International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 8-15 http://medscipublisher.com/index.php/ijmms 13 CRISPR-Cas9 editing system may be problematic, as it can lead to non-specific editing due to the presence of similar sequences or homologous genes in the genome. Non-specific edits have the potential to impact the function of other genes, resulting in potential adverse effects.There are also limitations in the selection of target sequences for CRISPR-Cas9 technology. Designing appropriate single-guide RNA (sgRNA) is a crucial factor in ensuring the correct editing of the target gene. However, due to the complexity and diversity of the genome, identifying an accurate and specific sgRNA is not always straightforward. Moreover, incorrect binding during the repair process and repair errors can lead to inaccurate gene editing. The CRISPR-Cas9 system typically relies on the natural repair mechanisms within cells to mend DNA breaks. However, this repair process may introduce unpredictable insertions, deletions, or other erroneous modifications, resulting in unexpected outcomes.In terms of clinical applications, gene editing and gene therapy have made significant strides. Clinical trials for some genetic diseases, such as cystic fibrosis and hereditary retinal disorders, have been conducted using gene editing or gene therapy, yielding promising results. These studies have laid the groundwork for further exploration of the clinical applications of gene editing and gene therapy. To address the accuracy issues of CRISPR-Cas9 technology, researchers are actively pursuing various improvements and innovations. For instance, the introduction of modified Cas9 protein variants or strategies, such as the use of dual RNA forms, aims to enhance specificity. Additionally, the development of better sgRNA design and specificity analysis methods contributes to improved accuracy. Despite the challenges faced by CRISPR-Cas9 technology in terms of accuracy, ongoing efforts within the scientific community are focused on refining and optimizing the technique. With advancements in technology and deeper research, it is believed that the accuracy of CRISPR-Cas9 technology will continue to improve, presenting more opportunities and prospects for gene editing research and applications.. 4.2 Non-specific cleavage and safety concerns The non-specific cleavage and safety issues of CRISPR-Cas9 technology are crucial considerations in its application. Non-specific cleavage refers to the potential cutting action of the CRISPR-Cas9 system at locations outside the target DNA sequence. This may result in unintended genome editing, potentially affecting the functionality of other genes. The side effects of non-specific cleavage may lead to unexpected genetic mutations or other chromosomal abnormalities, potentially causing harm to the normal functioning of cells or organisms. The repair processes introduced by CRISPR-Cas9 technology in genome editing can lead to unexpected outcomes. After the Cas9 protein cuts the target DNA, cellular repair mechanisms come into play to mend the DNA breaks. However, errors may occur during the repair process, resulting in insertions, deletions, or other changes in genome structure. These changes can have unforeseen effects, including the introduction of unstable genomic alterations, activation of undesirable gene expression, or potential pathogenic mutations. CRISPR-Cas9 technology also faces safety considerations. Ensuring the accuracy and specificity of the CRISPR-Cas9 system is crucial before direct application in the human body. As CRISPR-Cas9 technology is still in development, precise control and monitoring of the effects of target gene editing are essential to minimize potential adverse consequences and unexpected outcomes. To address these issues, researchers are making efforts to enhance the specificity and safety of CRISPR-Cas9 technology. For instance, reducing the occurrence of non-specific cleavage events can be achieved by introducing plasmid restriction enzymes or using specific Cas9 variants. Furthermore, a more comprehensive evaluation and preclinical studies of the CRISPR-Cas9 system to assess potential side effects and long-term safety are crucial steps in ensuring the safety of its application. 4.3 Ethical and moral issues and public acceptance CRISPR-Cas9 technology has raised numerous ethical and moral concerns, impacting on its public acceptance. The gene-editing capability of CRISPR-Cas9 technology has sparked ethical and moral controversies regarding human genetic modification. The genetic alteration of human embryos or reproductive cells, resulting in modified and improved genes transmitted through the individual's genetic lineage, has triggered concerns about the
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