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 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 system:
3. What we offer: LentiCRISPR libraries
We offer the packaging of your CRISPR libraries into our lentiviral vectors. LentiCRISPR libraries are packaged into highly pure and concentrated lentiviral particles, either from an existing lentiviral plasmid library, or from a custom plasmid library that we construct starting from a provided oligonucleotide pool, corresponding to your guide sequences. The generation of mutant cell lines by transduction is then the responsibility of the customers.
4. Pooled or arrayed format
We can provide scientists with lentiviral vectors libraries in arrayed or pooled format for low- or high-throughput screening. We will be assisting you in technical choices related to lentiviral particles (purification/concentration, titer, and options).
Custom lentiCRISPR Libraries: 2 formats, 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. We manufactures customized lentiviral vector batches carrying your 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. We manufacture 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 12 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, We have shown that it is possible to transduce cells at low MOI to obtain one sgRNA per cell for gene functions studies. We can help you 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 fully maintained in the plasmid library is to perform a NGS sequencing. To this end, a sample of the final plasmid library can be provided to the customer or to the NGS provider of its choice, before lentiCRISPR vector production.
5. Fully customizable lentiviral vectors carrying 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.
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.
- Want to start a crispr library project ? Please contact us.