Abstract

CRISPR knockout screens in primary human T cells can help to
address clinically relevant biological questions in vitro. Any
potential new target or biological behaviour identified in these screens could translate
more effectively from bench to bedside, saving time and money. Pooled and
arrayed CRISPR screens address different questions: pooled screens are
effective for analyzing impacts of gene loss at a population level, whereas
arrayed screens allow analyzes of individual genes on a well-by-well basis and
are more applicable to complex phenotypic endpoints. Our proof-of-concept
studies demonstrate the utility of pooled and arrayed CRISPR knockout screens
in primary human immune cells.

Using a combined lentivirus and electroporation approach
developed in house, we analyzed drug-gene interactions evident in a pooled CRISPR
knockout screen in ex vivo human CD3+ T cells exposed to phenformin, an
electron chain inhibitor. A short single guide RNA (sgRNA) library of over 7,000
sgRNAs was used to target 482 metabolic genes and 152 control genes. Our resulting
dataset passed all NGS and screening standard QC on a donor per donor basis and
confirmed loss of the gene GOT1 as a key sensitising factor to
phenformin in primary T cells. This finding is in agreement with Jurkat T cell
data published by Birsoy et al., (2015), and we also identified
additional genes which on deletion increased phenformin cytotoxicity.

Although pooled CRISPR screens are useful for analyzing the
impact of gene loss in a population of cells, sometimes well-by-well readouts
are needed to address more complex biological endpoints, such as co-culture
endpoints. We have used a semi-automated electroporation approach to carry out arrayed
CRISPR knockout screens in human T lymphocytes and human myeloid cells. For
CD4+ T cells, monocyte derived dendritic cells and monocyte-derived
macrophages, we have identified electroporation conditions that enable a high
percentage of cells in every well to remain viable after successful gene
knockout, verified using molecular- and protein-based approaches. We have also carried out functional assays to
look at the impact of gene loss. In CD4+ T cells we assessed the impact of CD3ε
knock out by co-culturing CRISPR-Cas9 edited T cells with unedited monocyte-derived
dendritic cells. Multiplexed HTRF and FACS analyses showed that CD3ε-edited CD4+ T cells had reduced proliferation,
decreased IFN-γ secretion and lower
CD25 expression compared to cells treated with non-targeting guides, as
expected.  

In summary, pooled and arrayed CRISPR-Cas9 screens in
primary T cells will provide useful methods for identifying novel potential
hits, which could translate into therapeutic targets. Pooled CRISPR screens are
applicable to primary immune cells that can be produced in large numbers,
whereas arrayed CRISPR screens can be used to assess the impact of gene
knockout in immune cells that are less abundant and to assess physiological,
co-culture endpoints that are not suited to a pooled population approach.

Birsoy, K. et al. (2015) An essential role of the
mitochondrial electron transport chain in cell proliferation is to enable aspartate
synthesis. Cell 162, 540.