Skip to content
Snippets Groups Projects
Commit ac458c21 authored by Devmc's avatar Devmc
Browse files

fix

parent 6f97fadc
No related branches found
No related tags found
No related merge requests found
Pipeline #126720 passed
Stage: build
Stage: deploy
Hide whitespace changes
Inline Side-by-side
wiki/pages/description.html
+ 173
− 81
View file @ ac458c21
......@@ -9,107 +9,199 @@
<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,
Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu E, Gu W, Miller S, Pan CY, Guevara H, Wadford DA, Chen JS, Chiu
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 Meta‐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.
350, no. 9070, 1997, p. 31., https://doi.org/10.1016/s0140-6736(05)66240-x.
</li>
<li>Sakitani, Kosuke, et al. “Early Detection of Gastric Cancer after <i>Helicobacter Pylori</i> Eradication
Due to
Endoscopic Surveillance.” <i>Helicobacter</i>, vol. 23, no. 4, 2018, https://doi.org/10.1111/hel.12503.
</li>
<li>Li, Shi-Yuan, et al. “CRISPR-CAS12A-Assisted Nucleic Acid Detection.” <i>Cell Discovery</i>, vol. 4, no.
1, 2018,
https://doi.org/10.1038/s41421-018-0028-z.
</li>
<li>Broughton, James P., et al. “CRISPR–CAS12-Based Detection of SARS-COV-2.” <i>Nature Biotechnology</i>,
vol. 38,
no. 7, 2020, pp. 870–874., https://doi.org/10.1038/s41587-020-0513-4.
</li>
<li>Plummer, M., et al. “Helicobacter Pylori Cytotoxin-Associated Genotype and Gastric Precancerous Lesions.”
<i>JNCI Journal of the National Cancer Institute</i>, vol. 99, no. 17, 2007, pp. 1328–1334.,
https://doi.org/10.1093/jnci/djm120.
</li>
<li>Szymczak, Aleksander, et al. “Application of 16S Rrna Gene Sequencing in <i>Helicobacter Pylori</i>
Detection.”
PeerJ, vol. 8, 2020, https://doi.org/10.7717/peerj.9099.
</li>
</ol>
</section>
</div>
......
0% or .
You are about to add 0 people to the discussion. Proceed with caution.
Finish editing this message first!
Please register or to comment