CRISPR/Cas9 is a revolutionary genome-editing technology that enables precise and targeted genome modifications in both eukaryotic and prokaryotic cell lines. Offering high efficiency, accuracy, and simplicity in experimental design, CRISPR/Cas9 was initially discovered in prokaryotes and archaea and later adapted for use in eukaryotic systems, becoming one of the most widely used genome-editing technologies. Before performing CRISPR/Cas9-mediated gene knock-out or knock-in experiments, researchers must first understand the fundamental concepts of CRISPR/Cas9 and its components. This course offers a comprehensive overview of CRISPR/Cas9 technology, detailing the protocols for creating gene knock-out and knock-in cell lines, followed by validation techniques. The course also covers the applications of CRISPR knock-out and knock-in, offering valuable tips to help ensure successful laboratory experiments. Furthermore, the course discusses common challenges and reasons for unsuccessful experiments.