From ac458c21d091963d2af611ee502ea03cc3f573e5 Mon Sep 17 00:00:00 2001
From: Devmc <dev.mcrf@qq.com>
Date: Tue, 11 Oct 2022 16:52:31 +0800
Subject: [PATCH] fix
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<div class="article">
<h1 class="content-header2">Description</h1>
+ <p class="text-center">
+ <u>
+ Detection of Helicobacter pylori based on CRISPR Cas12a Technology
+ </u>
+ </p>
+
<section>
- <h2 class="c-green">1. Background</h2>
- <p>
- Gastric cancer is the second incidence of cancer in China, and the infection rate of Helicobacter pylori (H.
- pylori) in the high incidence of gastric cancer is more than 60%. <i>H. pylori</i> infection has been identified as a
- major carcinogenic factor causing gastric cancer, so its detection has also been included in a common item in
- physical examination. Early detection and effective treatment of <i>H. pylori</i> infection can effectively reduce
- the incidence of a series of gastric diseases, such as gastric ulcers and gastric cancer. In recent years, H.
- pylori infection has emerged as an especially big problem in China. The current infection rate of HP stands at
- 49.6%, which is slightly higher than the global infection rate of 48.5%. Nowadays, the methods of detecting H.
- pylori are mainly based on the C-13/c-14 test, Blood and fecal antigen testing, et.al, which is not convenient
- for daily life.
- </p>
+ <h2 class="c-green">Overview: </h2>
<p>
- Based on CRISPR pathogenic microbial detection technology was developed in recent years, and was applied to a
- series of pathogenic microorganism detection, such as HPV and nCov2, has the advantages of being fast and
- efficient, so the development of CRISPR rapid detection of <i>H. pylori</i> can effectively prevent or find <i>H. pylori</i>
- infection as soon as possible, so as to reduce the incidence of gastric cancer. This CRISPR rapid detection
- technology mainly includes two types, one is the Cas13a-based SHERLOCK system, the former for detecting
- microorganisms with RNA as genetic material, and the Cas12a-based DETECTR system with DNA as genetic material.
- These methods have the advantages of being fast, accurate, and easy to operate.
- </p>
- <p>
- In order to develop a self-diagnostic box that could be used in daily life or even be used in under-developed
- countries with poor medical conditions, we employed FnCas12a protein, and build up an in vitro reaction system
- for <i>H. pylori</i> detection (Figure 1).
+ Helicobacter pylori (H. pylori) is a spiral, gram-negative bacterium. Recently identified as a gastric
+ carcinogen, its infection requires rapid and accurate detection methods; however, current mechanisms either
+ lack in conveniency due to their reliance on complex equipment, or in sensitivity due to their tendency to
+ produce false positive results. In our project, we developed an inexpensive, non-invasive, and reliable
+ screening method for H. pylori that can be implemented in both domestic households and hospital settings.
+ Using E. coli as a vector, we successfully synthesized oligo DNA segments to simulate H. pylori. We then
+ designed a CRISPR Cas12a-sgRNA complex to identify and bind to our target DNA sequences, which would result in
+ the non-specific cleavage of our ssDNA fluorescent reporter. To assess its performance, we conducted gel
+ electrophoresis and fluorescence tests. Experimental evidence suggests that our diagnostic test expressed high
+ sensitivity, selectivity, and rapidity.
</p>
<div class="imager">
- <img class="rw-100" src="https://static.igem.wiki/teams/4304/wiki/description/t-ykpao-description-01.jpg"
+ <img class="rw-100" src="https://static.igem.wiki/teams/4304/wiki/description/t-ykpao-description-02.jpg"
alt="">
<span class="figure">
- Figure 1. the principle of our in vitro <i>H. pylori</i> detection platform
+ The principle of our in vitro H. pylori detection platform
</span>
</div>
</section>
<section>
- <h2 class="c-green">2. Experiment Design</h2>
+ <h2 class="c-green">Background and Inspiration:</h2>
<p>
- Our team aims to develop an in vitro reaction system for <i>H. pylori</i> detection with FnCas12a. We synthesized
- four DNA fragments of <i>H. pylori</i> characteristics genes as targets and used the purified FnCas12a protein to
- recognize and cut those target genes.
