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To meet increasing demands for productivity, quality, and complex molecule expression, genetic engineering, particularly clustered regularly interspaced short palindromic repeats (CRISPR)\u2010mediated gene knockout (KO), is widely used to optimize host cell performance. However, systematic screening of KO targets remains challenging due to the labor\u2010intensive process of generating and evaluating individual clones. In this study, we present a robust, high\u2010throughput CRISPR workflow using stable KO pools in CHO cells. These pools maintain genetic stability for over 6 weeks, including in multiplexed configurations targeting up to seven genes simultaneously. Compared to clonal approaches, KO pools reduce variability caused by clonal heterogeneity and better reflect the host cell population phenotype. We demonstrate the utility of this approach by reproducing the beneficial phenotypic effects of fibronectin 1 (FN1) KO, specifically prolonged culture duration and improved late\u2010stage viability in fed\u2010batch processes. This workflow enables efficient identification and evaluation of promising KO targets without the need to generate and test large numbers of clones. Overall, screening throughput is increased 2.5\u2010fold and timelines are compressed from 9 to 5 weeks. This provides a scalable, efficient alternative to traditional clonal screening, accelerating discovery for CHO cell line engineering for biopharmaceutical development.<\/jats:p>","DOI":"10.1002\/biot.70033","type":"journal-article","created":{"date-parts":[[2025,5,16]],"date-time":"2025-05-16T04:29:50Z","timestamp":1747369790000},"source":"Crossref","is-referenced-by-count":5,"title":["Utilizing Stable Gene\u2010Edited Knockout Pools for Genetic Screening and Engineering in Chinese Hamster Ovary Cells"],"prefix":"10.1002","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-1683-2023","authenticated-orcid":false,"given":"Jannis Peter","family":"Marzluf","sequence":"first","affiliation":[{"name":"Department of Gene Therapy University of Ulm  Ulm Germany"},{"name":"Sartorius Stedim Cellca GmbH  Ulm 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