Genome Editing in Drug Discovery. Группа авторов
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4.3.2 Considerations for Practice of in vivo CRISPR Screening
There are practical considerations to bear in mind with regard to reagents and materials for the design and execution of in vivo CRISPR screens. Due to the limited number of cells that can be injected into each animal, it is challenging to perform genome‐wide In vivo screens with appropriate library coverage. Therefore, most researchers have chosen to use a focused mini‐pool gRNA library to ensure high screen quality. While off‐the‐shelf sub‐pool gRNA libraries are available from major commercial vendors, custom‐designed gRNA libraries can be constructed by CROs like Merck, Horizon Discovery, and GenScript. To make your own gRNA libraries, Twist Bioscience, CustomArray (now part of GenScript), Agilent, and IDT are the major suppliers for oligonucleotide library synthesis. It is easier to conduct pooled in vivo CRISPR screens using cells from transgenic mice constitutively expressing Cas9, as this bypasses the need for endonuclease delivery/selection into target cells. Several different Cas9 knock‐in mice are available from The Jackson Laboratory, but licenses for research use must be obtained from the original inventors for industry/biotech institutions. T cell receptor transgenic mice from The Jackson Laboratory and other suppliers are commonly used for adoptive T cell transfer experiments, which allow for T‐cell‐based in vivo CRISPR screening. Major CROs that offer in vivo genetic screens are included in Table 4.2.
Table 4.2 Genome editing service providers.
Company | Location | Technologies | Services |
---|---|---|---|
Applied Stem Cell | Milpitas, CA | CRISPR/Cas9 | Cell engineering: cell lines, ES/iPSCs |
ATCC | Manassas, VA | CRISPR/Cas9 | Cell engineering: cell lines |
BioGene | Shirley, NY | CRISPR/Cas9 | Cell engineering: cell lines, iPSCs, primary cells; rodent models; plants models |
Canopy Biosciences | St. Louis, MO | CRISPR/Cas9 | Cell engineering: cell lines, ES/iPSCs |
Cellecta | Mountain View, CA | CRISPR/Cas9 | genetic screening |
Cellular Dynamics International | Madison Wisconsin | CRISPR/Cas9 | ES/iPSC engineering |
Charles River Laboratories | Wilmington, MA | CRISPR/Cas9 | Cell engineering; rodent models |
Crown Bioscience | Santa Clara, CA | CRISPR/Cas9 | Rodent models |
DefiniGEN | Cambridge, UK | CRISPR/Cas9 | iPSC engineering |
Evotec | Hamburg, Germany | CRISPR/Cas9 | In vitro genetic screening |
genOway | Lyon, France | CRISPR/Cas9 | Cell engineering: cell lines, rodent models |
GenScript | Jiangning, China | CRISPR/Cas9 | Bacterial engineering; cell line engineering |
Horizon Discovery | Cambridge, United Kingdom | CRISPR/Cas9 | Cell engineering: cell lines, ES/iPSCs; in vitro and in vivo genetic screening |
Merck KGaA | Darmstadt, Germany | CRISPR/Cas9, ZFN | Cell engineering: cell lines, iPSCs, primary cells |
Oxgene | Oxford, United Kingdom | CRISPR/Cas9 | Cell line engineering |
Synthego | Redwood City, CA | CRISPR/Cas9 | Cell engineering: cell lines, iPSCs, primary cells; in vitro genetic screening |
System Biosciences | Palo Alto, CA | CRISPR/Cas9 | Cell line engineering |
ThermoFisher | Carlsbad, CA | CRISPR/Cas9, TALEN | Cell line engineering |
Ubigene | Guangzhou Science City, China | CRISPR/Cas9 | Bacterial engineering, fungal engineering, cell line engineering |
WuXi AppTec | Shanghai, China | CRISPR/Cas9 | Rodent models; in vitro genetic screening |
4.3.3 Next‐Generation in vivo CRISPR Screening
The CRISPR toolkit for genome editing has been rapidly evolving and next‐generation CRISPR technologies have been applied for genetic screening in vivo. For example, in vivo screens based on CRISPRa (Braun et al. 2016; Ebright et al. 2020), CRISPRi (Li et al. 2020), exon excision (Thomas et al. 2020), and Cas12a/Cpf1‐mediated double gene knockout (Chow et al. 2019) have been reported. Most recently, pooled in vivo CRISPR screening has been combined with single‐cell RNA‐seq analysis so that the relationships between genotypes to in vivo phenotypes can be mapped at single‐cell resolution (Giladi et al. 2018; Jaitin et al. 2016). We anticipate this approach to be coupled with diverse functional readouts across a broad spectrum of animal models and continue to transform biomedical research and drug discovery.