+ Helicobacter pylori, or H. pylori for short, is a gram-negative microaerophilic bacterium that acts as a
+ pathogenic factor for several gastroduodenal diseases, such as peptic ulcers and chronic gastritis [2]. It was
+ identified as a Group 1 carcinogen of gastric cancer on the 15th list of known carcinogens distributed by the
+ U.S. Department of Health and Human Services [3]. As of right now, infection with H. pylori remains the
+ strongest risk factor for two types of stomach cancer: gastric adenocarcinoma and gastric lymphoma [1, 4].
+ </p>
+ <p>
+ In recent years, H. pylori infection has emerged as an especially big problem in China. China’s current
+ infection rate of HP stands at 49.6%, which is slightly higher than the global infection rate of 48.5% [2].
+ Simultaneously, gastric cancer is the second most frequently occurring cancer in China, with H. pylori
+ infection as one of the biggest contributing factors [5].
+ </p>
+ <p>
+ Moreover, the prevalence of group dining cultures in China makes our population all the more susceptible to H.
+ pylori infections. Due to HP’s oral-oral transmission via saliva, Chinese people’s habit of using chopsticks
+ eases the HP bacterium’s transfer between family members and friends [6].
+ </p>
+ <p>
+ Research has proven that the prognosis of gastric cancer patients who underwent early detection and treatment
+ of H. pylori infections was significantly higher than those who haven’t [7]. Therefore, to minimize the cases
+ of gastric cancer caused by H. pylori in our community, a rapid, convenient, and reliable detection method is
+ urgently required.
+ </p>
+ <p>
+ However, current diagnostic methods all contain notable disadvantages that hinder their effectiveness within a
+ larger population. For example, the urease exhalation test, which is the major detection method used in
+ hospitals, is inconvenient due to its requirement of sophisticated equipment like HUBT-01, limiting its
+ application to clinical settings only. Another diagnostic practice utilized by doctors involves conducting a
+ biopsy of the patient’s stomach lining tissues. The invasive nature, expensive price, and inconvenience of
+ this method hamper people’s willingness to get tested. Finally, the blood and stool antigen tests lack
+ severely in accuracy and timeliness as it tests for the patient’s body’s reactions to H. pylori infection
+ instead of detecting the bacterium directly.
</p>
-
- <section>
- <h3>General Experiment Procedure</h3>
- <p>
- First, we synthesized four target genes of <i>H. pylori</i>, 16S, cagA, ipaH, and invA, as our target genes of
- FnCas12a, and the genes were synthesized into the pUC57 plasmid by a gene synthesis company. What’s more, we
- inserted the FnCas12a gene fragment into the pET28a vector for protein expression.
- </p>
- <p>
- Next, we transformed the recombinant plasmids pET28a-FnCas12a into BL21(DE3), inoculated the strain and
- induced the expression of FnCas12a with IPTG when the OD<sub>600</sub> was around 0.6-1.0, and cultured at 16℃ for 12h.
- Subsequently, we used nickel affinity purification to purify the acquired Cas12a proteins from other
- proteins in <i>E. coli</i>.
- </p>
- <p>
- Then, we obtained the sgRNAs through an in vitro transcriptional method and extracted the target sgRNAs
- fragments. We mixed the purified FnCas12a protein, the sgRNAs, the corresponding plasmids containing DNA
- fragments, and the reaction buffer, and we incubated the reaction system at 37°C for 2 hours, and we
- verified the result by gel electrophoresis.
- </p>
- <p>
- Finally, we designed a reporter system to easy detection the activity of FnCas12a, and then we measured the
- fluorescence intensity.
- </p>
- </section>
</section>
<section>
- <h2 class="c-green">3. Expected Results</h2>
- <ol class="l-top-05">
- <li>Successfully construct 16S, cagA, ipaH, and invA cantaining plasmids, and pET-28a-FnCas12a plasmids.</li>
- <li>Expressed and purified FnCas12a protein.</li>
- <li>Set up an in vitro reaction platform for FnCas12a activity detection.</li>
- <li>Measure the fluorescence intensity of the reaction system containing the reporter system.</li>
- </ol>
+ <h2 class="c-green">Our Design and Product:</h2>
+ <p>
+ To resolve this issue, we aim to develop a non-invasive, portable, accurate, and inexpensive test kit for the
+ rapid detection of H. pylori infection. Drawing inspiration from Zhang Feng’s Lab and Jin Wang’s Lab’s works,
+ we designed a CRISPR Cas-12a system for the specific recognition of target DNA sequences on H. pylori.
