Genome Engineering for Crop Improvement. Группа авторов

Чтение книги онлайн.

Читать онлайн книгу Genome Engineering for Crop Improvement - Группа авторов страница 32

Genome Engineering for Crop Improvement - Группа авторов

Скачать книгу

(tracrRNA) to activate and guide Cas9 nuclease (Barrangou et al. 2007). The Cas9 cleaves the homologs double‐stranded DNA sequence (protospacer) in the foreign DNA. The availability of PAM downstream of the target DNA is a key for the successful cleavage with frequent 5′‐NGG‐3′ and less frequent 5′‐NAG‐3′ (Hsu et al. 2013). Seed sequence approximately 12 bp upstream of the PAM are integral for pairing between RNA and target DNA (Bortesi and Fischer 2015). Multiplex editing by the introduction of double‐stranded breaks (DSBs) on various sites and ability to edit several genes at the same time is a unique feature of CRISPR/Cas system (Zhou et al. 2014). It only requires monomeric Cas9 protein with sequence‐specific gRNA meanwhile, ZFNs and TALENs requires dimeric proteins with reference to target site. In the CRISPR/Cas system, the recognition of target site is based on the Watson and Crick model enabling the off‐target detection through sequencing analysis along with off‐target mutation can be fixed by careful design of gRNA. On the contrary, ZFNs and TALENs target specificity depends on protein‐DNA interaction with unpredictable and function‐specific properties (Cho et al. 2014). Moreover, the scientific community equipped with CRISPR expertise had contributed a lot to disseminate the fresh information to newcomers which is opposite to ZFN proprietary nature. Several online platforms are now available and are assisting researchers for all concerns related to CRISPR (Table 3.1; Figure 3.1).

      Source: Extracted from (Bortesi and Fischer 2015; Chira et al. 2017).

sgRNA design platform Webpage link References
CRISPR Design http://crispr.mit.edu Hsu et al. (2013)
E‐CRISP http://www.e‐crisp.org/E‐CRISP Heigwer et al. (2014)
CRISPR Multi Targeter http://www.multicrispr.net/basic_input.html Prykhozhij et al. (2015)
sgRNA designer: CRISPRko http://portals.broadinstitute.org/gpp/public/analysis‐tools/sgrna‐design Doench et al. (2016)
Off‐Spoter https://cm.jefferson.edu/Off‐Spotter Pliatsika and Rigoutsos (2015)
CCTop http://crispr.cos.uni‐heidelberg.de/index.html Stemmer et al. (2015)
CHOPCHOP http://chopchop.cbu.uib.no/index.php Montague et al. (2014)
Plasmid free access
Add gene https://www.addgene.org/search/advanced Bortesi and Fischer (2015)
Expert discussion forum
OXfCRISPR https://www.dpag.ox.ac.uk/research/liu‐group/liu‐group news/oxfcrispr/ Bortesi and Fischer (2015)

Скачать книгу