Part 1: Introduction
A biosensor is a particular type of chemical sensor that uses biological components' recognition properties.[1] The interaction between the analyte and the bioreceptor produces a quantifiable signal, enabling the biosensor to identify target molecules and deliver rapid, precise, and dependable data.[2] This method is more efficient and user-friendly compared to conventional analytic technique (e.g., immunoassays, biochemical assays, etc.), which necessitate specific expertise and time.[3]
However, contemporary biosensors are unable to detect the sequence of analyte occurrences over extended periods, a capability that is essential for certain applications. For instance, monitoring a specific molecule in the human gut would require more than a biosensor that merely provides a binary (Yes/No) signal.[4] Given that the concentration of molecules in the intestine fluctuates constantly, only a long-term sensor capable of continuous recording would prove beneficial.
Despite ongoing efforts by researchers and scientists to develop such a biosensor, few have been successfully implemented in real-world scenarios. Following extensive brainstorming and literature review, we identified a recording system known as a "DNA memory device" that meets our specifications (Fig 1).[5]
DNA serves as a high-density storage medium that can be preserved stably and accessed easily via molecular biological techniques. Furthermore, gene editing tools can convert signals into DNA changes, thereby enabling both biosensing and recording. These characteristics have motivated us to develop a DNA-based biosensor.
Part 2: Description of CRISPReporter
CRISPReporter is a novel multi-level DNA memory device we designed in Escherichia coli that utilizes CRISPR (clustered regularly interspaced short palindromic repeat) -related technology. It can record the temporal order of the signal in DNA for a long duration. When required, the recorded information can be read out by simple molecular biological methods, such as qPCR.
Signal input
We choose blue light and relevant promoters as a proof of concept. To optimize the system, we perform directed evolution on the blue light promoter, EL222.
The light signal represents the presumptive pattern of the stimulation. It can be easily switched on/off and its intensity can be adjusted. Therefore, we investigated several light-inducible promoters and selected EL222, a blue-light inducible promoter. It can be stably toggled between ON- and OFF-states by repetitive pulses of blue light.[6] It can also regulate the level of gene expression precisely by modulating the dosage of light pulses or intensity.[6] Moreover, we performed directed evolution on its threshold in three directions: improving specificity (reduce leakage), avoiding noises (rise minimum threshold), and increasing sensitivity (lower minimum threshold). By substituting EL222 with other specific promoters and applying directed evolution on them, our system can be readily applied to various fields, sensing their specific signals.
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Recording
We build up a cascade editing system to achieve the core function of a recorder. Guided by our drylab model, our system could operate with high efficiency and high data deciphering activity.
The central part our project employs a cascade system that consists of a CRISPR Cas9 editing system, a Lambda Red homologous recombination system, and multiple levels of self-target guide RNAs (stgRNAs) with corresponding homologous arms. With our smart design, fragment knockout will take place sequentially under specific signal stimulation.
CRISPR/Cas9 system has been widely used in E. coli as an effective tool for strain optimization, metabolic engineering, and synthetic biology.[7] It comprises of CRISPR-associated (Cas) protein and crRNA (CRISPR RNA): tracrRNA (trans-activating crRNA).[8] Dual crRNA: tracrRNA (which can be engineered as a single guide RNA) can direct the Cas9 protein to introduce double-stranded breaks (DSB) in target DNA.[8] Through homologous recombination repair Homology directed repair (HDR), donor DNA fragments can be knocked in.
Over the past decade, Lambda Red recombination has been used as a powerful technique for making precisely defined editing in E. coli, requiring as few as 35 bp of homologous arm on each side of the desired alteration.[9] It contains three proteins that are necessary for carrying out dsDNA recombination: Gam, which prevents degradation of linear dsDNA; Exo, which degrades dsDNA and leaves single-stranded DNA; Beta, which facilitates recombination by promoting annealing.[10] Combined with CRISPR/Cas9, it can achieve highly efficient and precise gene editing,[11] which matches our needs.
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Signal output
It can be reported by qPCR, probes, or other molecular biology methods.