+ </p>
+ <p>
+ Cas12a belongs to the class 2 type V-A CRISPR-CAS system. Different from its predecessors CRISPR Cas9 and
+ Cas13a systems which are only able to bind to RNA sequences, Cas12a can recognize DNA sequences. With the aid
+ of a guide RNA sequence (sgRNA), the CRISPR Cas12a-sgRNA complex identifies and binds to the complementary
+ target DNA sequence, forming a Cas12a-sgRNA-target DNA ternary complex [8]. The formation of a target-bound
+ Cas12a complex unleashes a string of non-specific single-stranded DNA (ssDNA) trans-cleavage. As the ssDNA
+ strands can act as fluorescent reporters, a significant light-up reaction can be detected, enabling the
+ reliable measurement of the Cas12a-sgRNA complex’s target DNA recognition [9].
+ </p>
+ <div class="imager">
+ <img class="rw-85" src="https://static.igem.wiki/teams/4304/wiki/description/t-ykpao-description-03.jpg"
+ alt="">
+ <span class="figure">
+ Recognition Mechanism of our CRISPR Cas12a System
+ </span>
+ </div>
+ <p>
+ To obtain Cas12a proteins, we constructed Cas12a plasmids and transferred them into BL21 E. coli colonies.
+ After culturing overnight, Cas12a proteins were extracted via nickel affinity purification.
+ </p>
+ <p>
+ Four sgRNA sequences were then designed to monitor the target sequences of H. pylori, S. typhimorium, and S.
+ flexneri bacteria. Specifically, cagA and 16S were chosen as the target sequences for H. pylori, while invA
+ was chosen for S. typhimorium and ipaH for S. flexneri. cagA is a gene that is originally located in a
+ chromosomal region named the cag pathogenicity island (PAI) within H. pylori. It codes for an effector protein
+ cagA (cytotoxin-associated antigen A) that facilitates the bacterium’s entry into host cells through a type IV
+ secretion system (T4SS) [4]. Research has proven that the presence of cagA in H. pylori notably heightens the
+ development of malignant cancer cells and that the risk of contracting gastric cancer is significantly higher
+ in patients with cagA-containing H. pylori strains [10]. Simultaneously, the 16S sequence is characteristic of
+ all H. pylori strains, thus making it an ideal target for detection [11].
+ </p>
+ <p>
+ Plasmids were constructed and cultured for all four guide sgRNA sequences. After its extraction and isolation,
+ the plasmids underwent a polymerase chain reaction. The target DNA sequences of the plasmids were then
+ transcribed into sgRNA strands with a T7 transcription kit and purified via an RNeasy spin column.
+ </p>
+ <p>
+ Finally, the effectiveness of our Cas12a proteins in detecting H. pylori’s target DNA sequences was
+ investigated by assembling and incubating a Cas12a-sgRNA-oligoDNA system, a Cas12a-sgRNA-plasmid system, and a
+ Cas12a-sgRNA-E. coli culture system. The efficacy of these systems was quantified by the fluorescence
+ responses of ssDNA, as measured by the multiskan ascent.
+ </p>
+ <p>
+ Our experimental results proved the efficiency and reliability of our CRISPR Cas12a system. With further
+ developments and modifications, our product can be made into a self-diagnostic box that can be applied in both
+ medical and domestic settings. In our design, saliva samples will be taken by running a cotton swab against
+ the insides of the patient’s mouth. The swab will then be dipped and stirred in a tubule containing a lysis
+ buffer solution and a cas12a protein buffer solution, which are separated by a membrane. This arrangement
+ ensures the easy breakdown of the sample’s cell membranes and the cas12a-sgRNA system’s recognition of the
+ target DNA sequence. The solution will then be applied onto a lateral flow strip and placed within a device
+ containing a UV light switch and UV light strips. The patient can thus determine whether they are infected
+ with H. pylori through the fluorescence response of the CRISPR Cas12a system.
+ </p>
+ <div class="imager">
+ <img class="rw-100" src="https://static.igem.wiki/teams/4304/wiki/description/t-ykpao-description-04.jpg"
+ alt="">
+ <span class="figure">
+ Working Mechanism of our H. pylori Self-Diagnostic Kit
+ </span>
+ </div>
+ <p>
+ The potential and possibilities of our H. pylori self-diagnostic box range far and wide. Not only can it be
+ used in households and communities to promote the prevalence of H. pylori screening, but it can also be used
+ in hospitals to fasten the diagnostic time. As the manufacturing process and production costs of our device
+ are both very low, we believe its usage can be extended to a broad range of users and truly achieve the goal
+ of aiding the entire population’s health situation. As our product has already demonstrated potential in
+ accommodating screening methods for other bacteria, we wish to enhance it by establishing Cas12a systems with
+ different guide sgRNAs and creating a multi-function detection device. Ultimately, our goal is to expand the
+ frontiers of synthetic biology and to change the world for the better, step by step.
