CRISPR-Cas9-Mediated Genome Editing for Protease Engineering

Proteases represent one of the most important classes of industrial enzymes, with pivotal roles in detergents, food processing, leather treatment, and therapeutic development. However, naturally occurring proteases often lack the optimal properties, such as specific activity, stability, and substrate specificity, required for harsh industrial processes or precise medical applications. Traditional protease engineering methods, including rational design and directed evolution, have achieved significant success but are often hampered by their labor-intensive nature, limited mutational scope, and reliance on in vitro systems. The emergence of CRISPR-Cas9 as a powerful, precise, and programmable genome-editing technology has revolutionized the field of metabolic engineering and synthetic biology. This review provides a comprehensive examination of the application of CRISPR-Cas9 for protease engineering. We begin by outlining the fundamental biology of proteases and the molecular mechanics of the CRISPR-Cas9 system. We then contrast CRISPR-based strategies with traditional engineering approaches, highlighting the transformative advantages of in vivo, multiplexed genome editing. A detailed analysis of specific applications, including gene knockouts to create clean genetic backgrounds, precise point mutations to refine catalytic properties, and multiplexed editing to generate complex combinatorial libraries, is presented. Supported by key case studies, we demonstrate how CRISPR-Cas9 accelerates the engineering of proteases with enhanced thermostability, altered substrate specificity, and improved functional expression. Finally, we discuss the prevailing challenges, such as off-target effects and low HDR efficiency, and explore future perspectives, including the integration of base editing, artificial intelligence, and high-throughput screening to usher in a new era of designer protease creation for advanced biotechnological applications.