Bt Research 2024, Vol.15, No.1, 53-64 http://microbescipublisher.com/index.php/bt 60 7 Ethical and Regulatory Considerations 7.1 Ethical issues in genome editing The advent of CRISPR-Cas9 technology has revolutionized genome editing, offering unprecedented precision and efficiency. However, it has also raised significant ethical concerns. One of the most contentious issues is the potential for human germline modifications, which could lead to unforeseen and undesirable effects, including the risk of eugenics and the ethical dilemma of informed consent. The possibility of creating permanent changes in the human genome that can be passed on to future generations necessitates a thorough ethical review and stringent oversight. Additionally, the use of CRISPR-Cas9 in agriculture and the environment also poses ethical questions, such as the potential impact on biodiversity and ecosystem balance (Barman et al., 2019). Figure 3 CRISPR toolkit (Adopted from Mollashahi et al., 2023) Image capton: (A). CRISPR technology was originally used to create double-strand breaks in eukaryotic DNA (with a bacterial origin (Streptococcus pyogenes). 1. In bacteria, crRNA and tracrRNA guide Cas9 to target the intended region. These RNAs are artificially synthetized as a unique sgRNA to be more applicable in other creatures (yellow) 2. crRNA and tracrRNA are widely used in multiple experimental systems (e.g., mouse embryo microinjections, RNP electroporation into mammalian cell lines, etc.) [91,92] 3. Twenty nucleotides complementary to the target site are used to identify the target area (these nucleotides are designed in a targeted manner). 4. Before these 20 nucleotides, there are three PAM nucleotides (5′-NGG-3′ in Streptococcus pyogenes Cas9 system) which are necessary for CRISPR/Cas9 function. (B). 1. In order to modify the bases in a targeted way, the Cas9 protein was altered to cut only one strand of DNA by changing one amino acid in Cas9 protein (nickase Cas9 [nCas9]). 2. Additionally, they coupled the different base editor domains to the Cas9 protein. (C). 1. Prime editing, the subsequent iteration of this technique, cuts a DNA strand by creating a cut at the intended location. 2 and 3. The sgRNA is made in such a way that its 3′-end complements the two sides of cut site, and its 5′-end can recognize the target site. 4. The reverse transcriptase enzyme turns 3′ sgRNA into cDNA using this 3′ end as a primer. 5. In the cut region, bases are designed for knock-in to produce highly accurate results (Adopted from Mollashahi et al., 2023)
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