construct XFPs

The XFPs miRFP670nano, mScarlet-I, and mTagBFP2 that were tested in previous experiments were not codon-optimised, so additional testing for the final sequences was needed. As explained in our Model page, we deleted any possible stop-codons inside the sequences of our tapes, XFPs and other components by codon-optimisation with a self-developed software. Other things that were considered for the optimization were non-RFC compliant restriction sites, splice sites, repeats, GC-content or any other sequences that could negatively affect mRNA stability. An important thing to regard in this process were the XFPs. Their functionality needed to remain independent from the codon-optimisation. The new XFPs, called miRFP670nano3 (BBa_K5102001), mScarlet3 (BBa_K5102002) and mTagBFP2 (BBa_K5102003) were fused to the small fluorescent protein eUnaG. As eUnaG functions as our constitutive translation control, it needs to be expressed in every reading frame. Depending on the reading frame, eUnaG was optimised differently (BBa_K5102009, BBa_K5102010, BBa_K5102011). XFPs and eUnaG were connected by P2A (BBa_K5102015, BBa_K5102016, BBa_K5102017). The fusion products (BBa_K5102081, BBa_K5102082, BBa_K5102083) were tested for their fluorescence, as their sequence got changed in the process of codon optimization. The sequences were transfected with the jetOPTIMUS® protocol into HEK293T cells.

As shown in the figure, there is a basal expression of our construct (indicated in the observed eUnaG signal). Additionally, fluorescence is only present in the expected channels for each individual XFP, indicating no cross-excitation between them.

XFPs Tape-Switching

To demonstrate the functionality of our RNA-based molecular recording system, we designed a proof-of-concept experiment that validates the system’s ability to switch between different recorded states using our tapes 2.0 and 3.0.

The experiment aimed to test whether sequential RNA edits, guided by overlapping gRNAs, could reliably induce specific modifications and produce distinct fluorescent readouts in living cells. For that, each tape has three possible states depending on its ORF:

State	Fluorescent Protein	Reading Frame (ORF)	Adenosine Modification in START Codons
0	miRFP670nano3	ORF1	No modifications
1	mScarlet-3	ORF2	First adenosine modified
2	mTagBFP2	ORF3	First and second adenosines modified

To validate the recording of our tapes, HEK293T cells were transfected at 50-60% confluency with three plasmids in a 1:5:5 molar ratio, as described in the table below.

Plasmid	Name	Contents
Plasmid 1	Recording Tape	Tape 2.0 or 3.0 and XFPs
Plasmid 2	Editor	REPAIR v2 editing system
Plasmid 3	pU6 plasmid	gRNAs

Results

Transfection efficiency was evaluated by assessing XFP expression using a CX7 imager. Initial observations at 24 hours post-transfection revealed detectable fluorescence levels, indicating successful construct expression. Subsequent imaging was performed every 24 hours for a total of 72 hours. The figure below presents a compilation of the fluorescence imaging results.

In both tapes, we are able to detect signals from states 0 and 1 (miRFP670nano3 and mScarlet3, respectively). Notably, we also appreciate a blue signal that appears only at 72 hours. This could be due to minimal basal ribosome slipping, suggesting a low likelihood of leaky expression but only over extended periods of time.

Furthermore, when observing cells transfected with gRNA for switching to state 2 as well as state 1 from previous experiment we observe a slight increase in blue signal from mTagBFP2. This suggests that our tape can record into state 2 as well as state 1.

Post Wiki-freeze results

All iGEM projects can be pushed even further after wiki-freeze. After the freeze of the pages, we proceeded to review our data and perform more analysis. We implemented Principal Component Analysis (PCA) on the image data set from our tape-switching experiments. Images from four experiments were used. Conditions were with presence of tape 2.0 without the editor and gRNA (green), tape 2.0 with REPAIR editor to acknowledge off-target effect of the editor (black) and recording conditions with either gRNA 1 only (purple) or combination of gRNA 1 and 2 (orange) to simulate the recording progression.

As we can see from the figure above, the progression corresponds with the shift of the clusters to the right, along the X-axis. Further along the axis we go, we will see higher abundance and intensity of mTagBFP2 signal from our cells.

Apart from showing that we can detect the progression of tape recording we also are able to observe that each addition of the component of our system creates a distinct cluster allowing for distinction of more than just three states. This allows for signal multiplexing and should allow to record even more states in the future