Evaluating Capture Sequence Performance for Single-Cell CRISPR Activation Experiments

• Published in: ACS synthetic biology Vol. 10; no. 3; pp. 640 - 645
• Main Authors: Choo, Xin Yi, Lim, Yu Ming, Katwadi, Khairunnisa, Yap, Lynn, Tryggvason, Karl, Sun, Alfred Xuyang, Li, Shang, Handoko, Lusy, Ouyang, John F, Rackham, Owen J. L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 19.03.2021
• Subjects: Clustered Regularly Interspaced Short Palindromic Repeats - genetics; Human Embryonic Stem Cells; Humans; RNA, Guide, CRISPR-Cas Systems - metabolism; RNA, Messenger - metabolism; Single-Cell Analysis - methods; Transcription Factors - genetics; Transcription Factors - metabolism; feature barcoding technology; single-cell RNA-seq; CRISPR activation; overexpression; transcription factors; hESC
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.0c00499

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.