cross icon
Left Gear
Right Gear
Cassette Sticker

Description

In biological systems, countless molecular events occur simultaneously, driving the biochemical processes that sustain life. Recent advances in measuring time-dependent transcriptomic and proteomic changes within living cells have revealed complex expression networks governing cellular function and development. These dynamic patterns are vital not only for understanding how life evolves from a single cell but also for advancing fields like therapeutics, diagnostics, and toxicology. However, traditional methods such as sequencing, reporter assays, and mass spectrometry, while providing valuable snapshots, often disrupt the cellular environment and struggle to capture multidimensional events, requiring continuous effort for data acquisition and limiting our ability to observe cellular dynamics as they naturally unfold.

As a solution to this challenge, molecular recording appears as a biological field that allows the tracking of complex biological signals in the native cellular environment (Sheth & Wang, 2018; Farzadfard & Lu, 2018). It aims to reimagine study of living cells by turning them into recording devices on their own by detecting specific signals (sensing), encoding them in a biomolecule (writing), and later retrieving this information through DNA sequencing or related readout methods (reading).

At its current state, molecular recording is mostly oriented to DNA-based methods (Sheth & Wang, 2018; Choi et al., 2022) stemming from this biomolecule’s inherent ability to serve as a high-density, long-term biological information storage system. This reliance on DNA-based methods has caused most molecular recording techniques to operate over slow timescales and require invasive readout techniques like sequencing, which necessitate downstream processing and cell lysis to retrieve the information that has been written via permanent genetic modifications. As a result, they provide only a snapshot of the recorded data, showcasing the necessity of in vivo readout approaches.

We believe that with a novel, simple shift to RNA as the basis for molecular recording, we can transform the way we study living cells and significantly enhance both the setup speed and information writing efficiency. With that in mind, we are excited to introduce this year’s iGEM Munich project: ProgRAM, which stands for Programmable RNA Access Memory. The underlying idea is inspired by the function of RAM (Random Access Memory) in electronic devices like the one you are using to read this, which manages data temporarily without affecting long-term storage. Similarly, RNA allows for the processing, storage, and reading of cellular data without altering the DNA in the nucleus, avoiding permanent modification of genomic information.

DNA recorders typically rely on slow repair processes, such as homologous double-strand break repair, which are constrained by the timing of mitosis and the accessibility of molecular writers to DNA, which depends on the stage of the cell cycle. In contrast, RNA-based systems can function independently of these limitations, offering greater cytosolic availability, faster response times, and the ability to record events in both dividing and non-dividing cells, making them particularly suited for capturing transient biological signals.

Mechanism

We re-engineered the logic of established DNA-editing tools (Farzadfard et al., 2019) to develop a novel RNA-based recording system, marking a new approach that has not been explored previously. The core of our system uses dCas13, a dead RNA-targeting Cas13 protein coupled with an ADAR deaminase domain. Specifically, we employ the Prevotella species-derived dPspCas13b enzyme, which has shown high specificity and minimal off-target effects in RNA-editing systems such as “REPAIR” (Cox et al., 2017).

Upon sensing a cellular event, dCas13 guides ADAR to the target RNA, catalyzing the single base conversion of adenosine (A) to inosine (I), which is recognized as guanine (G) during translation (Rees & Liu, 2018; Booth et al., 2023). This process enables RNA to serve as a dynamic recording tape, where each modification represents a logged event. Unlike traditional DNA-editing systems that require specific recognition sites (PAM sites) (Farzadfard et al., 2019), Cas13’s independence from PAM allows for a more compact, flexible RNA recording system.

We drew inspiration from the REPAIR system to design a novel RNA-based recording mechanism by adapting the principles of DNA recorders. Since no pre-existing template could be directly applied, we constructed the RNA “writing” tape from scratch. This involved extensive research on the efficiency, accuracy, and off-target effects of RNA editing systems, through which we identified optimal positions for inducing base modifications. In our design, we accounted for optimal REPAIR recognition and deamination sites, Cas binding capability, and deamination environment, i.e. flanking nucleotides as a part of the so-called central base triplet, ensuring high editing efficiency and fidelity of the system (Cox et al., 2017).