Part 3: Wide Application
The CRISPReporter, due to its remarkable recording capacity and potential to sense diverse signals, holds promise for a multitude of applications. We have explored its use in various domains such as diagnostics, environmental monitoring, and bioinformatics.
Diagnosis
Environmental monitering
Part 4: Significance
Our multi-level marker response model was initially conceived with the aim of diagnosing Inflammatory Bowel Disease (IBD), a condition known for its recurrent nature. However, challenges encountered in achieving this goal, like the lack of a proven biosensor model, led us to delve into a more fundamental field. While this may seem removed from practical applications, it holds immense potential.
The multi-level editing system in E. coli serves as an excellent model for information recording and response.
As a biological system
It has a compact size, high genetic stability, enormous information storage capacity, and a rapid rate of reaction and reproduction.
Depending on various kinds of signal input designs, it can record a range of different signals.
As a CRISPR-based recorder
It has extraordinary compatibility, novelty, accuracy, stability, and readability.
There are quite a few iGEM teams that have endeavored to develop these recorders using E. coli as chassis and applied their systems into different fields, like bioinformatics, ecology, and diagnosis. However, none have tried to record the temporal order of stimulations, which is of vital importance.
Highly multiplexable and innovative system
Based on the in-silico sgRNA generator and our advanced software, theoretically, our product is compatible with thousands of levels, each recording the intensity of biomarkers.
Unlike traditional biosensors that directly sense and report signals, CRISPReporter stores information in DNA, enabling multi-level recording.
Easy to manipulate
Firstly, we developed two software programs: one to generate the sgRNA for every level of recording and another to convert experimental data into interpretable results, based on functional correlation and refractory-period mathematical assessment.
Secondly, we constructed a blue light regulation system using the EL222 promoter to actively control and debug the recording process.
Reliable storage approach
As the storage approach is knocking out DNA fragments, the result is quite reliable to avoid being influenced by any random mutation.
The approximately 200bp fragment can be quantified by qPCR, significantly reducing the cost of reading out.
References
- Ziegler C, Göpel W. Biosensor development. Current Opinion in Chemical Biology. 1998;2(5):585-591. doi:10.1016/S1367-5931(98)80087-2.
- Zou ZP, Du Y, Fang TT, Zhou Y, Ye BC. Biomarker-responsive engineered probiotic diagnoses, records, and ameliorates inflammatory bowel disease in mice. Cell Host & Microbe. 2023;31(2):199-212.e5. doi:10.1016/j.chom.2022.12.004.
- Ma Z, Meliana C, Munawaroh HSH, et al. Recent advances in the analytical strategies of microbial biosensor for detection of pollutants. Chemosphere. 2022;306:135515. doi:10.1016/j.chemosphere.2022.135515.
- Goode JA, Rushworth JVH, Millner PA. Biosensor Regeneration: A Review of Common Techniques and Outcomes. Langmuir. 2015;31(23):6267-6276. doi:10.1021/la503533g.
- Sheth RU, Wang HH. DNA-based memory devices for recording cellular events. Nat Rev Genet. 2018;19(11):718-732. doi:10.1038/s41576-018-0052-8.
- Jayaraman P, Devarajan K, Chua TK, Zhang H, Gunawan E, Poh CL. Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic Acids Res. 2016;44(14):6994-7005. doi:10.1093/nar/gkw548.
- Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Kelly RM, ed. Appl Environ Microbiol. 2015;81(7):2506-2514. doi:10.1128/AEM.04023-14.
- Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012;337(6096):816-821. Doi:10.1126/science.1225829.
- Pyne ME, Moo-Young M, Chung DA, Chou CP. Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli. Kivisaar M, ed. Appl Environ Microbiol. 2015;81(15):5103-5114. doi:10.1128/AEM.01248-15.
- Mosberg JA, Lajoie MJ, Church GM. Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate. Genetics. 2010;186(3):791-799. doi:10.1534/genetics.110.120782.
- Zhao D, Yuan S, Xiong B, et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact. 2016;15(1):205. doi:10.1186/s12934-016-0605-5.