Lantern: Programmable Logic Framework Based on Recombinase and CRISPR Interference
Overview
Programmable Logic Devices (PLDs) have become pivotal in developing digital circuits. Unlike traditional digital chips, which have fixed internal circuits that cannot be modified post-production, PLDs allow for manual reconfiguration after manufacture, enabling them to perform various logic functions.
One of the key concepts of synthetic biology is to apply the mindset of electronic engineering to biological systems, allowing cells to perform tasks similar to digital devices. However, once traditional gene circuits are established, they are set in stone and cannot be easily modified.
By integrating PLD-like circuits, we can program cells for multiple purposes. In-depth understanding of recombinases and CRISPR interference (CRISPRi) facilitates this endeavor by providing vast variability of circuits and a robust NOT gate.
Therefore, we designed LANTERN (Programmable Logic Framework Based on Recombinase and CRISPR interference). By leveraging recombinase and CRISPRi, we can construct various logic gates and signaling pathways to achieve precise control of cellular behaviors across 16 distinct scenarios defined by two inputs and one output.
CRISPRi and recombinase
CRISPRi
CRISPR interference (CRISPRi) employs an RNA-based approach to precisely silence gene transcription in bacteria and mammalian cells. Originating from the bacteria CRISPR system, CRISPRi operates by co-expressing a dead Cas9 protein, known as dCas9, along with a user-defined single guide RNA (sgRNA)(Larson et al., 2013). The dCas9-sgRNA complex targets DNA sequences that match the sgRNA, creating a physical barrier that prevents RNA polymerase from initiating or elongating transcription, thus repressing the target gene(Ghavami et al., 2021).
Studies of CRISPRi-mediated gene silencing in E. coli have demonstrated that it is highly effective and capable of inhibiting gene transcription by up to 99.9%. This makes CRISPRi an ideal option for constructing NOT gates, facilitating the implementation of A (active) and non-A (inactive) inputs in our genetic circuit designs. To prevent CRISPRi from inadvertently silencing upstream promoters in our engineered plasmid, where some promoters are arranged in series, we strategically employ dcas9 during the transcription initiation phase. This approach is part of our precision engineering to ensure that dcas9 affects only the intended targets, not the upstream promoters.
Recombinase
Recombinases are enzymes that facilitate precise DNA recombination at specific locations. They are categorized into two main types based on the nature of their active amino acid in the catalytic site: serine and tyrosine recombinases. For our project, we have selected a subset of serine recombinases known as "large recombinases." These enzymes are characterized by their larger size and ability to interact with dissimilar DNA sequences, referred to as attP and attB sites. They mediate irreversible DNA inversions, excisions, and integrations; however, reversible reactions can be induced with the help of a second protein, termed the excisionase.
In the genetic pathways we have engineered, each recombinase input can alter DNA segment composed of a pair of corresponding recombinase recognition sites, called attP and attB(Roquet et al., 2016). According to the mechanism of the recombinase, each recombinase can have multiple pairs of orthogonal recombinase binding sites and catalyze inversion (when the sites are anti-aligned, Fig2.A) or excision (when the sites are aligned, Fig2.B). By inserting multiple pairs of recombinase binding sites into the target DNA fragment and expressing different recombinase combinations, the programming of the target DNA fragment can be achieved(Fig.2C, 2D).
What kinds of situations are LANTERN-similar devices suitable for?
Synthetic biology is an engineering discipline — there is a desire to build things that do not yet exist (Church et al., 2014). We reflect on the essence in the realm of "engineering", seeking modular methods for gene circuit synthesis. Drawing inspiration from digital circuits, we completed the design of LANTERN, a programmable logic framework.
Through LANTERN, we can generate 16 different output mappings from two input molecules. In LANTERN we use IPTG and rha as input small molecules and GFP fluorescence as the output signal. This represents a new paradigm—— elements like LANTERN should be able to evolve to accept any molecule as input and produce any signal as output. Based on this concept, we have considered the potential application scenarios for LANTERN-similar devices.
The detection of multiple biomarker combinations
Based on the feature of LANTERN, which allows different inputs to correspond to different outputs, it is highly suitable for applications in biomarker detection. For example, for biomarkers A and B:
- When both biomarkers A and B are at high concentrations, it may indicate a special condition, and the logic should be programmed as follows:
| Biomarker A | Biomarker B | Output |
|---|---|---|
| 1 | 1 | 1 |
| 1 | 0 | 0 |
| 0 | 1 | 0 |
| 0 | 0 | 0 |
- When biomarker A is at a high concentration and B is at a low concentration, it may also indicate a special condition, and the logic should be programmed as follows:
| Biomarker A | Biomarker B | Output |
|---|---|---|
| 1 | 1 | 0 |
| 1 | 0 | 1 |
| 0 | 1 | 0 |
| 0 | 0 | 0 |
Moreover, if the two inputs could be expanded to multiple inputs or multiple components connected in parallel, this element would have even greater application potential.
