Abstract 4123: CRISPR-Cas9 and siRNA screening in primary human immune cells
Authors: Cristina Ghirelli, Thibault Laurent, Simon Scrace, Kim Hoenderdos, Chris Lowe, Nicola McCarthy, Jonathan Moore
Journal: Cancer Research (2017)

Abstract 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-tumour responses. Screens using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9-mediated genome editing seem well placed to identify new targets. However, although CRISPR-Cas9 gene editing works well in primary T cells using electroporation, use of a lentivirus one vector system has proved challenging in primary T cells compared with cancer cell lines. We have used several different approaches to identify the most useful method for transduction of primary human T cells with CRISPR components. Electroporation of sgRNAs and mRNA encoding Cas9 into proliferating T cells efficiently generate T cells with specific gene knock-outs or knock-ins, with targeting rates of around 37% for gene knockout. Thus, primary T cells are amenable to CRISPR-Cas9 gene editing, and the capacity to rapidly modify loci 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 in cancer cell lines 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. However, use of the same approach in primary T cells has not resulted in efficient transduction of the library. Specifically, isolated CD3+ T cells stimulated in vitro with anti-CD3 and anti-CD28 antibodies in the presence of recombinant IL-2 resulted in no expression or low level expression of GFP after cells were transduced with a one vector CRISPR-Cas9 sgRNA library. Our experiments indicate, in line with published data, that T cells can be transduced effectively with lentivirus, thus we are examining the use of a two vector CRISPR-Cas9 system and the use of CRISPRi to idealise CRISPR screening in primary T cells. We are also carrying out target identification and validation in myeloid derived suppressor cells (MDSCs). We are using an siRNA approach in these cells, which are generated by PBMC co-culture with cancer cell lines for 7 days, or by culture in the presence of recombinant GM-CSF and IL-6 for 7 days. Our initial data indicate that these MDSCs can effectively suppress autologous, as well as allogeneic, CD8 T cell proliferation mediated by anti-CD3 and anti-CD28 stimulation and that siRNA knockdown is effective in MDSCs. We will use our druggable genome plus arrayed siRNA library to identify targets that when knocked down inhibit the capacity of MDSCs to suppress T cell proliferation. We anticipate that these data will be useful in identifying new targets that are involved in regulating an immune response to tumour development and progression. Citation Format: Cristina Ghirelli, Thibault Laurent, Simon Scrace, Kim Hoenderdos, Chris Lowe, Nicola McCarthy, Jonathan Moore. CRISPR-Cas9 and siRNA screening in primary human immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4123. doi:10.1158/1538-7445.AM2017-4123