In contemporary times, cosmetics have become a vital element of our daily routine. Yet, the inclusion of specific ingredients or improper storage can lead to the proliferation of harmful bacteria within cosmetics, such as Staphylococcus aureus and Pseudomonas aeruginosa, which elevates bacterial levels. The presence of Staphylococcus aureus and Pseudomonas aeruginosa in cosmetics can disturb the skin's microecology, especially in those with skin damage, resulting in aggravated conditions such as inflammation, suppuration, and other severe outcomes. And the "Cosmetic Safety Technical Code" also states that Staphylococcus aureus and Pseudomonas aeruginosa are two types of pathogenic bacteria that can’t be found in certain cosmetics. However, the current detection methods typically require a time-consuming process of 3 to 5 days, and the procedures involved are complex. As a result, these methods do not meet the practical requirements for rapid and accurate microbial detection. Therefore, there is an urgent need to establish new rapid detection techniques and methods.

 

With the development and expansion of gene editing technology, CRISPR-related technology represents the third generation of gene editing technology, following TALENs, ZFN, and other gene editing technologies.[1] The CRISPR technology has undergone extensive research and development, and it has reached a level of maturity that enables its application in experimental design. Currently, CRISPR-based detection systems have been developed and widely used in pathogen detection, including rapid identification of human papillomavirus (HPV) and novel coronavirus.[2] These mainly include two types of technologies: SHERLOCK system based on Cas13a and DETECTR system based on Cas12a, the former is often used to detect microorganisms with RNA as genetic material, and the latter is often used to detect microorganisms with DNA as genetic material.[3] 

 

Figure1 The mechanism of DETECTR system based on Cas12 and SHERLOCK system based on Cas13[4]

 

The CRISPR-Cas12a system has advantages over other systems, for example CRISPR Cas9. CRISPR-Cas12a expands the editing sites where CRISPR-Cas9 system is not available and produces interleaved cuts at the 5 'end of the sequence. Compared with CRISPR-Cas9 system, this significantly increases the probability of cells to choose homologous recombination repair (HDR) mode and improves the efficiency of gene editing.

Cas12a recognizes thymidine rich PAM sequences and is capable of genome editing in organisms with AT-rich genomes, providing a broader range of sequence editing than the CRISPR-Cas9 system. Only the simple recombination of crRNA and Cas12a protein but not tracrRNA is required, and the crRNA of Cas12a is shorter, generally about 40-44nt, which is more conducive to chemical synthesis.

So CRISPR/Cas 12a is one of the most efficient, cost-effective, and user-friendly options. Our focus was on utilizing Cas12a, a multifunctional protein sourced from the microbial CRISPR-cas immune system. When the target DNA is detected by the specific sgRNA, Cas12a is activated and has endonuclease activity. When the target DNA is cleaved, Cas12a will also cleave the probe. The cleaved probe can be detected by corresponding means to identify whether the sample contains the target DNA[5]. This approach will allow us to achieve the desired results for our products and meet the convenience and ease-of-use requirements of our product.

 

In order to achieve the function of detecting the Staphylococcus aureus and Pseudomonas aeruginosa by Cas12a, we choose genes femA and GbcA as the target sequences for Staphylococcus aureus and Pseudomonas aeruginosa respectively[6]. We will construct two plasmids: pUC57-femA and pUC57-GbcA. Next, we will transform these two plasmids into chemically competent cells, E. coli DH5α, and allow these bacteria to grow and replicate. After we get E.coli containing these two plasmids respectively, we will conduct PCR to ensure that the bacteria containing the plasmids.

 

Besides, we also need to construct a plasmid of Cas12a and design sgRNA for femA and GbcA. Plasmid pET28a-FnCas12a containing Cas12a will be transformed into E. coli BL21 and IPTG induction will be carried out. Next Cas12a protein will be extracted and purified. SDS-PAGE and Coomassie brilliant blue will be carried out to confirm the expression of Cas12a. Then sgRNA will be cultured with Cas12a protein. Finally, the effectiveness of our Cas12a proteins in detecting Staphylococcus aureus and Pseudomonas aeruginosa target DNA sequences will be investigated. The efficacy will be quantified by the fluorescence responses.

 

All the above processes are shown in figure 2.

 

Figure 2 General concept of the experiment.

 

 

1. The plasmid pUC57-femA and pUC57-GbcA are successfully constructed, which are identified by bacteria PCR.

2. Cas12a protein products with biological functions could be extracted from E. coli BL21. And the protein can be purified correctly. SDS-PAGE and Coomassie brilliant blue staining show that the protein has good antigenicity.

3. sgRNA target the femA and GbcA sequences of Staphylococcus aureus and Pseudomonas aeruginosa can have good efficiency.

4. We hope that our product is safer, more convenient and easier to detect Staphylococcus aureus and Pseudomonas aeruginosa.

 

[1]Gupta, R. M., & Musunuru, K. (2014). Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9. The Journal of clinical investigation, 124(10), 4154–4161. https://doi.org/10.1172/JCI72992

 

[2]Fang, L., Yang, L., Han, M., Xu, H., Ding, W., & Dong, X. (2023). CRISPR-cas technology: A key approach for SARS-CoV-2 detection. Frontiers in bioengineering and biotechnology, 11, 1158672. https://doi.org/10.3389/fbioe.2023.1158672

 

[3]Swarts, D. C., van der Oost, J., & Jinek, M. (2017). Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a. Molecular cell, 66(2), 221–233.e4. https://doi.org/10.1016/j.molcel.2017.03.016

 

[4]Almeida, R. S., Wisnieski, F., Takao Real Karia, B., & Smith, M. A. C. (2022). CRISPR/Cas9 Genome-Editing Technology and Potential Clinical Application in Gastric Cancer. Genes, 13(11), 2029. https://doi.org/10.3390/genes13112029

 

[5] Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262–1278. https://doi.org/10.1016/j.cell.2014.05.010

 

[6] 佚名.环介导等温扩增技术检测铜绿假单胞菌的研究[J].安徽医科大学学报, 2017, 52(3):4.DOI:10.19405/j.cnki.issn1000-1492.2017.03.033.