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Evaluating Capture Sequence Performance for Single-Cell CRISPR Activation Experiments
- Source :
- ACS Synthetic Biology. 10:640-645
- Publication Year :
- 2021
- Publisher :
- American Chemical Society (ACS), 2021.
-
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.
- Subjects :
- 0106 biological sciences
Computer science
Human Embryonic Stem Cells
Cell
Biomedical Engineering
Computational biology
01 natural sciences
Biochemistry, Genetics and Molecular Biology (miscellaneous)
03 medical and health sciences
010608 biotechnology
medicine
Humans
CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Messenger
Guide RNA
Gene
030304 developmental biology
Sequence (medicine)
0303 health sciences
RNA
General Medicine
medicine.anatomical_structure
Single-Cell Analysis
RNA, Guide, Kinetoplastida
Transcription Factors
Subjects
Details
- ISSN :
- 21615063
- Volume :
- 10
- Database :
- OpenAIRE
- Journal :
- ACS Synthetic Biology
- Accession number :
- edsair.doi.dedup.....f702721e1d2f5bb5419fa0f82b4e3037