Objective

There is a strong need for advanced cellular models in drug discovery, particularly for reliable target validation for novel therapies. The CRISPR/Cas9 system (adjacent) has become fundamental in our ability to modulate genes to generate such cellular models.

However, precise genome-editing of certain cell types such as primary cells can prove challenging. Furthermore, the generation of knock-in (KI) cell lines can be difficult in cell lines that favour non-homologous end joining (NHEJ) DNA repair over homologous recombination (HR). Such NHEJ-prone cell lines incorporate the donor template (with the desired mutation) at very low frequencies, making KI cell line generation difficult.

Here we present a successful workflow for the generation of 1) A HPK1 kinase-dead KI Jurkat T cell line using an inhibitor to reduce NHEJ and 2) The in-house isolation of primary immune cells and establishment of efficient downstream CRISPR/Cas9 genome-editing.

This methodology allows for the generation of unique pre-clinical models, further cementing the role of the CRISPR/Cas9 system as a hallmark of future genome-editing in drug discovery.