We designed overlapping guide RNAs (gRNAs) that target sequential regions of the RNA tape, ensuring edits occur in a precise order and minimizing off-target effects. This allows ProgRAM to maintain the correct sequence of recorded events, addressing a key limitation of previous systems that struggle to capture the temporal sequence of multiple signals (Lear & Shipman, 2023). To increase deamination efficiency and accuracy we incorporated a deliberate cytosine-to-adenosine mismatch in the guide RNAs to induce base flipping, along with the mismatch-binding disruption (Cox et al., 2017). For more details on our in silico modeling and guide RNA design, please visit our Model page, while all relevant tape design considerations are highlighted on our Parts page.

Readout

One of the main features of our project is a programmable in vivo readout system that relies on a series of sequentially modifiable adenosine sites in our RNA tape design. This creates a dynamic “traffic light” system of fluorescent protein expression aligned with the current state of our tape that enables precise in vivo visualization of recording events without disrupting the cells.

Here, each adenosine on the tape is part of a translation initiation sequence (Kozak), where, upon deamination, the start codon is disrupted, shifting translation to the next initiation site. Due to our precise arrangement of modifiable adenosines, this shift corresponds with a +1 frameshift on the open reading frame (Hinnebusch et al., 2016). As a result, each base modification triggers the expression of one of three distinct fluorescent proteins (XFPs): near-infrared (mRFP670nano3), red (mScarlet3), or blue (mTagBFP2). This “traffic light” system not only indicates the current state of the recording but also enables seamless tracking of RNA modifications. This enables researchers to visualize and analyze the recording in vivo, without the need for cell lysis and subsequent sequencing.

To ensure proper protein formation, each fluorescent protein is encoded downstream of the RNA tape and preceded by a 2A peptide (Liu et al., 2017), which promotes ribosomal skipping during translation and ensures proper production of XFPs without upstream sequences. For continuous and reliable translational-level control of total protein expression across our system, we incorporated eUnaG (Truong et al., 2024), a small green fluorescent protein codon-optimized for all three ORFs. Due to the complex nature of our frameshift-driven translation of multiple XFPs from a single construct, we needed to optimize codon usage and eliminate premature stop codons across all three reading frames in silico, which you can read about on our Model page.

In summary, ProgRAM introduces a novel and transformative approach to molecular recording, offering several key advantages over traditional methods. First, it provides a highly accessible platform, using RNA as the recording medium for an in vivo tracking of cellular events without requiring complex sequencing or invasive processes. Additionally, the transient nature of RNA modifications means our system can log short-lived, dynamic biological signals, capturing cellular activity without altering the long-term genetic code, making it an ideal tool for studying both stable and rapidly fluctuating processes in ways that do not raise typical concerns about genetic privacy present with current methods (see more on our Human Practices page).

gRNA delivery

Our system offers a novel approach to molecular recording, providing an innovative output through an in vivo fluorescent readout. However, it’s equally important to highlight the flexibility of the system’s input mechanism. The core functionality relies on guide RNA-targeted deamination, meaning that effective gRNA delivery is essential to the system’s operation. There are three general strategies we have envisioned, each encompassing a variety of specific methods, that can be employed for gRNA delivery:

