Abstract B086: CRISPR-Cas9 engineering and screening in primary human T cells
Authors: Ghirelli Cristina , Laurent Thibault , Hoenderdos Kim , Lowe Chris , McCarthy Nicola , Moore Jonathan 
Published: November 2016

Abstract
Recent clinical data indicate immunotherapy can be an effective treatment for cancer patients, yielding dramatically increased survival times in some cases. As not all patients benefit from treatments such as anti-CTLA4 and anti-PD1 antibodies, a major focus in immuno-oncology research is finding new immuno-oncology targets, including those that alter the character and frequency of T-cell-mediated anti-tumor responses.
We have had great success using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 mediated genome editing to probe gene function in cancer cells via the generation of knock-out and knock-in mutants. We are now applying this approach to primary immune cells and are deploying both CRISPR–Cas9 cell engineering and CRISPR–Cas9 screens to better understand T cell biology and to find new therapeutic targets.
We use T cells negatively purified by magnetic sorting from peripheral blood mononuclear cells taken from healthy donors. Our initial work in primary human T cells has focused on the capacity to knock out and knock in genes using the Neon™ transfection system. Our data indicate that primary T cells are amenable to gene editing, and the capacity to rapidly modify loci, such as PDCD1, which encodes PD-1 enables generation of primary T cell models suitable for comprehending the function of modified receptor–ligand pairs involved in an immune checkpoint response.
Our pooled sgRNA–Cas9 screens have used our in-house sgRNA libraries, which include a modified tracrRNA component improving Cas9 affinity and subsequently the performance of a typical sgRNA for promoting gene editing and modifying phenotype. Briefly, isolated CD3+ T cells are stimulated in vitro with anti-CD3 and anti-CD28 antibodies in the presence of recombinant IL-2. Next, the proliferating T cells are co-cultured with a GFP expressing Lentivirus that directs expression of Cas9 and an sgRNA drawn from a 3900 member sgRNA library targeting genes involved in the regulation of metabolism and a control library of 2442 guide RNAs. After prolonged Lentivirus and T cell co-culture, transduced T cells are sorted based on their GFP expression and periodically re-stimulated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 to allow their proliferation and expansion over several weeks. Representative cell pellets are taken after GFP sorting and then at specific time points throughout the screen. For each time point, gDNA is extracted and PCR is carried out to isolate the gRNAs that are present in each cell and these are analysed using NGS. NGS results are interpreted using algorithms from the previously published model-based analysis of genome-wide CRISPR–Cas9 knockout (MAGeCK) and Bayesian normalisation of gene expression levels (BAGEL) approaches. These screens are currently ongoing and are being run in T cells isolated from five independent donors to assess the impact of donor variability.
We will present results from these screens assessing guide drop-out kinetics and reproducibility by comparing the performance of specific guides over multiple time points in each of the donors. We anticipate that these data will be useful in building more complex screens that assess T cell biology in the presence of additional cells, such as myeloid derived suppressor cells, involved in regulating the immune response to tumor development and progression.
Citation Format: Cristina Ghirelli, Thibault Laurent, Kim Hoenderdos, Chris Lowe, Nicola McCarthy, Jonathan Moore. CRISPR-Cas9 engineering and screening in primary human T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B086.