+ </p>
</section>
<section>
- <h2 class="c-green">4. Reference</h2>
+ <h2 class="c-green">References:</h2>
<ol class="l-top-05 text-justify">
- <li>Polk, D., Peek, R. Helicobacter pylori: gastric cancer and beyond. Nat Rev Cancer 10, 403–414 (2010).
- <a href="https://doi.org/10.1038/nrc2857">https://doi.org/10.1038/nrc2857</a></li>
- <li>Li, M, Sun, Y, Yang, J, et al. Time trends and other sources of variation in Helicobacter pylori infection
- in mainland China: A systematic review and meta-analysis. Helicobacter. 2020; 25:e12729.
- <a href="https://doi.org/10.1111/hel.12729">https://doi.org/10.1111/hel.12729</a></li>
- <li>Cover TL. Helicobacter pylori Diversity and Gastric Cancer Risk. mBio. 2016 Jan 26;7(1):e01869-15. doi:
- 10.1128/mBio.01869-15. PMID: 26814181; PMCID: PMC4742704.</li>
- <li>Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA. CRISPR-Cas12a target binding
- unleashes indiscriminate single-stranded DNase activity. Science. 2018 Apr 27;360(6387):436-439. doi:
- 10.1126/science.aar6245. Epub 2018 Feb 15. Erratum in: Science. 2021 Feb 19;371(6531): PMID: 29449511;
- PMCID: PMC6628903.</li>
- <li>Li SY, Cheng QX, Wang JM, Li XY, Zhang ZL, Gao S, Cao RB, Zhao GP, Wang J. CRISPR-Cas12a-assisted nucleic
- acid detection. Cell Discov. 2018 Apr 24;4:20. doi: 10.1038/s41421-018-0028-z. Erratum in: Cell Discov. 2019
- Mar 12;5:17. PMID: 29707234; PMCID: PMC5913299.</li>
- <li>Broughton JP, Deng X, Yu G, Fasching CL, Servellita V, Singh J, Miao X, Streithorst JA, Granados A,
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- CY. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020 Jul;38(7):870-874. doi:
- 10.1038/s41587-020-0513-4. Epub 2020 Apr 16. PMID: 32300245; PMCID: PMC9107629.</li>
+ <li>Polk, D. Brent, and Richard M. Peek. “Helicobacter Pylori: Gastric Cancer and Beyond.†<i>Nature Reviews
+ Cancer</i>, vol. 10, no. 6, 2010, pp. 403–414., https://doi.org/10.1038/nrc2857.
+ </li>
+ <li>Li, Mengmeng, et al. “Time Trends and Other Sources of Variation in <i>Helicobacter Pylori</i> Infection
+ in
+ Mainland China: A Systematic Review and Metaâ€Analysis.†<i>Helicobacter</i>, vol. 25, no. 5, 2020,
+ https://doi.org/10.1111/hel.12729.
+ </li>
+ <li>United States, Congress, National Toxicology Program. <i>15th Report on Carcinogens</i>, 15th ed.,
+ National
+ Toxicology Program, Department of Health and Human Services, 2021. <i>Report on Carcinogens</i>.
+ </li>
+ <li>Cover, Timothy L. “Helicobacter Pylori Diversity and Gastric Cancer Risk.†<i>MBio</i>, vol. 7, no. 1,
+ 2016,
+ https://doi.org/10.1128/mbio.01869-15.
+ </li>
+ <li>He, Yuxin, et al. “Chinese and Global Burdens of Gastric Cancer from 1990 to 2019.†<i>Cancer Medicine</i>,
+ vol.
+ 10, no. 10, 2021, pp. 3461–3473., https://doi.org/10.1002/cam4.3892.
+ </li>
+ <li>Leung, WK, et al. “Does the Use of Chopsticks for Eating Transmit Helicobacter Pylori?†<i>The Lancet</i>,
+ vol.
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+ </li>
+ <li>Sakitani, Kosuke, et al. “Early Detection of Gastric Cancer after <i>Helicobacter Pylori</i> Eradication
+ Due to
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+ </li>
</ol>
</section>
</div>
--
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