  1. External Delivery: External delivery methods offer rapid and flexible solutions for gRNA delivery. The simplest form, external pipetting, utilizes in vitro transcribed gRNAs, which eliminates the transcriptional delay, making it ideal for fast recording applications. This approach works best when the Editor construct is the primary focus of regulation, not the gRNA expression itself. A more advanced variation, VLP-mediated delivery, leverages virus-like particles (VLPs) to package both gRNA and Editor constructs. This system excels in scalability, offering a pre-packaged, “ready-to-use” recording kit format. The VLP approach also presents exciting commercial potential by reducing preparation requirements and streamlining the distribution process.
  2. In situ Expression: cellular expression offers a longer-term and more controlled solution for gRNA delivery. Plasmid-driven expression is the most straightforward here, using DNA plasmids to transcribe gRNA under a promoter, either constitutive or conditional. This approach is particularly useful for experiments involving transcription factors or optogenetic control, and it has applications in gene therapy and CAR-T cells. For longer timeframes, stable cell lines can be engineered to express gRNA in response to specific cellular conditions. This method reduces variability and eliminates the need for repeated transfection, providing a robust solution for sustained experiments.
  3. Conditional Release: Conditional release strategies offer a unique method for gRNA delivery by utilizing cellular (endogenous) or exogenous RNA and applying conditional logic to excise or release gRNAs at specific time points. This can be achieved through various mechanisms, such as RNA splicing, endogenous transcription-gated switches (ENTS), or intron-encoded cistronic transcripts (INSPECT) (Truong et al., 2022). These methods enable the programmed release of gRNAs, allowing them to be cleaved off from existing RNA molecules, such as the tape itself. By triggering the release of gRNAs only under specific conditions, these systems provide a powerful tool for temporal and spatial control over editing processes.

Add-ons

ProgRAM is a highly versatile tool that can be easily expanded with various add-ons designed to further enhance the system’s functionality. We have extensively discussed various add-ons to enable and improve the inducibility, robustness, localization, and accessibility of our system as outlined in the STIR protocols described on our Human Practices page.

A notable enhancement of our existing framework can be realized by incorporating an inducible Cas13 enzyme. Control of enzyme activity could be mediated both through transcriptional (e.g. Tet-On 3G system; Michalec-Wawiórka et al., 2021) and functional inducibility. The latter is achieved with the help of split Cas13, assembly of which can then be induced with several methods, including optically or chemically (Xu et al., 2023; Yu et al., 2024; Bottone et al., 2023)

In addition, we have devised strategies to improve the robustness of our system by increasing the turnover of the editor. By optimizing the degradation rate of the Cas13-ADAR complex, we can sharpen the temporal resolution of editing events, enabling faster and more dynamic responses. This could be achieved by incorporating a PEST sequence or a ubiquitin-dependent degron (Chassin et al., 2019).

A further add-on for our system lies in its RNA aptamer-mediated localization capability. By incorporating a PP7 phage aptamer downstream of the coding region, which serves as an anchor for our tape, the system can be precisely localized to specific regions or organelles within the cell through the interaction with the aptamer-binding protein, PP7 coat protein (PCP; Wu et al., 2014). This allows for the concentration of specific interactions, such as reading and writing, in designated cellular locations. Moreover, particular interaction partners can also be localized directly to the tape. In one such scenario, we envision equipping the Cas13-ADAR base editor with PCP to potentially minimize global off-target effects by directing the enzyme specifically to the tape. For this purpose, the PP7 aptamer has already been incorporated into our composite parts BBa_K5102072–BBa_K5102080.

We envision further extending our in vivo readout system by integrating it with sequencing techniques without the need for cell lysis. This could be achieved through the programmable export of our RNA tape via exosomes (O’Grady et al., 2022). By engineering the system to package the RNA tape into exosomes, including with the help of RNA aptamers, we can enable the real-time collection and sequencing of the tape from the extracellular environment. This method would provide an additional non-destructive way to monitor cellular processes.

Finally, to enhance the longevity and stability of our mRNA-based tape, we propose the integration of a viral replicon system designed to maintain the tape in the cytosol in the form of mRNA (Nordström et al., 2005; Nakamura et al., 2022). By leveraging the self-replicating properties of retroviral elements, this system would allow continuous propagation of the mRNA, ensuring its persistence without the need for constant transcription from a DNA template. This approach could significantly improve the system’s durability and enable long-term recording of cellular events. You can read more about how we explored replicon-based approaches with different experts on our Human Practices page.

Overall, the modular nature of our system, with its numerous potential add-ons and extensions, greatly enhances its versatility. By allowing for the integration of various components, our system can be adapted to address a wide range of challenges that scientists face today, broadening the system’s applications.

Applications

progRAM offers the potential to transform how we monitor and manipulate cellular processes by enabling cells to autonomously record and report their own biological states. While still in early stages of development, the system presents numerous future applications across various fields of research, diagnostics, and therapeutics.

References

Footer with Ad Roller