Detection of Helicobacter pylori based on CRISPR Cas12a Technology
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].
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].
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].
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.
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.
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.
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].
Recognition Mechanism of our CRISPR Cas12a System
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.
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].
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.
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.
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.
Working Mechanism of our H. pylori Self-Diagnostic Kit
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.
