Helicobacter pylori 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 H. pylori infection can effectively reduce the incidence of a series of gastric diseases, such as gastric ulcers and gastric cancer. Based on CRISPR pathogenic microbial detection technology was developed in recent years, and was applied to a series of pathogenic microorganism detection, has the advantages of being fast and efficient, so the development of CRISPR rapid detection of H. pylori can effectively prevent or find H. pylori infection as soon as possible, so as to reduce the incidence of gastric cancer.
In our project, we applied FnCas12a to develop an in vitro detecting platform. We synthesized four genes as targets and designed sgRNAs according to their DNA sequences. Then we mixed the sgRNAs, FnCas12a protein, and corresponding plasmid, and detected if FnCas12a could be used for H. pylori detection. We also designed a reporter system to measure the activity of FnCas12a. Therefore, our product will provide a candidate protein for H. pylori detection.
Because the T7 promoter and T7 RNA polymerase have strong ability in translation and usually be used as protein expression, we choose pET28a-vector and E. coli BL21(DE3), to express our target protein FnCas12a. To achieve this, we designed the DNA sequences of FnCas12a to be inserted into the pET28a vector and transformed the recombinant plasmid into E. coli BL21(DE3) for protein expression.
We designed another 2 plasmids: 16S, cagA transcription plasmids, and the DNA fragments 16S and cagA are amplified from the genome of Helicobacter Pylori. These two genes’ DNA fragments were inserted into pUC57 vector.
In order to construct our plasmids, we let the company synthesize the DNA fragments, FnCas12 was inserted into the pET28a vector, and the fragments 16S and cagA were inserted into the pUC57 vector. The constructed plasmids were contained in E. coli strains, we streak inoculated them on LB solid medium plates containing corresponding antibiotics, and incubate them at 37℃ overnight (Figure 1).
Figure 1. incubate the plasmids containing strains. We transformed the pET28a-FnCas12a expression plasmid into E. coli BL21(DE3) competent cells, and cultured at 37℃ overnight (Figure 2A). we inoculated a single colony into LB (Kana+) culture medium, incubated overnight, and then transferred the cultured medium into 1L fresh LB (Kana+) culture medium. We induced the expression of FnCas12a with IPTG when the OD600 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 E. coli (Figure 2B).
Figure 2. Expression and purification of protein FnCas12a. Cas12a protein has a size of 130kDa. The SDS-PAGE electrophoresis result indicates that the Cas12a protein is present in the solution we collected at 130kDa, and not present in the nonspecific protein impurities. Thus, Cas12a proteins were expressed and purified with high quality.
Then, we tested the concentration of Cas12a protein by Bicinchoninic Acid Assay (BCA), using SpectraMax i3x Multi-Mode Microplate Reader with the absorption peak at 562nm (Figure 3).
Figure 3. BCA method standard linear regression line for calculation of protein concentration.
| Absorbance (L/(g·cm)) | Protein Concentration (µg/ml) | |
|---|---|---|
| Cas12a 1 | 0.2057 | 10.9358974 |
| Cas12a 2 | 0.1775 | 7.32051282 |
With this BCA standard curve, we measured the concentration of two samples of Cas12a protein, they are 10.9 µg/mL and 7.32 µg/mL respectively. This result indicated that we obtained a sufficient concentration of Cas12a protein.
In order to verify if FnCas12a we purified could precisely recognize and cut the target DNA sequence, we developed an in vitro reaction platform. Firstly, we obtained the sgRNAs through an in vitro transcriptional method and extracted the target sgRNAs fragments. Next, we mixed the purified FnCas12a protein, the sgRNAs, the corresponding plasmids containing DNA fragments, and the reaction buffer together. Then we incubated the reaction system at 37°C for 2 hours, and we verified the result by gel electrophoresis (Figure 4).
Figure 4. Gel electrophoresis comparing before and after cleavage of oligo DNA. After Cleavage is constituted of oligo DNA after cleavage by cas12a protein and sgRNA. In contrast, NC (negative control) contains oligo DNA before cleavage only. Compared to NC, the oligo DNA band is significantly diminished after cleavage. This displays that the in vitro cutting experiment is successful.
We have already collected the data from our experiments. The DETECTR technology is based on CRISPR-Cas12a. When target DNA is detected under the specific sgRNAs, the endonuclease activity of Cas12a is activated and cleaves the target DNAs. From our result, we can find that the FnCas12a could recognize the target genes and precisely cleave the target DNA.
H. pylori was originally isolated from the body of gastric ulcer patients and has been listed as a well-defined carcinogen. The detection of H. pylori has become an important physical examination project for the prevention of gastric cancer. This project aims to develop a fast and convenient detection program for H. pylori based on CRISPR to help people facilitate an efficient detection of H. pylori to contribute to the prevention of gastric cancer. We believed that we could apply FnCas12a in H. pylori detection in the future. When the technology developed, it could be easier for people to diagnose H. pylori.