This has many practical application potentials:
- Previous researchers proposed detecting a customizable set of endogenous microRNA expression levels and triggering a cellular response only when the expression levels match a predetermined target pattern. They also designed a HeLa cancer cell classifier that can selectively recognize HeLa cells and output the hBax protein to trigger apoptosis, without affecting non-HeLa cell types(Zhen et al., 2011). This inspired us to envision using a single device to recognize a pattern of high- and low-expression biomarkers, thereby specifically identifying a type of tumor cell and inducing apoptosis.
- Locust plagues threaten agricultural and environmental safety throughout the world. 4VA (4-vinylanisole) showed significantly higher emissions in gregarious locusts than in solitary locusts but PAN (phenylacetonitrile) showed significantly lower emissions(Guo et al., 2020). Compared with detecting a single marker, our system can simultaneously detect the above two markers. The output is high when the PAN concentration is low and the 4VA concentration is high in the environment. Therefore, the ratio of gregarious locusts to solitary locusts can be directly reflected through the output signal, and the probability of locust plague can be directly characterized through the output signal.
Detection of biomarker changes over time
Due to LANTERN's programmable and controllable characteristics, it is highly suitable for detecting changes in biomarkers over time. Its advantages are particularly evident when the concentration of a certain biomarker exhibits significant changes over time and follows a logistic curve. This gives LANTERN a notable advantage in applications such as fermentation engineering. For example:
In a dual-strain fermentation, if the two intermediate metabolites are dominant, then it can be divided into five phases. Edit LANTERN to output these four stages as 0, 1, 0, 1,0 respectively, and the effect of detecting the degree of fermentation can be achieved.
LANTERN's simple display also gives it the potential for everyday applications, such as home food fermentation, like making pickles.
we can use Lantern to program the detection of any harmful substances, thereby achieving the effect of simply detecting harmful substances.
Synthetic circuits integrating logic
Since the rise of synthetic biology, scientists have been searching for a strategy for efficiently assembling synthetic genetic circuits to achieve Boolean logic functions with stable DNA-encoded memory of events(Siuti et al., 2013). Our LANTERN element is a powerful component of genetic circuits.
In the future, LANTERN-like devices could be expressed in cells, attempting to enable memory storage within cells, with effective inheritance passed on to offspring. These could be applied to larger cell factories, such as biological memory storage systems.
Synthetic Biology System Stability Regulation Module
We hope that LANTERN-similar devices can contribute to the synthetic biology community. We soon realized that they could play a role in preventing mutations and detecting the normal operation of genetic pathways.
As an example, we can look at the UCAS-China 2023 project "NOX: Neo-quOrum sensing-based Xpression biosensor platform." NOX provides a highly compatible and robust platform with impressive performance. Chimeric receptors are assembled for optimal compatibility, and the orthogonal quorum sensing module is responsible for luminescence, optimized through modeling (see: https://2023.igem.wiki/ucas-china/). In the experimental design, NOX used 4-HBA, a metabolic byproduct of E. coli, as the detection target and luciferase as the output. We can monitor any two intermediates to see if the pathway is valid, just like using an ammeter to test two points in a circuit to see if it is functioning properly. This also provides new ideas for the igem project in the space village to see if there are any mutations.
Drylab
Software
For project pre-research, we developed a natural language query program for the Biobricks database called Ask Lantern (See Software for more details), aimed at reducing the pre-research time finding suitable Biobricks from the massive Biobricks database.
In addition, we developed a bioinformatics circuit visualization tool to show how our genetic circuit works. Click here to use it.
Model
Our model consists of three parts. The first is predictions of productivity and efficiency for Register&Patch and Logic AND Gate. The second is evidence of inhibition of CRISPRi with Simbiology. The third is an estimation of time consumed by the Boolean logic circuit system.
We use modeling to predict and complement the project, including the three components shown in Figure 5. (See model for more details)
References
- Larson, M. H., Gilbert, L. A., Wang, X., Lim, W. A., Weissman, J. S., & Qi, L. S. (2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols, 8(11), 2180-2196.
- Ghavami, S., & Pandi, A. (2021). CRISPR interference and its applications. Progress in molecular biology and translational science, 180, 123-140.
- Roquet, N., Soleimany, A. P., Ferris, A. C., Aaronson, S., & Lu, T. K. (2016). Synthetic recombinase-based state machines in living cells. Science (New York, N.Y.), 353(6297), aad8559.
- Church, G., Elowitz, M., Smolke, C. et al. Realizing the potential of synthetic biology. Nat Rev Mol Cell Biol 15, 289-294 (2014).
- Zhen Xie et al. ,Multi-Input RNAi-Based Logic Circuit for Identification of Specific Cancer Cells.Science333,1307-1311(2011).
- Guo, X., Yu, Q., Chen, D., Wei, J., Yang, P., Yu, J., Wang, X., & Kang, L. (2020). 4-Vinylanisole is an aggregation pheromone in locusts. Nature, 584(7822), 584-588.
- Siuti, P., Yazbek, J. & Lu, T. Synthetic circuits integrating logic and memory in living cells. Nat Biotechnol 31, 448-452 (2013).
