gms | German Medical Science

Introduction: Neutrophils are essential mediators of host defense and pathologic inflammation. We recently described that neutrophils display high plasticity, enabling them to acquire both tissue as well as disease-specific signatures. Now, new approaches are needed to mechanistically study the role of individual factors identified through omics approaches on neutrophil functions.

Methods: For this purpose, we dsigned a custom CRISPR library. Putative targets were identified based on differentially expressed genes in neutrophils from synovial fluid compared to blood in inflammatory arthritis. Furthermore, upstream transcription factors mediating expression of these genes were included. To enable translational approaches, we focused exclusively on targets with structural orthologs and conserved differential expression across murine and human inflammatory arthritis.

In total, 269 genes fit these criteria. Using bioinformatic modeling, we designed six single-guide RNA molecules per target and included control sequences, resulting in a total of 1,652 sg-RNA molecules. Two libraries were designed, targeting the exact same genes – one targeting the human genome and the other targeting the mouse genome. The sg-RNAs were cloned into the lentiviral vector system lentiCRISPR v2 using enzymatic digestion and Gibson-assembly. We amplificated the two CRISPR libraries using electrocompetent E.coli and validated library coverage and guide-distribution via Next-generation-sequencing (NGS) and bioinformatical analysis.

Results: Our approach resulted in two custom CRISPR-libraries with pre-selected genes including internal positive and negative controls. Sequencing revealed that the resulting libraries achieved full coverage across all sgRNAs with a balanced quantitative distribution of guide RNA sequences. The library was then successfully transduced into human HL-60 cell lines and stably expressed. Thereafter, HL-60 cells could be differentiated into neutrophil-like cells with typical nuclear segmentation, the capacity to generate reactive oxygen species and phagocytose bacteria.

Conclusion: This study demonstrates the principal feasibility of creating a custom CRISPR-Cas9 library to study neutrophil function in vitro. Future work will apply this platform to in vitro-functional experiments for neutrophil key functions including ROS-production, migration and phagocytosis as well as differentiation, apoptosis and aging experiments to investigate the functional impact of specific genes and transcription factors in human und murine neutrophils.

Disclosures: There are no conflicts of interest in the context of the work shown.