Drug Discovery 2022: driving the next life science revolution

Arrayed CRISPR screening in primary human Tregs using a multi-colour flow cytometry endpoint


CR Dunlop1; S Pavlou1; V Premkumar2; N Popov1; W Chiang1; D Baker1; R Cimbro2; S Engberg2; M Nordberg2; A Will2; Z Guo2; H Yu2; E Whittam2; C Wiggins1; M Delahaye2; K Bednar2; L Escudero-Ibarz1
1 Discovery Sciences, R&D, AstraZeneca, Cambridge, UK;  2 AstraZeneca, UK


Regulatory T cells (Tregs) are a subset of CD4+ T lymphocytes, characterised by high expression of FOXP3 transcription factor. Tregs are responsible for maintaining immune homeostasis and self-tolerance by suppressing the effector function of conventional CD4+ and CD8+ T cells, as well as inhibiting other immune cell subsets. Therefore, dysfunction/loss of Tregs can lead to a break in tolerance and drive autoimmunity. Gene engineering of patient Tregs for improved stability and function aims to provide a potentially curative treatment for severe autoimmunity in inflammatory bowel disease, rheumatoid arthritis, and more, while differentiating from current Treg therapies. To develop an engineered-Treg cell therapy, we must first identify novel genes for Treg stabilisation through a whole genome screening approach. To this aim, we have built a new capability for arrayed CRISPR screening in primary human Treg cells that will validate hits arising from a whole genome pooled screen. We have developed an automation workflow that allows primary Tregs to be CRISPR-edited by Cas9-sgRNA ribonucleoprotein electroporation at scale in 384-well plates. To test the editing workflow, we targeted both neutral and control genes important to Treg function, achieving ≥87% KO efficiency at all loci. We then combined this editing workflow with a newly-developed 384-well flow-staining protocol that uses the iQue Flow Cytometer. We optimised a 10-colour flow panel that allows us to assess markers of Treg stability and function, and achieved robust staining with no signal spill-over between channels. Having achieved high editing efficiencies and generated reliable flow data, this capability has enabled arrayed CRISPR screening in primary Tregs for the first time in AstraZeneca (AZ). This new capability will be utilised as part of our cross-functional collaboration in AZ, with the aim to identify novel targets for an autologous ex-vivo CRISPR-edited Treg cell therapy in our future portfolio.

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