PhD Scientific Days 2022

Molecular Sciences V.

Targeted genetic editing of putative neutrophil precursors in vitro

Text of the abstract

Introduction • Neutrophils are crucial players in host defence and inflammatory disease pathogenesis alike. Their short-lived nature poses a significant challenge to the field of neutrophil biology, importantly, also precluding genetic manipulations on them following isolation.
Aims • We report successful CRISPR-based gene editing in conditionally immortalised HoxB8 progenitor cells followed by differentiation of mutant cells to functional neutrophils.
Methods • HoxB8 cells were generated from murine bone marrow-derived myeloid progenitors by transduction of a HoxB8 transcription factor – estrogen receptor fusion gene. Immortalised state is maintained while cultured in the presence of estrogen; differentiation to neutrophils commences upon estrogen withdrawal in vitro or transplantation to irradiated recipients in vivo. CRISPR editing was achieved by gRNA transduction into Cas9-expressing HoxB8 cells.
Results • We generated HoxB8 progenitors from bone marrow of Cas9 expressing mice and confirmed the presence of the endonuclease in the cells as well as the progenitor’s ability to differentiate to neutrophils in vitro and in vivo. Next, gRNA targeting the Itgb2 gene (encoding the CD18 surface marker) was designed, cloned into a lentiviral backbone, packaged into infectious particles and transduced into progenitors. Cells were monitored for CD18 expression that drastically decreased following selection for successful lentiviral integration showing the activity of Cas9 when provided with suitable guide. Kinetic analysis of CD18 abundance suggested that disappearance of the target protein is close to complete after four days in the transductants. Editing was also confirmed by Sanger sequencing at genetic level.
Conclusion • The above results highlight the viability of a HoxB8-based system for targeted genetic manipulations of in vitro readily expandable neutrophil progenitors, the subsequent differentiation of which to polymorphonuclear cells would allow for the characterisation of the resulting phenotype in virtually any in vitro or in vivo neutrophil assay.
Funding • This work was supported by the Hungarian National Scientific Research Fund (NKFIH-OTKA No. K119653 and NVKP_16-1-2016-0039).