Publications
Molecular Therapy AdvancesJan 2026 |
201678
DOI:
10.1016/j.omta.2026.201678

Overcoming lentiviral delivery limitations in hard-to-transduce suspension cells for genome-wide CRISPR screening

Napoleone, Antonino; Sebastian, Ivy Rose; De Marco, Federico; Molin, Alexander; Hussein, Mohamed; Kramer, Lovro; Jostock, Thomas; Kelly, Thomas; Borth, Nicole
Product Used
Variant Libraries
Abstract
Lentiviral vectors are a widely used delivery modality, combining broad cell-type versatility and stable transgene integration into the host genome for long-term genetic manipulation. These properties make them indispensable tools for delivering molecular components in functional genomics and genome engineering applications. However, challenges such as unpredictable integration profiles and variable cell susceptibility to transduction may introduce experimental biases. Considering the impact of intrinsic cell line properties and transduction methodology, we optimized lentiviral delivery specifically in suspension-adapted Chinese Hamster Ovary (CHO)-K1 and Human Embryonic Kidney (HEK)293-6E cells by systematically comparing two procedures: spinoculation and static transduction. We achieved significantly higher library delivery efficiencies by implementing a two-step static transduction protocol, while minimizing cellular stress, streamlining workflows, and eliminating scalability limitations encountered in large scale applications like genome-wide CRISPR screening. To characterize the variation in lentiviral integration, we used droplet digital PCR (ddPCR) to quantify copy number variation (CNV) both at the pooled-cell level and within individual clonal isolates. Our approach established a robust, enhanced lentiviral delivery strategy in difficult-to-transduce suspension cells and highlights the importance of modulating transduction rates to limit multiple integrations to ensure the consistency required for large-scale functional genomics applications.
Product Used
Variant Libraries

Related Publications