By Magnus Lundgren, Emmanuelle Charpentier, Peter C. Fineran
This quantity provides a listing of state-of-the-art protocols for the examine of CRISPR-Cas protection structures and their purposes on the genomic, genetic, biochemical and structural degrees. CRISPR: equipment and Protocols publications readers via innovations which have been built particularly for the research of CRISPR-Cas and strategies tailored from normal protocols of DNA, RNA and protein biology. Written within the hugely winning Methods in Molecular Biology series structure, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, easily reproducible laboratory protocols, and pointers on troubleshooting and fending off recognized pitfalls.
Authoritative and cutting-edge,CRISPR: equipment and Protocols offers a wide checklist of instruments and strategies to review the interdisciplinary facets of the prokaryotic CRISPR-Cas security systems.
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Extra resources for CRISPR: Methods and Protocols
4 Notes 1. This protocol is applicable to type I and III Cas endoribonucleases: any of the Cas6 enzymes (including Cas6f and Cas6e) as well as Cas5d. 2. We use a synthetic RNA modeled on the full-length CRISPR repeat. 3. To radiolabel RNA see ref. 28. 4. The pre-CRISPR RNA used in this protocol can be produced by chemical synthesis or in vitro transcription using T7 RNA polymerase. We use synthetic RNA (Integrated DNA Technologies) which comes bearing a free 5′ hydroxyl. In vitro-transcribed RNA must first be treated with alkaline phosphatase (Invitrogen) before 5′ radiolabeling.
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E) Processing of the model pre-crRNA substrate is inhibited by a deoxyG at position 26 consistent with the CRISPR endoribonuclease cleavage mechanism. (f) Proposed mechanism of pre-crRNA processing by the type I and III Cas endoribonucleases. The 2′ OH of the nucleotide 5′ to the cleavage site attacks the scissile phosphate generating cleavage products with a 2′–3′ cyclic phosphate on the 5′ cleavage product and a free 5′ hydroxyl on the 3′ cleavage product Cas5d-dependent cleavage of the RNA (Fig.