Fig. 3

Overview of gRNA library NGS data collection. (a) Data collection during cell and KO library generation. The CHO genome-wide gRNA library, containing 111,651 unique gRNAs targeting 21,585 genes, was designed, cloned, and introduced into CHO-K1 host and recombinant cells via RMCE. Cell libraries underwent genome-wide KO (Cas9 short-term library) and prolonged cultivation (Cas9 long-term library) for data collection. Genome-wide gRNA representation in plasmid and cell libraries was validated, and gRNA distribution in the Cas9 short-term and long-term libraries was analyzed. Core and conditional essential genes can be identified as significantly depleted genes, depending on the specific cell lines and culture durations. (b) Data collection during positive selection. The highly productive recombinant Cas9 short-term library was enriched using the cold capture secretion assay. A control recombinant cell library underwent the same experimental procedure to exclude false positive hits. Functional genes associated with the phenotype of interest were identified as significantly enriched gRNAs. The generated NGS dataset is indicated by asterisks, with the number of asterisks representing the biological replicates: black for the plasmid library, blue for CHO-K1 host, and red for recombinant cells. In total, 13 NGS datasets were collected. S, short-term; L, long-term; FP, false positive; HP, highly productive; -, significantly depleted gRNAs; and + , significantly enriched gRNAs.