Optical Pooled Screening Infrastructure

The Optical Pooled Screening Infrastructure enables high-throughput microscopy-based screening with single cell genotyping resolution

What is CRISPR?

The CRISPR system has originated in bacteria as a means of defence against bacteriophages millions of years ago. Nowadays, in the lab, the RNA-guided CRISPR-associated nuclease Cas9 can be utilized to carry out targeted mutations in the genome and can be harnessed as a powerful tool for genome-scale genetic screening in human cells. Beyond the classic loss-of-function mutations, different versions of the system such as base editing (BE) and prime editing (PE) have been developed to carry out precise genome editing on the nucleotide level. In addition, epigenetic modulation of genomic loci to suppress (CRISPRi) or activate (CRISPRa) transcription is also possible. Together, these novel applications of CRISPR-Cas systems offer tremendous potential for uncovering the genetic components behind fundamental and disease-related cellular processes in the form of unbiased genetic screens, performed in physiologically relevant model systems such as non-transformed and primary cells.

What is Optical Pooled Screening?

Optical Pooled Screening (OPS) is a recently developed technique which enables high-throughput microscopy-based screening with single cell genotyping resolution. Compared to arrayed screening formats, a key advantage of OPS is the flexibility to fully integrate novel CRISPR technologies and libraries into the screening pipelines. The facility enables users to apply this technology for large-scale screens using validated experimental workflows, automated microscopy and data analysis pipelines supported by the staff members.

Who are we?

Dimitriya supports the experimental design and set up as well as day to day hands-on training. She is also responsible for implementation of technological developments such as in situ sequence automation, integrating OPS with emerging CRISPR technologies.

Alicia supports users all steps of post-screen image analysis and data storage. She is responsible for building and maintaining the data and computational infrastructures and implementing novel analysis pipelines.

What facilities/infrastructures are available?

The users will have access to lab bench, tissue culture labs (GMO1 and 2), and the equipment needed for performing OPS including dedicated microscopes. The facility has relevant vectors for CRISPR KO, BE and OPS screens as well as relevant cell lines required to perform different screens.

Our facility will maintain a dedicated computational infrastructure optimized for Optical Pooled Screening (OPS) analysis. This will include high-performance servers equipped with multi-core CPUs, GPUs for deep learning–based image analysis and high-throughput storage systems for managing raw and processed imaging data. All computation will be performed on internal resources managed by our analysis team.

OPS screens

For new users planning to conduct an OPS experiment, an initial meeting will involve the staff. Here, the library design will be planned. The experiment will be discussed in depth, and a smaller pilot screen will be planned that tests image readout parameters and allows power calculations to determine the number of cells to be imaged in the full-scale screen. Once a successful pilot screen has been performed, the staff will help with the planning, execution, and analysis of the large-scale screen.

During the entire course of the experiment the user will work closely with the staff and get the required hands-on training required for executing screens independently. Once the user has acquired this experience, support by the infrastructure staff will mainly entail consultation and guidance. If needed, the staff can work with the experienced user to implement novel CRISPR techniques into the screening flow

Computational Analysis

Our facility will provide end-to-end computational analysis for Optical Pooled Screening (OPS) experiments, handling all data processing and interpretation in-house. We will manage large-scale image datasets from acquisition through final analysis (including but not limited to illumination correction, cell segmentation, decoding of in-situ sequencing reads). The user will be provided with single-cell-resolved tables linking guide identities with quantitative morphological features. Downstream analysis will include quality control, normalization, feature extraction, dimensionality reduction, and perturbation-level statistical modeling.