High-throughput CRISPR-library screening has emerged as a transformative approach to identify functional genes in diverse biological processes—from drug resistance to cell viability and immune evasion. By leveraging pooled single-guide RNA (sgRNA) libraries to systematically knock out genes across the genome, researchers can uncover genetic dependencies with unprecedented speed.

However, the power of CRISPR screening lies in experimental nuance. Seemingly minor parameters—such as infection efficiency, sgRNA coverage, or cell passage timing—can dramatically influence results. This article outlines seven key technical variables that critically determine screening accuracy, statistical robustness, and biological interpretability.

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