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).

Visual representation of Cas9 Proteins in a lab dish for CROP-Seq. Illustration of Cas9 Proteins in a laboratory dish for CROP-seq. Cells and Cas9 protein illustrated in the lab dish

LENTIVIRAL GUIDE RNA LIBRARY

Cells are infected with a pooled lentiviral single-guide RNA (sgRNA) library.

Visual representation of a lentiviral sgRNA library for performing  perturb-seq. Illustration showing a lentiviral sgRNA library utilized in a perturb-seq. Illustration of lentiviral sgRNA library for perturb-seq.

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.

Illustration depicting multiple cells undergoing CRISPR perturbations. Illustration portraying numerous cells subjected to CRISPR perturbations. Illustration showing multiple cells in CRISPR perturbations.

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.

Visual representation of cell encapsulation for single-cell library preparation and RNA sequencing Illustration of cells encapsulated in lipid droplets for single-cell RNA sequencing Illustration of cell encapsulation in lipid droplets with barcoded bead in single-cell RNA sequencing.

GUIDE RNA MAPPING

Mapping of the guide RNAs will connect each single-cell transcriptome to the guide RNA perturbation that caused the transcriptomic phenotype.

Visualization of how RNA guides are mapped to to each single cell in single-cell RNA sequencing. Visualized guide RNA Mapping: Linking single-cell transcriptomes to perturbations. Illustrated single cellsmapped to three individual guide RNAs in single-cell RNA sequencing.

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.

Illustration of a computer screen showing single-cell RNA sequencing datasets from CROP-seq. Computer screen displaying single-cell RNA sequencing datasets for CROP-seq, as illustrated. Illustrated computer screen with single-cell RNA sequencing datasets derived from CROP-seq.

Videos about Myllia's single-cell CRISPR screening technology

How do CRISPR screens work?

Explore how we can use CRISPR screens to support drug target identification

About us

Discover the CRISPR screening portfolio at Myllia Biotechnology

Phoenix Award 2022

Myllia Biotechnology wins the Phoenix award 2022 in the "Start-up" category!

A genome-scale CROP-seq screen reveals mediators of T-cell signaling

In a 10x Genomics webinar, Tilmann Buerckstuemmer, CSO at Myllia Biotechnology, describes the use of genome-wide CRISPR screens in target discovery for immuno-oncology. Using targeted sequencing of markers of T cell activation, this CRISPR interference screen represents the largest CROP-seq/ Perturb-seq dataset created to date.

What’s the difference between CRISPRko and CRISPRi screens?

Learn more about the differences of CRISPRko and CRISPRi screens

CROP-Seq: CRISPR screens at single-cell resolution

Learn how the CROP-Seq technology at Myllia can help to elucidate novel drug targets at single-cell resolution

Targeted sequencing

Would you like to know more about targeted sequencing applications? Explore how tailored PCR panels can boost the NGS read-outs of your next CRISPR screens

Primary human T cell CRISPR screening

At Myllia, CRISPR screens are performed in primary human T cells to explore factors of T cell activation, differentiation and exhaustion. Learn more about our CROP-seq technology to boost CAR-T and TCR-T cell potency!

Resources about Myllia's CROP-Seq (Perturb-Seq) screening platform

CRISPR technology, cellular models and omics read-outs

Our Factsheet

Check out Myllia's technology platform enabling drug target discovery and functional genomics at single-cell resolution

A Genome-Scale PARPi CRISPR KO Screen for Synthetic Lethality

CRISPR KO screen at genome-scale in HeLa cells to identify novel or well-known genes that are involved in DNA damage repair to explore PARPi synthetic lethality gene pairs

A Genome-Wide CROP-Seq CRISPRi Screen for Factors of TCR Signaling

At Myllia, the Perturb-seq (CROP-seq) workflow has been adapted to enable genome-scale CRISPRi (CRISPR interference) screens in Jurkat cells at single-cell resolution. The first-of-its-kind genome-scale CRISPRi screen was conducted to verify factors involved in TCR signaling pathways.

Single-cell CRISPR screening at Myllia - fully customized screens for target discovery

Our Portfolio

At Myllia, pooled CRISPR screens are performed at large scale, and our unique platform involves a broad portfolio of CRISPR/Cas9 technologies

CRISPR-ready cancer cell lines for CRISPRn & CRISPRi screens

Browse through our catalogue of CRISPR-ready cancer cell lines for your next tailor-made CRISPR screens

Large-Scale CROP-Seq Screening in Primary Human T Cells

At Myllia, we can identify rare subsets, transitional states, and distinct functional profiles that may be masked in bulk analyses, providing a more comprehensive understanding of T cell activation and differentiation. The CROP-seq experiment in pan T-cells involved a targeted single-cell RNA sequencing of 300 mRNAs.

A CROP-Seq Screen for Modulators of Th2 Cell Polarization

In a CROP-Seq experiment with CD4+ T cells, Th2-skewed cells were studied using a sgRNA library targeting 102 genes with a targeted read-out of 300 mRNAs.

Poster: A Genome-Scale PARPi Screen for Synthetic Lethality

At Myllia, we’ve performed a genome-scale CRISPR KO screen in HeLa cells to identify novel or well-known genes that are involved in DNA damage repair and would resemble gene pairs with PARP.