The CRISPR-Cas9 genome editing system has transformed the landscape of molecular biology, enabling researchers to disrupt gene function with unparalleled precision. One of the most common applications of this technology is the creation of knockout (KO) cell lines, which serve as essential tools for studying gene function, validating therapeutic targets, and modeling human diseases.
Although CRISPR tools are now widely accessible, generating a reliable KO cell line remains a multi-step process that requires thoughtful planning and methodical execution. In this guide, we’ll break down the major stages of KO cell line development, provide insights into best practices, and highlight common pitfalls to avoid.

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