LentiCRISPR library partnership:
Desktop Genetics, Vectalys and Horizon Discovery Group plc (LSE: HZD), have entered into a collaboration agreement for the design, the production and the commercialization of custom-made lentiCRISPR gRNA libraries.
The partnership provides our customers with a fully licensed custom-made CRISPR library, by combining know-how and intellectual property portfolios of the three companies.
1. What is a CRISPR library ?
The production of a CRISPR library is based on the cloning of hundreds or thousands gRNAs, targeted toward a panel of genes of interest. Target cells are then transduced or transfected, with the library, to generate genetically engineered cells which are then screened and analyzed.
2. Applications of CRISPR libraries
The CRISPR library tool facilitates functional genomics screening for gene or target discoveries. There are various applications for this recent system:
3. What we offer: LentiCRISPR libraries
LentiCRISPR libraries are designed, cloned and packaged into highly pure and concentrated lentiviral particles. After this, cells are transduced by the customer with the library in order to generate mutant cell lines. After experimentations, Desktop Genetics can support the customer for a comprehensive data analysis.
3.1 Custom library design
Using its own powerful algorithm, Desktop Genetics can identify and design guides with the highest on-target activity and the best off-target prediction. This allows Desktop Genetics to provide scientists with the most appropriate guides for specific applications. High specificity of the guides allows to save time and money thanks to a lower number of guides needed per gene of interest than with a low specificity design.
3.2 Custom library manufacturing
Once Desktop Genetics has designed your guides, Vectalys will package them inside lentiviral particles.
3.2.1 Pooled or arrayed format
With this new offer, Vectalys can provide scientists with ready-to-use libraries in arrayed or pooled format for low- or high-throughput screening. Vectalys will be assisting the customer in technical choices related to lentiviral particles (purification/concentration, titer, and options).
Custom lentiCRISPR Libraries: 2 offers, depending on your research application:
The lentiCRISPR libraries in an arrayed format are ideal to identify the function of hundreds of genes inside a subfamily or a specific pathway. Vectalys manufactures customized lentiCRISPR libraries in arrayed format for low-throughput screening of permissive immortalized cell lines only.
Quality control and process for the arrayed format:
The lentiCRISPR libraries in pooled format are ideal to identify the function of a large number of genes when studying, for example, a subfamily of genes or the whole genome. Vectalys manufactures flexible volumes (starting from 1 ml) of lentiviral vector batches carrying the number of guides you need, with various possible concentrations according to your target cells.
With over 10 years of know-how in lentiviral vectors production, Vectalys is able to provide highly pure and highly concentrated lentiCRISPR libraries to transduce with high efficiency any type of cells. Note that the process of production and the quality of the lentiCRISPR vectors in pooled format are the same as for our classic lentiviral vectors.
For High-throughput screening, Vectalys has shown that it is possible to transduce cells at low MOI to obtain one sgRNA per cell for gene functions studies. Vectalys can advise the customer to find the best transduction conditions to reach this sgRNA per cell ratio.
Quality control and process for pooled format:
Note: the cloning process is calibrated to maintain the library's complexity, but the only way to ensure that the guide complexity is maintained in the library is the NGS control. This technology is available on demand and is performed on the final plasmid library before lentiCRISPR vector production.
3.2.2 Fully customizable gRNA libraries
For a custom lentiCRISPR gRNA library in a pooled or arrayed format, the customer can choose between two options:
- All in one construct (gRNA guides + Cas9, Tet inducible or not, + a reporter or an antibiotic resistance gene of choice)
- Two transfer constructs (Cas9, Tet inducible or not, to generate Cas9 cell lines + a reporter or an antibiotic resistance gene of choice) + (gRNA guides + a reporter or an antibiotic resistance gene of choice)
One transfer construct
The first option is the design of one transfer construct including one guide, Cas9 and several added custom options on the same sequence:
- Cas9 under a Tet-on 3G promoter: recommended option to avoid off-target effect of constitutive Cas9 on your cells of interest (Cao et al., 2016).
- A fluorescent reporter or antibiotic selection with a polycistronic system (IRES, 2A peptide) for a modified cell selection.
Note: the large size of this type of construct causes low titers in crude harvests, which can be compensated by further concentration.
Two transfer constructs
The second option is the design of two transfer constructs. The first lentiviral particle carries the Cas9 under a constitutive or a Tet-inducible promoter for the generation of a Cas9 expressing cell line, prior to sgRNA transductions. Note that a fluorescent reporter or an antibiotic resistance gene can be added. The second lentiviral particle will carry the sgRNA guides, with several options as well: a H1 or U6 promoter, a fluorescent reporter or an antibiotic resistance gene.
3.3 Screening and analysis
After screening experiments, Desktop Genetics can analyze sequencing data to provide a comprehensive post-experimental analysis.
4. Who does what ?
5. Intellectual property
Combined, the collaboration provides our customers with IP rights for research use of third generation lentiviral vectors, guides and the CRISPR-Cas9 technology.