Working with Service Providers for Outsourcing CRISPR Studies
This section covers CRISPR outsourcing solutions, but the same principles apply to other forms of gene editing technologies like TALENs and ZFNs. Although the tools and methods for application of genome editing technologies are becoming more accessible, some labs may choose to outsource partial or entire experiments on a fee‐for‐service basis. Genome editing CROs have invested considerable efforts on internal development and have amassed great knowledge on how the technology works, which translates to a high competence to design genome editing projects and troubleshoot problems. In addition, CROs enable researchers to conduct research without the need to purchase and maintain expensive equipment (e.g. flow sorter, or next‐generation sequencing instruments). CROs are also used when there is time pressure to deliver while internal resource is in short supply, or in cases where there is limited or no internal expertise. Such a business model has several advantages and disadvantages which need to be considered before proceeding with this option (Pichler and Turner 2007).
4.4.1 Critical Steps for Outsourcing
A number of companies and academic institutions offer genome editing services. Providers can be found through websites including Science Exchange (https://www.scienceexchange.com) and Biocompare (https://www.biocompare.com) and we provide a non‐exhaustive list in Table 4.2. It is important to base your choice on the reputation and track record of the company, otherwise you run the risk of contracting a CRO that will use your project for its own capability development (Pichler and Turner 2007). If possible, it is advisable to visit the CRO facility to inspect their laboratory set‐up, view the equipment used, and discuss case studies that the company has completed. Also, you will need to perform due diligence checks. For example, discuss if the CRO holds foundational CRISPR licenses and understand their workflows with regard to data integrity (i.e. how they record and track their data). The next step is to set up a Confidential Disclosure Agreement (CDA) so that you are able to freely exchange information about the project you want to conduct and codevelop a detailed experimental plan. As in the case of sourcing reagents, when discussing genetic screens or cell line generation experiments with a CRO, it is important to focus on the formula Model × Assay × Perturbation (M × A × P), as clearly defining all three will ensure success. Following, discussions on the science, economics, and timelines need to be considered. Sometimes, providers may offer optimistic timelines that work if everything goes well according to plan and no repeats need to be made. However, this may not always be the case, so it is important to discuss this and ensure that timelines are realistic. If possible, reach out to other groups that have used the same provider and learn how long the execution of their project took. Alongside timeline discussions, you will need to build milestone checkpoints as part of the service agreement. The importance of milestone checkpoints is to provide you (the client) with the opportunity to review the data at appropriate progress checks and terminate the programme if it is unsatisfactory. Also, one should mutually agree on the format and frequency of updates on the project and ensure that the CRO is willing to provide not only analyzed, but also raw data, so that you can independently review and assess the quality of the results. If material transfer is needed (i.e. a host cell line), a material transfer agreement (MTA) should be part of the service contract. Figure 4.1 outlines key steps involved in working with CRO on CRISPR genome editing or screen projects.
4.5 Considerations on Experimental Design and Controls Required when Outsourcing
For most experiments, CRISPR outsourcing falls into three broad categories: (i) CRISPR genetic screening; (ii) custom cell model generation; (iii) animal model generation. Animal model generation is out of scope for this chapter. For several commonly used cell lines, KO clones may already be commercially available, e.g. the human haploid HAP1 collection comprising over 7500 knockout cell lines. However, in the majority of cases, a custom edit will need to be generated. So far, we have provided a general description of the overall process of working with CROs. The remaining discussion will focus on specific aspects of the experimental design that are critical to discuss when outsourcing cell line generation or CRISPR genetic screens.
Figure 4.1 Process workflow for project externalization of genome editing to CROs.
4.5.1 Considerations on Selecting the Appropriate Cellular Host
Selecting appropriate host cells for genetic manipulation is a key aspect of experimental design and applies both to CRISPR screens and custom cell model generation. A trade‐off may need to be considered between physiological relevance (tissue origin and/or differentiation state) and technical feasibility. This is because most primary cells can only be cultured in the laboratory for a limited number of days and therefore researchers often choose immortalized cell lines as the next best alternative to conduct their research. For example, Yeung and colleagues used the THP‐1 cell line as a proxy for macrophages and performed a genome‐wide screen to identify loss‐of‐function mutations conferring resistance to Salmonella uptake (Yeung et al. 2019). Shang and colleagues used Jurkat cells as proxy for primary T cells and performed a genome‐wide screen to identify genes that regulate T cell activation upon anti‐T cell receptor (TCR) stimulation. Recently, strategies like gRNA lentiviral infection with Cas9 protein electroporation (SLICE) were developed to overcome the requirement for stably expressing Cas9 cells and have enabled genetic screens to occur in primary human cells (Shifrut et al. 2018). However, not many CROs have the ability to conduct such technically demanding types of genetic screens.
When work is to be carried out in non‐primary cells, several aspects should be considered to help you choose the right cellular host. These have been extensively discussed in a review by Kimberland and colleagues (Kimberland et al. 2018). They include information on chromosome ploidy, genetic stability, and clonality. For most cancer cell lines, multi‐omic information can already be accessed through datasets, such as the Cancer Cell Line Encyclopedia (https://portals.broadinstitute.org/ccle/about) (Ghandi et al. 2019), the Cell Model Passports portal (https://cellmodelpassports.sanger.ac.uk/) (van der Meer et al. 2019), or the Cancer Dependency Map portals (https://depmap.org/portal/ & https://depmap.sanger.ac.uk/). This information enables researchers to bioinformatically interogate their cell line of interest and make informed decisions on how similar it is to their tissue of interest. Moreover, online tools such as Cellector faciliate the selection of the most appropriate model to use based on similarity to patient samples (https://ot‐cellector.shinyapps.io/CELLector_App/). We would also recommend discussing with the CRO what assays they routinely use to validate the cell lines used in their work, and if needed draft additional testing as part of the contract research. In our experience, array comparative genomic hybridization (aCGH) plus SNP genome analysis provides a cost‐effective and quick survey of the host cell lines. aCGH detects structural and numeric chromosomal alterations, while SNP analysis allows detection of polyploidy, loss of heterozygosity, and uniparental disomy (Wiszniewska et al. 2014). Such information ensures that the genetic makeup of the cell line used matches the parental cell line.
4.5.2 Considerations on the Gene/Locus to be Edited
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