CRISPR-Cas9 screening technology has provided functional genomics with a powerful tool, allowing scientists to unpack the fundamental connection between genotype and phenotype, elucidate cellular pathways, identify drug targets, and discover intricate biological relationships such as synthetic lethal interactions. Pooled library screening workflows using CRISPR technology are now increasingly popular—but to complete a CRISPR screen requires molecular biology, cell biology, and next-generation sequencing capacity, the facility to work with lentivirus, as well as the ability to perform powerful bioinformatic analysis to identify the resulting hits. In laboratories that do not possess all of these capabilities, outsourcing a CRISPR screen to a contract research organization (CRO) can offer a sensible and cost-effective alternative to setting up and performing a CRISPR screen in house.
How a CRO can help
Prior to engaging with a CRO, you should consider carefully where you may need help in the screening process. Perhaps you lack the necessary sequencing ability or downstream bioinformatic analysis capacity, or require assistance with delivering the library and performing the screening methodology. Some CROs can offer help at every step of the process—from performing next-generation sequencing or bioinformatic analysis, to helping set up and perform the screen—and some CROs will have expertise in specific steps of the process.
Once you have identified where you need help, you should ensure the CRO’s offering is in line with your requirements—for instance, will they perform the sequencing and bioinformatic analysis, or just provide you with a cell pellet following completion of the screening experiment? Being clear upfront about what you need and expect will help avoid any pitfalls and minimize the number of agencies you need to get involved to perform your screen and get the best out of the experimental process.
Here are some ways a CRO can help at each stage of the CRISPR screening process, and some things to consider when selecting a service provider.
Choosing a model system
When beginning a CRISPR screen your choice of model system or cell line will be key to the biological relevance of your results. A CRO will be able to help you select a model system that is most appropriate, and in some cases have access to unique genetically modified cell lines in which your screen can be performed. Some CROs will also have expertise in working with particular model systems—such as primary immune cells, organoids, or animal models—and can be selected based on these capabilities. Make sure whichever CRO you choose has the appropriate experience with the system you wish to work in.
Choice of library
As the CRISPR toolkit has expanded so has the choice of genetic manipulation possible—in addition to the original CRISPR-knockout, CRISPRi and CRISPRa libraries are now available, allowing gene inhibition and activation to be included in a functional genomic screen. Correct design of your library is crucial to maximize on-target editing and minimize off-target effects, and there are many best practice guidelines describing guide design. However, changes to genomic annotation and release of new algorithms means that guidance and library quality is always evolving. A CRO will be able to help you navigate best practices in terms of both guide selection and the size and scope of the library that is best for your screen. For example, a genome-wide screen will not always be appropriate, and so you may wish to design a custom library to address a specific family of genes. Some CROs will have pre-designed panels that can be utilized, thereby reducing costs and increasing speed to results. Just ensure that the CRO you select has the necessary experience in performing screens utilizing the different CRISPR systems.
Designing, optimizing, and executing your CRISPR screen
The basis of a CRISPR screen is the separation of cells according to the phenotype of interest, allowing those sgRNA that have been enriched or depleted after application of the chosen selection pressure (e.g. drug treatment or inhibitor) to be identified. Your CRO can help design your experiment—determine the best dosing with which to use your screening condition, identify optimal timepoints, and give advice on experimental methodology. For optimal CRISPR screening, a scientist will typically have to optimize multiple steps of the workflow to minimize bias and maintain representation of the sgRNA library. Steps that require optimization include transduction of lentiviral library, selection of infected cells, and during cell culture, seeding and passaging followed by isolation of genomic DNA and PCR amplification. By working with a CRO experienced in CRISPR screening, many of these optimization steps will have already been performed for workhorse cell lines or reagents, and in the case of bespoke models, the CRO will be familiar with running a raft of parameters, to identify optimal conditions quickly.
Bioinformatics and hit identification
Once the screen at the bench is complete, gDNA is recovered from the control and screen populations and sent for next-generation sequencing, at which point a data analysis bioinformatic pipeline is required. For many biology-focused labs, this capability will not be possessed in house, and will not be provided as standard by sequencing providers.
For this reason, should you require help with data analysis, having this conversation up front with your CRO, and discussing the level of support you require is key. The depth of analysis provided can vary between CROs—for example, some simply provide you with a list of hits, whereas others can help triage the hits and work with you to make recommendations for onward validation analysis.
Stop, collaborate, and listen
While the technical capabilities of the CRO will be a key factor in selection—whether they can work with your model of choice, their ability to execute the screen and their bioinformatic capabilities—it is also critical to choose a CRO with whom you can develop and maintain a good working relationship. As with any project involving multiple teams and stakeholders, communication and collaboration to achieve the best possible outcome is key. It minimizes waste as outcomes are clearly defined from the outset, maximizes the likelihood of success as best practices and ideas are clearly communicated—and if both parties are invested in the success of the project, the probability of obtaining a meaningful outcome increases proportionately.