Evaluating Capture Sequence Performance for Single-Cell CRISPR Activation Experiments

ACS Synth Biol. 2021 Mar 19;10(3):640-645. doi: 10.1021/acssynbio.0c00499. Epub 2021 Feb 24.

Abstract

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.

Keywords: CRISPR activation; feature barcoding technology; hESC; overexpression; single-cell RNA-seq; transcription factors.

Publication types

  • Research Support, Non-U.S. Gov't