Part 54 The world, from the past to the present, retold from the timelines.

Part 54

Article: Biotechnology My Blog Title: The world, from the past to the present, retold from the timelines.

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2023: [8.10] Mice-tested engineered bacteria to detect cancer DNA. Specifically they engineered non-pathogenic A. baylyi to detect donor DNA released from CRC cells, organoids, and tumours. Integration of tumour DNA into the bacterial genome conferred resistance to the antibiotic kanamycin; antibiotic kanamycin works by killing bacteria or preventing their growth. However, this medicine will not work for colds, flu, or other virus infections. Kanamycin is used to treat tuberculosis of the lungs and other organs upon resistance to other antituberculosis drugs. As such, kanamycin selection could be used as an easily measurable output for detecting colorectal tumours. Capitalising on the inherent ability of A. baylyi to take up foreign DNA through horizontal gene transfer (HGT), a phenomenon known as natural competence, this represented the first application of bacterial biosensors for targeted detection of cell-free DNA released from colorectal tumours. Dr Worthley explained that the natural competence of Acinetobacter baylyi, coupled with the fact that it was a very well-studied, non-pathogenic organism that can colonize the colorectum, colon and the rectum, were reasons why they chose to engineer this bacterial species as a biosensor for the detection of tumour DNA in the gut. CRISPR spacers were introduced into the bacteria to enable the detection of cancer mutations. Often, the sequences of mutant genes in cancer cells differred only slightly from those of wild-type genes. An oncogene is defined as a gene that encodes a protein that is capable of transforming cells in culture or inducing cancer. For example, in KRAS, an oncogene that was frequently mutated in CRC, a single guanine-to-adenine substitution was sufficient to produce a constitutively activated protein that promoted unrestrained cell growth. The authors assessed the ability of the biosensors to determine the mutation status of KRAS in vitro using coculture assays; assays is a laboratory test to find and measure the amount of a specific substance. The aim of coculture is to study the interactions between different types of cells. CRISPR-Cas systems store fragments of foreign DNA, called spacers, as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the most recent are known to be prioritized for immune defence. The sequence of the CRISPR spacers was designed in such a way that they could determine whether the KRAS gene that was integrated into the bacteria was wild-type or KRASG12D. KRAS mutations are one of the most common genetic abnormalities in cancer, especially lung, colon, and pancreatic cancers. KRASG12D, as the most common mutation (33%) in KRAS-mutant tumors, has its unique molecular mechanism and clinical features. This was a superb idea by the co-first author of the paper, Dr Rob Cooper. He designed an RNA guide that would cut the normal wild-type KRAS gene, but not the mutant one, Dr Worthley noted. The authors tested CATCH using engineered tumour models expressing an antibiotic resistance gene flanked by sequences from the KRAS oncogene. Both colorectal cancer CRC cell lines and 3D organoid models were engineered to release donor DNA. Biosensor bacteria took up and integrated tumour DNA, gaining antibiotic resistance. CRISPR spacers introduced into biosensors enabled single-base discrimination between mutant and wild-type KRAS. In this setup, only bacteria that had taken up mutant forms of KRAS could survive in the presence of antibiotics. Commenting on the advantages of cellular testing over traditional laboratory-based methods for the detection of tumour DNA, Dr Worthley said, “A cellular biosensor can diagnose and treat diseases at the time and place of disease onset without requiring the processing and analysis of the sample ex vivo, which is time consuming and complex.” In science, ex vivo refers to experimentation or measurements done in or on tissue from an organism in an external environment with minimal alteration of natural conditions. He added that this approach was particularly useful for disease screening in settings where access to resource-rich laboratories and sophisticated assays was limited. To validate the clinical relevance of their method, the team used CATCH to analyse the ability of sensor bacteria to detect KRAS mutations in CRC organoids and an orthotopic mouse model. In medicine, “orthotopic” is often used to describe tissue or an organ that is transplanted into its normal place in the body.

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Picture sources: Peakpx.com and Pexels, Pixabay in PowerDirector and other websites:

1:https://www.biorxiv.org/content/biorxiv/early/2022/10/04/2021.09.10.459858/F3.large.jpg

2:https://www.science.org/cms/10.1126/science.adf3974/asset/c55b14b5-f531-44e8-ab03-9ef39100a40c/assets/images/large/science.adf3974-f3.jpg

3:https://www.science.org/cms/10.1126/science.adf3974/asset/27245fc1-7012-41c5-81c4-f09fa2486163/assets/images/large/science.adf3974-f2.jpg

4:https://www.cancer.gov/news-events/cancer-currents-blog/2023/engineered-bacteria-detect-cancer

5:https://asianatimes.com/wp-content/uploads/2023/10/acinetobacter.jpg

6:https://i.pinimg.com/736x/e8/ae/aa/e8aeaab747f683edae94067fe7050473.jpg

7:https://www.creative-diagnostics.com/upload/image/study-on-the-combination-of-kanamycin-and-other-antibiotics-1.jpg

8:https://www.frontiersin.org/files/Articles/1410115/fcimb-14-1410115-HTML/image_m/fcimb-14-1410115-g001.jpg