OUR CROP-Seq (Perturb-Seq) TECHNOLOGY PLATFORM ENABLING CRISPR SCREENS LINKED TO SINGLE-CELL RNA SEQUENCING
Myllia Biotechnology combines CRISPR screening with single-cell RNA sequencing, leveraging two transformative technologies to enable single-cell CRISPR screens for complex cellular phenotypes.
CROP-Seq & CRISPR perturbation screening
CRISPR screening has revolutionized the unbiased annotation of gene function, but most screens done so far have been confined to rather simplistic read-outs (usually life/death of the target cells). Combining pooled CRISPR perturbation with single-cell RNA sequencing allows researchers to assess much more complex phenotypes, thus effectively broadening the scope of these single-cell CRISPR screens. At Myllia, we use the CROP-Seq (Perturb-Seq) technology to perturb cells with CRISPR/Cas9 and profile transcriptional outcomes at scale by RNA sequencing at single-cell resolution. Importantly, our technology is applicable across a wide range of cell types including primary immune cells. The latter include primary human T cells or monocyte-derived cells isolated from PBMCs which are of great interest for the discovery of novel targets in immuno-oncology.
CROP-Seq & CRISPR perturbation screening
CRISPR screening has revolutionized the unbiased annotation of gene function, but most screens done so far have been confined to rather simplistic read-outs (usually life/death of the target cells). Combining pooled CRISPR perturbation with single-cell RNA sequencing allows researchers to assess much more complex phenotypes, thus effectively broadening the scope of these single-cell CRISPR screens. At Myllia, we use the CROP-Seq (Perturb-Seq) technology to perturb cells with CRISPR/Cas9 and profile transcriptional outcomes at scale by RNA sequencing at single-cell resolution. Importantly, our technology is applicable across a wide range of cell types including primary immune cells. The latter include primary human T cells or monocyte-derived cells isolated from PBMCs which are of great interest for the discovery of novel targets in immuno-oncology.
CAS9-EXPRESSING CELLS
Every single-cell CRISPR screen starts in a cell line or primary cell that harbors a CRISPR/Cas9 machinery (e.g., Cas9, dCas9-KRAB, or dCas9-VPR).
LENTIVIRAL GUIDE RNA LIBRARY
Cells are infected with a pooled lentiviral single-guide RNA (sgRNA) library.
CRISPR PERTURBATIONS
Following perturbation with Cas9 and a suitable guide RNA, every single cell in the pool will carry a knockout for a different gene.
Single-cell RNA sequencing
The CROP-Seq (Perturb-Seq) technology measures transcriptome responses to CRISPR perturbation. It offers the flexibility and information content of arrayed CRISPR screens at the scale of pooled CRISPR screens, thus providing a synergy of the two widely popular genetic screening paradigms.
Single-cell RNA sequencing
The CROP-Seq (Perturb-Seq) technology measures transcriptome responses to CRISPR perturbation. It offers the flexibility and information content of arrayed CRISPR screens at the scale of pooled CRISPR screens, thus providing a synergy of the two widely popular genetic screening paradigms.
SINGLE CELL LIBRARY PREPARATION
Each single cell is then encapsulated in a lipid droplet together with a barcoded bead. Reverse transcription occurs on the surface of the bead, thus creating a unique transcriptomic fingerprint for each cell.
GUIDE RNA MAPPING
Mapping of the guide RNAs will connect each single-cell transcriptome to the guide RNA perturbation that caused the transcriptomic phenotype.
NGS AND BIOINFORMATIC ANALYSIS
Single-cell RNA sequencing datasets are typically large and complex. We are routinely analyzing these high-content datasets and are providing bioinformatic/computational analyses that are customized to the specific needs of our clients.