Genome Editing in Drug Discovery. Группа авторов

repair (HDR) of DSB, and transcriptional or epigenetic regulation via fusion of an inactivated CRISPR Cas protein (“endonuclease dead” dCas9) with a transcriptional activator (CRISPR activation, CRISPRa) for upregulation of gene expression, with a transcriptional repressor (CRISPR interference, CRISPRi) for downregulation of gene expression, or with an epigenetic modifying enzyme (CRISPRepi) for DNA methylation or post‐translational modifications of histone proteins. While these platforms were largely developed by academic labs, their rapid implementation in biomedical and pharmaceutical research settings has been greatly facilitated by reagent providers and contract research organizations (CROs) that offer state‐of‐the‐art reagents and contract services. In fact, it is now routine practice for scientists to use commercially available CRISPR reagents for in‐house studies and/or to consider working with a CRO on a specific genome editing project. This article aims to summarize our learnings and provide guidance on the selection of appropriate reagents and/or CROs for CRISPR‐based studies.

4.2 CRISPR Resources and Reagents for Bespoke Editing and Genetic Screening

      4.2.1 Publicly Available Resources

      CRISPR technologies have been quoted as being the “Swiss army knife” equivalent for the science world, due to the ease of their design and generation – as compared with zinc‐finger nucleases (ZFNs) and transcription activator‐like effector nucleases (TALENS) – and the diversity of their applications (Doench 2018; Mans et al. 2015). Furthermore, the “open‐source” culture of the CRISPR field has helped accelerate its continued innovation (Zhang 2019). This has been facilitated by numerous resources like web‐based tutorials, publicly available webinars, databases for data deposition, and annual CRISPR meetings. For example, a freely available resource called the Open Repository for CRISPR Screens (ORCS) (https://orcs.thebiogrid.org) was developed by the Biological General Repository for Interaction Datasets (BioGRID) (Oughtred et al. 2019), which enables researchers to search, filter, and download CRISPR screen datasets. Version 1.0.3 of the ORCS contains 895 CRISPR screens from 3 major model organism species and 629 cell lines. In addition, when novel methodologies are published, the associated constructs often become available through repositories like Addgene, an international nonprofit repository (Kamens 2015), to which over 350 labs have contributed. Reagent sharing has the advantage that it enables vigorous testing of the new technology and encourages rapid further improvements. However, Addgene is not accessible to for‐profit organizations; thus, for industry/biotech institutions, the only access to reagents is via commercial providers.

There are many commercial providers of CRISPR reagents and major suppliers will be discussed further below. However, it is important to highlight that for pharma and biotech companies, before any work can begin and reagents purchased, it is important to ensure that there is appropriate product use license agreement to allow freedom to operate. In the case of CRISPR, this poses challenges as the foundational CRISPR patents are still under dispute for the United States and Europe with Jennifer Doudna of the University of California, Berkeley, and Emmanuelle Charpentier (formerly of the University of Vienna) competing for patent rights against Feng Zhang of the Broad Institute of MIT and Harvard University (Cohen 2019). Moreover, according to a recent patent review, beyond the foundational patents, 2072 additional patent families associated with CRISPR were filed up to a 31 December 2017 priority date (Martin‐Laffon et al. 2019).

      4.2.2 Cas9 Enzymes

Commercial providers have developed tools and solutions for every step of the CRISPR genome editing workflow and these are listed in Table 4.1. The most popular form of editing involves use of the Streptococcus pyogenes Cas9 (SpCas9) enzyme, which can be delivered to cells in one of five formats: (i) as protein (in complex with gRNA), (ii) expressed through a plasmid expression vector, (iii) via lentiviral particles, (iv) via AAV particles, or (v) via mRNA. The format of choice depends on the cell type and type of experiment that is to be performed. For example, before conducting screening experiments in cancer cell lines, one needs to generate cells stably expressing Cas9. In this case, Cas9 lentiviral particles are the format of choice, as they facilitate Cas9 integration into the genome, which allows for stable, long‐term expression of Cas9 driven by promoter sequences contained within the vector. Multiple lentiviral construct options are available. For example, Horizon Discovery offers the choice of CMV, EF1a, PGK, and CAG promoters. Also, Merck offers a range of antibiotic resistance or fluorescent reporters in their lentiviruses (blasticidin, hygromycin, neomycin, zeocin, or GFP/RFP fluorophores). Other providers like Takara Bio offer the choice between constitutive or Tet‐inducible Cas9 expression. Therefore, the choice of vendor may depend on the type of construct that best suits your experimental design. In addition, if the desired construct is not available off‐the‐shelf, several companies offer custom vector generation (Table 4.1), thus there is a lot of flexibility. However, there is a trade‐off. Off‐the‐shelf products arrive quickly and tend to be less expensive, whereas custom reagents need time for production and will typically cost more.

Table 4.1 Major providers of CRISPR reagents.

Company	Location	Reagents
Agilent	Santa Clara, CA	Oligo library synthesis; synthetic gRNA and libraries
Cellecta	Mountain View, CA	Cas9 and gRNA expression vectors, lentiviral gRNA libraries
GenScript	Jiangning, China	Cas9 nuclease; Cas9 and gRNA expression vectors; lentiviral gRNA libraries
Horizon Discovery	Cambridge, United Kingdom	Cas9 nucleases; Cas9 and gRNA expression vectors; synthetic, plasmid, and lentiviral gRNA and libraries
Integrated DNA Technologies	Coralville, IA	Oligo library synthesis; Cas9 nucleases; Cpf1 nuclease; Cas9 and gRNA expression vectors; synthetic gRNA libraries
Merck KGaA	Darmstadt, Germany	Cas9 nucleases; Cas9 and gRNA expression vectors; synthetic, plasmid, and lentiviral gRNA and libraries
New England Biolabs	Ipswich, MA	Cas9 nucleases
Oxgene	Oxford, United Kingdom	Plasmid and lentiviral gRNA libraries
Synthego	Redwood City, CA	Synthetic gRNA and libraries
System Biosciences	Palo Alto, CA	Cas9 and gRNA expression vectors
Takara Bio	Mountain View, CA	Cas9 nucleases; Cas9 and gRNA expression vectors; synthetic, plasmid, and lentiviral gRNA and libraries
ThermoFisher	Carlsbad, CA	Cas9 nucleases; Cas9 and gRNA expression vectors; synthetic and lentiviral gRNA and libraries
Transomic Technologies	Huntsville, AL	Cas9 and gRNA expression Скачать книгу В начало < 27 28 29 30 31 32 33 34 35 36 > В конец