کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1993233 1541235 2015 5 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Optimization of a multiplex CRISPR/Cas system for use as an antiviral therapeutic
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Optimization of a multiplex CRISPR/Cas system for use as an antiviral therapeutic
چکیده انگلیسی


• Here we detail optimization and construction multiplex CRISPR/Cas9 AAV vectors.
• We describe screening for optimal Sau Cas9 sgRNAs using a dual luciferase assay.
• We outline assembly of a final dual sgRNA:Sau Cas9 scaffold.
• Lastly, we describe construction of multiplex AAV and lentiviral vectors.

RNA-guided endonucleases or CRISPR/Cas systems have been widely employed for gene engineering/DNA editing applications, and have recently been used against a variety of dsDNA viruses as a potential therapeutic. However, in vivo delivery to specific tissue reservoirs using adeno-associated virus (AAV) vectors is problematic due to the large coding requirement for the principal effector commonly used in these applications, Streptococcus pyogenes (Spy) Cas9. Here we describe design of a minimal CRISPR/Cas system that is capable of multiplexing and can be packaged into a single AAV vector. This system consists of the small Type II Cas9 protein from Staphylococcus aureus (Sau) driven by a truncated CMV promoter/enhancer, and flanked 3′ by a poly(A) addition signal, as well as two sgRNA expression cassettes driven by either U6 or ∼70-bp tRNA-derived Pol III promoters. Specific protocols for construction of these AAV vector scaffolds, shuttle cloning of their contents into AAV and lentiviral backbones, and a quantitative luciferase assay capable of screening for optimal sgRNAs, are detailed. These protocols can facilitate construction of AAV vectors that have optimal multiplexed sgRNA expression and function. These will have potential utility in multiplex applications, including in antiviral therapy in tissues chronically infected with a pathogenic DNA virus.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Methods - Volume 91, December 2015, Pages 82–86
نویسندگان
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