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Implications of CRISPR Technology in Biological Systems
Published in Jyoti Ranjan Rout, Rout George Kerry, Abinash Dutta, Biotechnological Advances for Microbiology, Molecular Biology, and Nanotechnology, 2022
Kikku Sharma, Souvik Sen Gupta
Antibiotics are known for their potential application to disrupt bacterial infection. In recent times, antimicrobials are threatened to undoing. The overuse and abuse of antimicrobials have led to the development of multi-drug-resistant microbes and also the beneficial amicrobial flora is destroyed (Beisel et al., 2015). As a result of this antimicrobial resistance, many common bacterial diseases are becoming increasingly difficult to cure. The development of modern antimicrobials without antimicrobial resistance requires more effort in the upcoming years. The CRISPR is an important part of bacterial immunity systems. Out of six types of the CRISPR system, type II CRISPR is structurally very simple and also it has a versatile application in genome editing and ecological engineering. Several years ago, it was shown that synthetic CRISPR may be used to eradicate certain microbes. Recent studies suggest that the CRISPR technique can be used as a potential tool for the disruption of multidrug-resistant bacterial strains. CRISPR/CAS9 technique is used to remove the antimicrobial-resistant gene from a selective strain of bacteria thus enabling the resensitization of particular bacteria to antimicrobials. To remove the specific strains from the mixed population of bacteria, CRISPR CAS9-mediated transformation has been done. For this purpose, CRISPR CAS9 has been incorporated into the host by phagemid-mediated delivery (Pursey et al., 2018). In this method, plasmids with selective phage packaging signals along with other components like Streptococcus pyrogens nuclease encoding sequence, artificially designed CRISPR RNA sequence and trans activating RNA sequence has been delivered into selective microbial strains. Here phage is used because phage is naturally evolved to inject their DNA into the host bacterial cell. After the packaging of phagemid and its delivery to the host, there is a rapid disruption of bacterial cells. These phagemids possess specific antimicrobial resistance plasmids, which result in the efficient removal of the plasmid (Beisel et al., 2014). Delivery of CRISPR CAS9 containing phagemids may cause sequence-specific killing of bacteria. In yet another study, it has been shown that the delivery of CRISPR CAS9 namely conjugative plasmid leads to the death of the bacteria that contain antimicrobial-resistant genes (Pursey et al., 2018). The traditional antibiotics have a poor range of application because most of the bacteria contain antimicrobial resistance genes. This serious problem can now be overcome by CRISPR-based editing of an antimicrobial gene. Besides, CRISPR CAS9 cannot only provide resistance to the undesired gene but can also target mobile genetic elements like transposons to disrupt it thus ensuring genetic homeostasis. CRISPR CAS9 can also be used for vaccination by targeting invasive genetic elements. The acquisition of virulence trait by natural DNA transformation is prevented when CRISPR technology creates a potent barrier for bacteriophages (Barrangou, 2015).
Antimicrobial, antibiofilm, and anticancer potential of silver nanoparticles synthesized using pigment-producing Micromonospora sp. SH121
Published in Preparative Biochemistry & Biotechnology, 2023
Birgül Mazmancı, Serpil Könen Adıgüzel, Yiğit Süha Sadak, Derya Yetkin, Hilal Ay, Ali Osman Adıgüzel
DNA cleavage activity of Mm-AgNPs was evaluated by monitoring supercoiled, nicked, and linearized forms of pBluescript SK II phagemid DNA. DNA forms separated in agarose gel (Figure 9) were quantified using Image J software. From the results, no significant change was detected in the intensity of supercoiled DNA after the phagemid was incubated with different concentrations of Mm-AgNPs. The intensity of the band, which corresponds to the linearized form of phagemid DNA, increased when Mm-AgNPs concentration was up to 16 µg/mL. On the other hand, a further increase in Mm-AgNPs did not cause an alteration in the band intensity. Briefly, the DNA cleavage activity of Mm-AgNPs was not statistically significant. Unlike our findings, some previous studies demonstrated that AgNPs cause breaks in plasmid DNA.[63–65] In such studies, it has been claimed that the main reason for the antimicrobial effect of AgNPs may be their DNA cleavage activity.[48]
Progress on electrochemical sensors for the determination of heavy metal ions from contaminated water
Published in Journal of the Chinese Advanced Materials Society, 2018
Xiangzi Dai, Shuping Wu, Songjun Li
Immunosensors are compact analytical devices based on specific antigen–antibody interactions and in which the immunochemical reactions are either directly or indirectly detected by means of a transducer. In electrochemical immunosensors, the event of immunochemical reactions is converted into an electrical signal such as an electric current, a voltage difference or a resistivity change.[21,74–76] The main principle of electrochemical immunosensor for the quantification of heavy metal ions is the change in the current on electrode surface due to oxidation and reduction of adsorbed metal ions. An ultrasensitive electrochemiluminescent (ECL) competitive immunoassay for mercury (II) was developed based on CdSe QDs, gold nanoparticles (GNPs) and specific monoclonal antibody (mAb) against Hg(II).[77] GNPs as substrate and electron transfer accelerator could load more number of coating antigen and magnify the electrochemical signal. Based on this method, Jing et al. developed a cheap and selective GNPs/Ovalbumin-MNA-CH3 Hg/mAb-QDs immunosensor based on the specific mAb against Hg(II). Competitive immunoassay was applied for the detection of Hg(II), and the ECL assay process is depicted in Figure 5(a). The actual size of the thioglycolic acid (TGA) modified CdSe QDs was about 4.6 nm (Figure 5(b)). The possible ECL mechanism could be expressed by the cyclic voltammetry (CV) of the immunosensor (Figure 5(c)). Furthermore, at the range of 0.01 to 50 ng/mL, the logarithm of the mercury (II) concentration change is linear to the decrease of the ECL intensity, with a much lower than previous methods detection limit of 2.6 pg/mL. The immunosensor also exhibit great selectivity to the mercury (II), the result was manifested in the (Figure 5(c)). This ECL immunoassay which applied GNPs as substrate combined with specific mAb detecting Hg(II), is presented for the first time. In addition, Zhu et al. had specifically generated and selected measured environmental uranium with an antibody-based sensor based on recombinant single-chain variable fragment antibodies (scvF).[78] The obtained scvF was complexed to 2,9-dicarboxyl-1,10-phenanthroline-acid (DCP) using genetic material obtained from the spleen cells of rabbits immunized with UO22+—DCP conjugated to keyhole limpet hemocyanin. Recombinant antibody library and phage-displayed antibodies were obtained by amplifying and cloning immunoglobulin light chain and heavy chain genes into the phagemid pSD3. The limit of detection to UO22+ was 2.2 nM, which is below the United States Environmental Protection Agency (US EPA) action limit of 126 nM. In the real sample, the recovery ranged from 84.9% to 124.5% and average sample recovery was 98.89%.