Genome-Editing Technologies Using CRISPR-Cas9
Abstract
The advent of CRISPR-Cas9 technology has revolutionized genome editing, establishing it as a cornerstone of modern molecular biology and genetic engineering. Initially discovered in bacteria and archaea as an adaptive immune system, CRISPR-Cas9 enables precise programmable modifications of DNA sequences across diverse organisms. This chapter presents a comprehensive overview of CRISPR-Cas9, focusing on its molecular mechanisms, components, and wide-ranging biological applications. Key advances such as single-guide RNA (sgRNA) design, protospacer adjacent motif (PAM) recognition, and Cas9-mediated double-strand DNA cleavage are explored to elucidate the fundamentals of genome targeting and editing. Further attention is given to emerging CRISPR platforms, including base editors, prime editing systems, and high-fidelity Cas9 variants, which have expanded the potential for accurate and safe genome manipulation. This chapter critically reviews CRISPR’s applications in functional genomics, disease modeling, gene therapy, crop improvement, and synthetic biology. It also addresses strategies to minimize off-target effects, enhance delivery methods, and improve editing efficiency. Ethical, biosafety, and regulatory concerns—particularly regarding ecological applications and human germline editing—are thoroughly discussed. By integrating current knowledge with future prospects, this chapter aims to provide researchers, students, and policymakers with an in-depth understanding of both the capabilities and challenges of CRISPR-Cas9-based genome-editing technologies.
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