Название: Genome Editing in Drug Discovery
Автор: Группа авторов
Издательство: John Wiley & Sons Limited
Жанр: Биология
isbn: 9781119671398
isbn:
For many editing experiments including one‐off editing and gene KO for arrayed screening, use of Cas9/gRNA ribonucleoprotein complex (RNP) is preferred, as it has been reported to demonstrate higher efficacy as compared with other delivery formats, together with simpler protocols and reduced toxicity, because RNP pipelines do not require delivery of foreign DNA, or enrichment/selection. Moreover, use of RNP has been reported to help reduce off‐target effects (OTEs), because unlike Cas9 delivered as plasmid or lentivirus which remains expressed beyond 72hours, Cas9 introduced as RNP is cleared rapidly by 48hours (Kim et al. 2014; Liang et al. 2015). For therapeutic genome editing applications, several improved Cas9 enzymes have been described which improve on‐target editing specificity and/or reduce off‐target editing. Two of these, eSpCas9 (Slaymaker et al. 2016) and high‐fidelity (HiFi) Cas9 (Vakulskas et al. 2018) are commercially available through Merck and IDT.
4.2.3 Cas9 Alternatives
Although SpCas9 is the most popular nuclease, Cas enzymes derived from other bacterial species have been described for editing applications. Commercially available alternatives to SpCas9 include the Acidaminococcus and Lachnospiraceae Cas12a (also known as Cpf1), from IDT and NEB respectively (Zetsche et al. 2015), the Staphylococcus aureus SaCas9, from Takara Bio (Ran et al. 2015), and the Francisella novicida FnCas9 from Merck (Acharya et al. 2019). The primary difference between Cas9 nucleases derived from different bacteria is in the protospacer adjacent motif (PAM) sequence that they require for binding and cleavage. For example, SaCas9 recognizes a longer PAM, 5'‐NNGRRT‐3', compared with 5'‐NGG‐3' for SpCas9. In addition, SaCas9 is about 1 kb smaller in size than SpCas9, so it can be packaged into viral vectors more easily, offering possibilities for non‐integrative AAV‐mediated gene therapy (De Caneva et al. 2019; Ginn et al. 2020).
Cas12a has recently emerged as an interesting alternative to SpCas9, due to its ability to target T‐rich motifs with the PAM, typically 5′‐TTTV‐3′, located upstream of the spacer. This makes Cas12a attractive as an epigenome editing platform, because it can target regions around transcription starting sites, which are inaccessible to SpCas9 (Tak et al. 2017). Although its potential in human research has yet to be fully realized, Cas12a has shown remarkable versatility in genome editing across a range of model organisms, including mice, porcine (female embryos), frogs (Xenopus), zebrafish, bacteria, and plants (Safari et al. 2019).
4.2.4 Guide RNA Formats and Reagents
Guide RNA (gRNA) is an essential part of the CRISPR system, as it serves to direct the Cas nuclease to specific genomic locations defined by its complementation to the target DNA sequence of interest. In the case of SpCas9, the gRNA is composed of two parts: a) a 20‐nucleotide sequence complementary to the target DNA named CRISPR RNA (crRNA), and b) a 67‐nucleotide sequence which serves as a binding scaffold for the Cas nuclease, named trans‐activating CRISPR RNA (tracrRNA). These two essential gRNA components can be kept separate as two‐piece reagents, or manufactured as a simpler alternative that combines both the crRNA and tracrRNA elements into a chimeric single‐guide RNA molecule (sgRNA).
Guide RNA design is critical to achieving efficient gene knockout. In the first few years after the emergence of CRISPR, multiple groups studied gRNA design and found that while many gRNAs will cut on‐target with a reasonably high rate, a substantial portion will produce a low or zero cutting rate, or alternatively bind promiscuously in the genome, which can lead to off‐target mutagenesis (Fu et al. 2013; Kim et al. 2019; Wienert et al. 2019). To address these issues, research focused on identifying the sequence and structural features that contribute to effective (and ineffective) gRNAs has led to noticeable improvements to the system (Filippova et al. 2019; Wu and Yin 2019). The production and use of chemically modified gRNAs, which are more resistant to degradation by cellular RNases, is now the norm, with different providers offering their own proprietary modification solutions. Major providers of gRNAs, for bespoke or library screening applications, are listed in Table 4.1.
As in the case of the CRISPR enzymes, there is high flexibility with regard to gRNA formats, and reagents can be purchased as synthetic single‐ or two‐component gRNA for use with Cas9 RNP complexes, or within a plasmid or lentiviral vector backbone usually expressed under the control of a human U6 promoter. Determining the most appropriate type of gRNA for your experiment will depend on your particular application and cell type. As a general rule of thumb, for single‐target KOs, we recommend the RNP approach and in our hands single‐ and two‐component gRNAs appear to work equally well. Choice of format is more dependent on delivery time, sample handling automation, and cost considerations. Selecting good‐quality gRNAs is critical for the success of your CRISPR experiments and now all major commercial providers offer predesigned gRNAs (single or pools). They have spent years optimizing their designs with proprietary algorithms and extensive internal R&D testing, so their gRNAs generally work well for simple gene KOs. It is still best practice though to purchase a minimum of 4 gRNAs and test their editing efficiencies before selecting the best two or three for your actual experiment, as demonstrated by Martufi and colleagues (Martufi et al. 2019). In addition, for gene KO experiments exploring target biology, to achieve a high level of editing we would recommend a strategy described by Seki and Rutz (Seki and Rutz 2018), where a combination of two or three gRNAs against the same target are added in one transfection. The rationale behind this is that it increases the chances of knocking down protein expression efficiently, as gene editing will occur in multiple regions of the gene in addition to the indel mutations induced by each gRNA. This strategy has been adopted by Synthego and Horizon Discovery as part of their arrayed CRISPR libraries portfolio and has the added benefit of alleviating the need for testing multiple gRNAs individually. Joberty and colleagues further demonstrated that if gRNA pairs target sequences СКАЧАТЬ