High-throughput genome-wide phenotypic screening via immunomagnetic cell sorting

• Published in: Nature biomedical engineering Vol. 3; no. 10; pp. 796 - 805
• Main Authors: Mair, Barbara, Aldridge, Peter M., Atwal, Randy S., Philpott, David, Zhang, Meng, Masud, Sanna N., Labib, Mahmoud, Tong, Amy H. Y., Sargent, Edward H., Angers, Stéphane, Moffat, Jason, Kelley, Shana O.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.10.2019; Nature Publishing Group
• Subjects: 45; 45/23; 631/1647/2163; 631/1647/2217; 631/1647/277; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Cancer immunotherapy; CD47 Antigen - metabolism; Cell Line, Tumor; Cell surface; Cell viability; CRISPR; CRISPR-Cas Systems; Flow Cytometry; Fluorescence; Gene Editing; Genetic screening; Genome; Genomes; Glutamine; High-Throughput Screening Assays - methods; Humans; Immunomagnetic Separation - methods; Immunomodulation; Immunotherapy; Lab-On-A-Chip Devices; Microfluidics; Modulators; Neoplasms - therapy; Oncology; Phagocytosis; Phenotype; Phenotypes; Regulators; Screening
• ISSN: 2157-846X; 2157-846X
• DOI: 10.1038/s41551-019-0454-8

Genome-scale functional genetic screens are used to identify key genetic regulators of a phenotype of interest. However, the identification of genetic modifications that lead to a phenotypic change requires sorting large numbers of cells, which increases operational times and costs and limits cell viability. Here, we introduce immunomagnetic cell sorting facilitated by a microfluidic chip as a rapid and scalable high-throughput method for loss-of-function phenotypic screening using CRISPR–Cas9. We used the method to process an entire genome-wide screen containing more than 10 8 cells in less than 1 h—considerably surpassing the throughput achieved by fluorescence-activated cell sorting, the gold-standard technique for phenotypic cell sorting—while maintaining high levels of cell viability. We identified modulators of the display of CD47, which is a negative regulator of phagocytosis and an important cell-surface target for immuno-oncology drugs. The top hit of the screen, the glutaminyl cyclase QPCTL, was validated and shown to modify the N-terminal glutamine of CD47. The method presented could bridge the gap between fluorescence-activated cell sorting and less flexible yet higher-throughput systems such as magnetic-activated cell sorting. Immunomagnetic cell sorting implemented in a microfluidic chip can perform loss-of-function CRISPR–Cas9-mediated phenotypic screening at higher throughput than fluorescence-activated cell sorting.