Lantern: Programmable Logic Framework Based on Recombinase and CRISPR Interference
Overview
Logic gates in Synthetic biology
Logic gates are fundamental components of computation, performing specific operations (such as AND, OR and NOT) on input signals to define outputs. Synthetic biology adapts this concept to biological systems by using molecular components to construct similar logic operations in living cells.
With Bio-logic gates, we aim to construct an inventory of well-characterized parts and engineer distinct gene and circuit behaviors that are externally controllable. This idealogy has been applied to numerous fields of bioengineering, including but not limited to:
- Complex multicellular computing (Tamsir et al., 2011)
- Delivery of stimuli-responsive nanocarriers (Zhang et al., 2020)
- Integration of multiple stress signals in plants (Anderson et al., 2023)
- Optimization of CAR-T cell engineering (Hamieh et al., 2023)
Programmable Logic Device
Programmable Logic Devices (PLDs) is a more versatile and flexible alternative to conventional logic gates, as it allows for the customization of logic functions through programming, whereas traditional logic gates perform fixed operations based on their design.
PLDs have been used to:
- build processors, control systems, complex computational logic, and create highly customized processors for different computational tasks in Computer Science and Engineering;
- create circuits that can be updated or modified post-manufacturing, which is essential in rapidly evolving technological fields in Electrical and Electronic Engineering;
- manage data flow, process signals, and control routing paths in network devices, routers, and communication systems in Telecommunications.
Their ability to be reprogrammed makes them incredibly versatile, allowing for constant innovation and optimization in diverse industries, including AI, robotics, telecommunications and beyond.
As concepts of logic gates are contiuously being introduced to Synthetic Biology and iGEM, we came up with this idea: How about building a programmable logic device in synthetic biology to provide reconfigurable solutions for implementing customized functions?
Therefore, we designed LANTERN (Programmable Logic Framework Based on Recombinase and CRISPR interference). By leveraging recombinase and CRISPRi, we built a genetic circuit in E.coli that can switch between different types of logic gates based on user requirements, thus transforming into any logic gate as desired. With LANTERN, we aim to develop a kit where users can use our biological programmable logic device to perform any 2-input, 1-output logic gate according to their specific needs.
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 and non-A inputs in our genetic circuit designs.
Recombinases
Recombinases are enzymes that catalyze the recombination of DNA molecules by recognizing specific DNA sequences and facilitating the rearrangement of genetic material. They can alter the DNA sequence between a pair of recombinase recognition sites, called attB and attP(Roquet et al., 2016). Different recombinases have different recognition sites, and each recombinase can have more than one pairs of orthogonal recognition sites. After recombinase binds to its recognition site, it will catalyze inversion (when the sites are anti-aligned, Fig2.A) or excision (when the sites are aligned, Fig2.B) of the DNA sequence between.
By inserting multiple pairs of recombinase recognition sites into the target DNA fragment and expressing different combinations of recombinases, the programming of the target DNA fragment can be effectively achieved (Fig. 2C, 2D).
What kinds of situations are LANTERN-similar devices suitable for?
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.
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-like 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.
Take the UCAS-China 2023 project "NOX: Neo-quOrum sensing-based Xpression biosensor platform." as an example." 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
To support our project's pre-research phase and give visualization about how our circuits work, we developed two tools: (1) a circuit visualization website, illustrating the functionality of our genetic circuits and making the programmable process of LANTERN easier; (2) Ask Lantern, a natural language processing model for searching Biobricks (See Software for more details), aimed at reducing the pre-research time finding suitable Biobricks from the massive Biobricks database.
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 comprises four components: 1) ensuring functionality of constant promoter, 2) characteristics of the logic AND Gate, 3) Demonstrating the inhibition induced by CRISPRi, and 4) estimating the time consumption of the boolean logic circuit system.
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).
