We also are actively involved in developing new gene editing methods and provide opportunities for collaborations on more extensive or challenging uses of CRISPR techniques such as development of new construct designs, application to whole organisms, development and use of pooled genome-wide CRISPR libraries.

Services/Equipment available

Engineered mammalian and bacterial cell lines

For this service, we will generate clonal cell lines* containing a mutation of interest (e.g. knock-out, knock-in, SNP, etc.) using the latest CRISPR-based gene editing tools, including Base Editing and Prime Editing. We also provide RNP-based (ribonucleoprotein; CRISPR enzymes pre-loaded with synthetic guides) for sensitive cell lines that do not tolerate plasmid-based gene editing.

Clients can choose from a range of standard immortalised cell lines stocked at GEO or can provide their own, provided they have been tested for mycoplasma. Alternatively, we can order in cell lines from a third party such as ATCC at an additional charge.

Commonly requested edits include:

• single KO
• double KO
• SNPs
• small insertions (e.g. small tags, loxP sites, etc.)
• large insertions (e.g. fluorescent markers, dTAG, etc.)
• small deletions (e.g. lncRNAs, miRNAs, enhancers, etc.)
• large deletions (e.g. haploidised alleles for Saturation Prime Editing)

*For custom-made cell line services, you will receive at least two clones containing the mutation of interest. We commonly edit the following cell lines: A549, HEK293(T), HeLa, HCT116, (e)HAP1, RPE-1 and U2OS. Please contact Joey Riepsaame if you wish to use a different cell line.

Pooled genome-wide CRISPR multiplex library screening

In November 2023, we will be launching our pooled genome-wide loss-of-function CRISPR/Cas12a multiplex library screening program. For this service, we exclusively use the state-of-the-art Humagne and IN4MER platforms designed for assays using limited cell numbers. Due to the multiplexed nature (2-4 guides expressed from a single construct) of Cas12a-based screens, these libraries only require 1/5th of cells required for conventional Cas9-based library screens.

In addition, we offer both guide-only (without enAsCas12a) and all-in-one (enAsCas12a and guides expressed from the same construct). See Table 1 for library composition details.

For genome-wide loss-of-function screens, clients have a choice between dropout (aka ‘negative selection’) and enrichment (aka ‘positive selection’) screens.

Dropout screens are used to identify genes that are essential for cell survival or proliferation. In these screens, cells missing certain genes due to CRISPR-mediated knockout are ‘dropped out’ of the population because they grow more slowly or die. Common uses of dropout screens include identification of novel drug targets or to understand the function of essential genes.

Enrichment screens are used to identify genes that, when inactivated, give cells a survival or growth advantage under certain conditions. In these screens, changes that confer an advantage lead to those cells becoming more ‘enriched’ in the population over time. Enrichment screens can be used to study drug resistance, adaptation to environmental changes, or any other condition where a genetic change may provide a selective advantage.

Table 1: pooled genome-wide multiplex CRISPR libraries available at GEO

Library screening at GEO is performed using established, standardised protocols from the Genetic Perturbation Platform (GPP, Broad Institute). Standard conditions include transduction of the library with a multiplicity of infection (MOI) of 0.3 and a coverage of 500 cells/gRNA construct and at least seven population doublings upon antibiotic selection. However, protocols can be tailored to client’s specific needs.

Access to facility

Services are available to all members of Oxford University, but those in DPAG, Biochemistry, Pathology and Pharmacology have preferential access and rates.

Our services are not exclusive to academics associated with the University of Oxford. Potential users should contact Joey Riepsaame to discuss the details of their projects.

Training required/available

No training is required apart from a basic knowledge of the CRISPR system and the genomic structure of the genes of interest. Collaborative projects will likely involve training in the design and implementation of genome engineering techniques and are assessed on an individual basis.

Facility costs

Engineered mammalian cell lines

Costs for custom-made cell lines range between £3000 and £8000, depending on the complexity of the introduced gene edit (single knock-out vs double knock-out, knock-in, copy number, locus accessibility, cell line, etc.). Please contact Joey Riepsaame to discuss the details of your project.

Pooled genome-wide CRISPR multiplex library screening

Please inquire.