Evaluating Capture Sequence Performance for Single-Cell CRISPR Activation Experiments

The combination of
single-cell RNA sequencing with CRISPR inhibition/activation
provides a high-throughput approach to simultaneously study the effects
of hundreds if not thousands of gene perturbations in a single experiment.
One recent development in CRISPR-based single-cell techniques introduces
a feature barcoding technology that allows for the simultaneous capture
of mRNA and guide RNA (gRNA) from the same cell. This is achieved
by introducing a capture sequence, whose complement can be incorporated
into each gRNA and that can be used to amplify these features prior
to sequencing. However, because the technology is in its infancy,
there is little information available on how such experimental parameters
can be optimized. To overcome this, we varied the capture sequence,
capture sequence position, and gRNA backbone to identify an optimal
gRNA scaffold for CRISPR activation gene perturbation studies. We
provide a report on our screening approach along with our observations
and recommendations for future use.