Evaluating capture sequence performance for single-cell CRISPR activation experiments

Abstract

The combination of single-cell RNA sequencing with CRISPR inhibition/activation provides a high-throughputapproach to simultaneously study the effects of hundreds if not thousands of gene perturbations in a single experiment. One recentdevelopment in CRISPR-based single-cell techniques introduces a feature barcoding technology that allows for the simultaneouscapture of mRNA and guide RNA (gRNA) from the same cell. This is achieved by introducing a capture sequence, whosecomplement 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 gRNAscaffold for CRISPR activation gene perturbation studies. We provide a report on our screening approach along with ourobservations and recommendations for future use.

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Identifiers

ORCID for Owen J. L. Rackham: orcid.org/0000-0002-4390-0872

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