CRISPR Functional Genomics
The CRISPR Functional Genomics unit (formerly High Throughput Genome Engineering, HTGE) provides affordable access to high throughput functional genomic screens using the CRISPR/Cas9 system in cell lines. Pooled CRISPR/Cas9 screening enables parallel interrogation of thousands to tens of thousands of genes for involvement in biological processes of interest. CFG provides access to verified lentiviral CRISPR guide libraries for whole genome and targeted loss- and gain of function studies (CRISPR knock-out, CRISPR inhibition, CRISPR activation).
Our service portfolio includes CRISPR gene perturbation followed by single cell RNASeq (together with Eukaryotic Single Cell Genomics (ESCG) and we are developing base-editing approaches for targeted mutagenesis screens to characterize protein-protein or protein-drug interactions.
Standard pooled CRISPR screen. Guides targeting the elements of interest are synthesized, cloned, and packaged into lentivirus in pool (1-3). Cas9-expressing cells (4) are then transduced at low multiplicity of infection to ensure that each cell obtains a single guide (5), and phenotypic selection is applied after genome editing has occurred (6,7). Guide representation in treatment and control populations is then determined by NGS, resulting in a list of genes involved in the biological process of interest (8).
Barcoded guide libraries.
For superior statistics and data analysis, all our libraries are barcoded, as described in our publication CRISPR/Cas9 Screening Using Unique Molecular Identifiers. PDF. Full text.
- Compared to the conventional method, inclusion of RSLs generates considerably more information at an identical experimental scale and enables additional ways of data analysis.
- RSL‐based analysis requires fewer cells per guide to reach a set statistical power. This is important if cell numbers are limiting, such as in very large, genome‐wide screens and/or screens in primary cells.
- RSLs can be used to track cell lineages throughout a pooled screen.
Services
- Cas-expressing cell line generation (Cas9, nCas9, dCas9, dCas9 fusions, Cas12a, Cas13d)
- CRISPR-ko, CRISPR-inhibition, CRISPR-activation screens
- Multiplexed Cas12a screens
- Base-editor screens
- RNA targeting screens with Cas13d
- Small pooled screens with single cell transcriptomic readout
- Experienced support for screen design
- Custom-made lentiviral guide libraries produced and provided
- Library transduction performed
- NGS libraries prepared
- NGS at NGI Stockholm included in the pipeline
- Data analysis: hit list generated
Applications
- Determine essential genes in specific cell types
- Screen for drug resistance/sensitivity genes
- Find novel genes/pathways involved in any cellular process
- Map novel genes/pathways regulating a reporter gene
Using the unit
CFG is open to the entire Swedish and international research community. Please contact bernhard.schmiererATki.se to schedule a free consultation meeting.
Our local branch, Karolinska Genome Engineering (KGE), performs precision edits in cell lines, such as knock-out, knock-in, deletions, point mutations, etc. KGE collaborate with the Karolinska Center for Transgene Technologies (KCTT) to offer CRISPR technology in mouse, and has an active pipeline for precision editing of iPSCs with the iPSC core facility at Karolinska Institutet.
Your project is out of the box? All the better, we would love to hear from you!
Shipping and Mailing Address
Biomedicum 9B
Tomtebodavägen 16
171 65 Solna
Visiting Address
Biomedicum 9B
Solnavägen 9
171 65 Solna