Abstract
Pooled genome-wide CRISPR screening allows high-throughput
identification of new genetic vulnerabilities or resistance mechanisms to drug
treatment. Validation and further characterisation of the effect of the genetic
perturbation using orthogonal arrayed methods is critical to build confidence
in the observed phenotype. However, long-term proliferation assays in plate
format are generally limited to short-term kinetics as introducing cell
splitting usually exacerbates background noise. This prevents the screening for
drugs which requires multiple cell cycles before showing an effect, such as
PARP inhibitors.
Here, we report the successful establishment of a miniaturised and scalable
growth competition assay workflow in 96-well plates using in-house automated
platforms. This lentiviral-based approach measures the cell fitness of
fluorescent CRISPR edited cells over non-fluorescent WT cells across multiple
rounds of cell splits and data acquisition/analysis by flow cytometry. For
example, if loss of a gene has a negative effect on proliferation, a decrease
in the edited population of cells will be detected, with
varying effect degrees over time depending on the underlying molecular
mechanism. The
workflow consists of three main independent modules:
(1)
high-throughput arrayed production of pseudo-lentiviruses co-expressing a guide
RNA of interest and a fluorescent marker,
(2) transduction of cells of interest with those
pseudo-viruses at low multiplicity of infection so that only a portion of the
cells have been transduced and compete with untransduced cells and,
(3) semi-automated cell split, cell harvesting and
preparation for automated iQue flow cytometer analysis at each time-point
(repeated for desired assay time).
Our
assay development work was successfully run up to 15 days (5 cell splits) and
confirmed known effect of knocking out control genes in presence of PARP
inhibitor in the colorectal cell line DLD1 stably expressing Cas9.
In the
future, this automated competition assay workflow will provide us with long
term kinetic data on resistance/sensitisation effects to treatment in a
screenable arrayed format – critically improving our ability to identify and
prioritise both clinical biomarkers and new potential drug targets.
