Genome engineering enables precise, targeted changes to be made in the genomes of essentially any organism, enabling genetic analysis in systems previously unavailable to such manipulation, and greatly simplifying and speeding up the process of generating genetic model organisms. The CRISPR/Cas9 system is a simple, programmable and efficient system that can be used to achieve this.
Genome Engineering Oxford (GEO) is a joint venture between the Dunn School of Pathology, DPAG, Biochemistry and Pharmacology. We provide a variety of CRISPR-based services to the Oxford research community. Our services implement the latest and most advanced CRISPR-based gene editing tools, including Base Editing and Prime Editing.
Quick links
Comparison between the conventional two-component CRISPR/Cas9 system and the more advanced Base Editing and Prime Editing genome editing tools.
Schematic overview of the IN4MER platform used for whole-genome screening. The IN4MER human whole-genome library consists of 44K all-in-one constructs (enAsCas12a and gRNA arrays expressed from the same lentiviral plasmid) and independently targets up to four genes from a single guide array. Targets not only include >19K single genes, but also >2K paralog pairs, triples, and quads. Each gene or gene family is targeted by two arrays encoding the same gRNA.
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.
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:
*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 | Genes targeted | Constructs | gRNAs | Lentiviral Backbone |
Humagne Set C | 19,755 | 20,355 | 40,710 | pRDA_052 guide-only expression vector |
Humagne Set D | 19,755 | 20,355 | 40,710 | pRDA_052 guide-only expression vector |
IN4MER all-in-one | 19,687 | 43,972 | 175,888 | pRDA_550 one-component vector |
IN4MER guide-only | 19,687 | 43,972 | 175,888 | pRDA_052 guide-only expression vector |
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.
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.
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.
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.