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Single-cell CRISPR Screening

CRISPR screening (CRISPR-Screen) technology quantifies the changes of sgRNA targeting target genes before and after screening based on sequencing means and is currently widely used to screen candidate genes associated with specific functional phenotypes such as cell growth, differentiation, immune tolerance, or drug resistance. Although CRISPR screening can study gene function to a certain extent, this technology still has great limitations in studying the regulatory mechanisms downstream of the target gene.

Single-cell CRISPR screening is a powerful way to study underlying developmental, disease, and therapeutic response mechanisms. By directly correlating CRISPR perturbations and single-cell gene expression data cell by cell, it is possible to analyze hundreds of different CRISPR perturbations and detect single guide Rnas (Sgrnas) with direct gene expression phenotypes in hundreds to tens of thousands of cells without knowing the cell type or markers.

10x single-cell sequencing combined with Feature Barcode technology enables simultaneous detection of CRISPR perturbations and the resulting multiple transcriptional signatures, greatly expanding the operability, scalability, and resolution of high-throughput functional screening, allowing us to further explore the mysteries of biology.

Traditional CRISPR hybrid screening can provide limited information about how a gene affects other genetic pathways, and also requires trade-offs between depth and scale of characterization. For example, when we have multiple genes of interest and want to study all the targets at once, if we use traditional CRISPR hybrid screening, we can directly mix together Sgrnas designed for these target genes and transfect them into cells, and we will end up with a group of cells that we do not know what has been transfected. The single-cell CRISPR screen is a very effective alternative to this problem, not only screening thousands of GRnas in a single experiment, but also obtaining perturbed full transcriptome data at the same time to provide the clearest understanding of cell type-specific gene function and pathway analysis.

Experimental procedures and principles of 10x single-cell CRISPR screening

Single-cell CRISPR screening requires first designing gRNA according to target gene, then assembling the designed gRNA sequence into lentiviral vector for subsequent cell transformation, then transfecting the cells with the assembled lentivirus, and screening the transfected cells by flow or antibiotic method.

Finally, the screened cells were captured through the 10x platform: In the oil-in-water reaction system, the oligo sequence in Gel Beads will capture the gRNA sequence in the cells, and the protospacer sequence on the gRNA will know which gRNA is transferred into the cells. In this way, the cell expression profile data can be examined at the same time to detect which gRNA is edited by the cell and the gene expression after editing.

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Figure 1: Experimental procedures of single-cell CRISPR screening

Application of 10x single-cell CRISPR screening

Single-cell CRISPR screening directly correlates gRNA within the same cell with full transcriptome gene expression through sequencing, helping us to fully understand cell type-specific gene function and opening up a new era of biological exploration. As a cutting-edge technology, single-cell CRISPR screening will gradually be widely used in many important research fields such as immunology, neuroscience, and cell development.

With the depth of information brought about by single-cell CRISPR screening methods, researchers are able to gain insight into disease mechanisms and new pathways for therapy development, not only for neurodegenerative diseases, but for all major types of disease. Gene function itself is no longer a simple reading of cell death or survival. Instead, technological innovations in CRISPR screening allow our experiments to match the true complexity of biology, resolving the perturbed transcriptomic effects at single-cell resolution, resulting in a rich picture of gene function in specific cell populations.

References

[1] Wu C A M , Roth T L , Baglaenko Y ,et al.Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins[J].Journal of Immunological Methods, 2018:S0022175918300073.DOI:10.1016/j.jim.2018.03.009.

[2] Rodríguez-Pinto, D. B cells as antigen presenting cells. Cell. Immunol. 238, 67–75 (2005).

[3] Amanna, I. J. & Slifka, M. K. Mechanisms that determine plasma cell lifespan and the duration of humoral immunity. Immunol. Rev. 236, 125–138 (2010).