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Chapters
- 00:05Title
- 01:37CRISPR/Cas System Overview
- 02:29Generation of Long Pre-crRNA Substrates via PCR
- 04:14Generation of Intermediate Pre-cdRNA Substrates via Annealing of DNA Oligonucleotides
- 06:10Generation of Short Cas RNA Substrates via Custom RNA Oligonucleotide Synthesis
- 06:41In vitro T7 RNA Polymerase Transcription
- 08:04Cas6 Endonuclease Assay
- 09:08Results: Representative Cas6 Endonuclease Assay
- 11:10Conclusion
CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.
Tags
Substrate GenerationEndonucleasesCRISPR/Cas SystemsVirusesProkaryotic HostsBacterial EvolutionArchaeal LifeRestriction ModificationAbortive InfectionAdaptive Immune SystemsCRISPR SequencesCas GenesCas ProteinsSpacer ElementCRISPR ClusterPrecursor-crRNACas6 FamilyCrRNAsViral DNA UptakeCascade ComplexBase ComplementarityNuclease Cas3Invader DNA Destruction
