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 which allows for the simultaneous capture of mRNA and gRNA from the same cell. This is achieved by introducing a capture sequence, whose complement can be incorporated into each gRNA, and which can be used to amplify these features prior to sequencing. However, with the technology in its infancy, there is little information available on how such experimental parameters can be optimised. 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. single cell RNA sequencing of hESC overexpressing either ASCL1 or SOX17 via CRISPR activation, cells are collected 3 days, 5 days and 7 days after introduction of gRNA and pooled into a single single-cell library, which is the sequenced for gene expression and CRISPR gRNA capture