CRISPR Editing Route, Not Just Target Genes, Reshapes CHO Cell Lines

Chinese hamster ovary cells are the workhorses of modern biopharmaceutical manufacturing, churning out the majority of approved monoclonal antibodies and countless other therapeutic proteins. For years, scientists have used CRISPR/Cas9 gene editing to knock out undesirable host cell functions in these cells, from enzymes that degrade product quality to proteins that sabotage formulation stability. But a new study raises an uncomfortable and fascinating question: does it matter how you edit a cell, not just which genes you edit? According to research published in Frontiers in Bioengineering and Biotechnology, the answer is a resounding yes.

A team at Biberach University of Applied Sciences and Boehringer Ingelheim systematically compared two CRISPR/Cas9 workflows for generating CHO cell lines lacking nine polysorbate-degrading hydrolases. Polysorbate is a surfactant added to therapeutic protein formulations to prevent aggregation, but host cell-derived hydrolases can enzymatically chop it up, generating free fatty acids that form visible and subvisible particles and compromise drug stability. Removing the offending enzymes has therefore become a promising strategy for stabilizing biologics during manufacturing and storage.

The researchers compared a sequential editing strategy, in which genes were knocked out one or a few at a time over repeated rounds of transfection and single-cell cloning, with a multiplex strategy that removed several targets simultaneously. The sequential lineage required 383 days of cultivation, seven rounds of single-cell cloning, and seven electroporations to reach the final genotype. The multiplex route took only 250 days and four cloning rounds, though it needed eleven electroporations because some guide RNAs worked inefficiently, forcing repeated transfections. Both approaches deleted the same nine hydrolase genes, including lipoprotein lipase, phospholipases, and two large carboxylesterase gene clusters excised with paired guide RNAs spanning 506 and 1,629 kilobases of genomic DNA.

Functionally, both strategies succeeded. A fluorescence-based micelle assay showed that polysorbate retention climbed from roughly 30 to 45 percent in parental controls to more than 75 percent in final knockout clones from either workflow. RNA sequencing confirmed transcript depletion at the targeted loci, and genomic analysis verified editing at every intended site. On paper, the two cell lineages were equivalent. In practice, they were strikingly different organisms.

In fed-batch bioreactor cultures, the sequentially edited clones deteriorated in a stepwise fashion as knockouts accumulated. Peak viable cell density fell from about 25 million cells per milliliter in the parental line to 15 to 17 million in final clones, while harvest viability collapsed from above 90 percent to between 50 and 60 percent. The multiplex clones also declined, but more gently, retaining peak densities of 17 to 21 million cells per milliliter and viabilities between 55 and 80 percent, albeit with greater clone-to-clone variability. Metabolic behavior diverged too: sequential clones showed escalating glucose consumption, reaching 42 to 53 grams per liter by day ten, whereas multiplex clones stayed closer to parental levels at 19 to 27 grams per liter.

Perhaps the most striking physiological change emerged in the sequential lineage after knockout of the Pla2g15 and Lipa genes: cells became larger and carried more DNA, with increased cell diameter correlating with elevated DNA content measured by flow cytometry and with reduced proliferation. This shift was never reversed by subsequent editing steps. Intriguingly, one multiplex clone, M-KO4, independently developed the same enlarged, high-DNA, slow-growing phenotype, suggesting that clone-specific disruptions during repeated manipulation, rather than the knockout targets themselves, drive these undesirable traits.

The transcriptomic data told an even sharper story. Final sequential knockout clones showed 425 upregulated and 415 downregulated genes relative to the parental line, while final multiplex clones displayed a far smaller footprint of 162 up and 109 down. Direct comparison between the two endpoints revealed 894 genes higher and 1,003 genes lower in the sequential clones. On a two-dimensional UMAP projection of global gene expression, sequential intermediates drifted progressively away from the parental state, while the multiplex lineage stayed within a confined transcriptional space. The two workflows ended in largely non-overlapping transcriptomic states despite engineering identical genotypes.

Stage-resolved analysis of when differentially expressed genes first appeared pointed to clonal selection, not the editing events themselves, as the dominant driver. Early sequential intermediates changed only modestly, but the Pla2g15 and Lipa knockout step coincided with a jump of 87 up and 83 down genes, matching the inherited increase in cell size and DNA content. The largest sequential shift, 346 up and 250 down genes, followed deletion of the second carboxylesterase cluster, yet analysis of genes flanking the deleted regions showed no broad local expression changes, arguing against simple position effects. Meanwhile, the multiplex lineage accumulated changes gradually, and even its final step involving seven electroporations produced a shift no larger than several sequential stages. The authors conclude that repeated single-cell isolation and clonal adaptation, consistent with the known genomic plasticity of CHO cells, primarily shape the final cellular state.

The team also probed whether eliminating nine hydrolases triggers compensatory upregulation of alternative polysorbate-degrading enzymes, a key concern for product stability engineering. Across a curated panel of candidate serine hydrolases defined by the conserved catalytic triad associated with active CHO-derived enzymes, most genes showed minimal change. Only Pla1a and Abhd14b rose above threshold in sequential clones, and only Liph in multiplex clones, indicating workflow-specific rather than universal compensation. Notably, Ppt2, a lysosomal thioesterase paralog of the knocked-out Ppt1, was consistently downregulated in both lineages, the opposite of what a simple compensation model would predict.

The practical implications for cell line development are substantial. For non-essential targets such as problematic host cell proteins, the data favor multiplex editing, which reached the same functional endpoint in less time, with fewer cloning rounds, and with less accumulated drift in growth, DNA content, and gene expression. Sequential editing retains value when target biology is uncertain or when individual knockouts might impair essential functions, since intermediate phenotypes can be monitored before proceeding. Either way, the study delivers a sobering message: the engineering process itself, particularly the gauntlet of electroporation, cloning, and selection, can sculpt cellular phenotype and transcriptome as powerfully as the intended genetic modifications, and unfavorable traits acquired early tend to persist through the entire lineage. Choosing an editing strategy, the authors argue, is a critical design parameter in its own right.

Subject of Research: Comparison of sequential versus multiplex CRISPR/Cas9 editing workflows for hydrolase knockout in CHO cells

Article Title: Same hydrolase knockouts, different outcomes: CRISPR/Cas9 editing workflow shapes resulting cellular phenotype and transcriptome in CHO

Article References: Weiß, L., Schneider, S., Otte, K., & Fischer, S. (2026). Same hydrolase knockouts, different outcomes: CRISPR/Cas9 editing workflow shapes resulting cellular phenotype and transcriptome in CHO. Frontiers in Bioengineering and Biotechnology, 14, 1923843. https://doi.org/10.3389/fbioe.2026.1923843

Image Credits: AI Generated

DOI: 10.3389/fbioe.2026.1923843

Keywords: CHO cells, CRISPR/Cas9, gene knockout, polysorbate degradation, hydrolases, cell line development, transcriptomics, multiplex editing, clonal selection, biopharmaceutical manufacturing, RNA-seq, host cell proteins