- The Question
- The Answer
- CRISPR Background
- Genome Editing
- Cas9 Mechanism
- Molecular Techniques
Guide RNA Binding
The Cas9 nuclease encloses the guide RNA and binds the structure with specific interactions in a number of domains. Specifically, the REC1 and REC2 domains bind the complementary region of the guide RNA, and eventually the guide RNA target DNA heteroduplex upon DNA binding. Mutations to the REC2 domain causes a small decrease in Cas9 activity, while mutations in the REC1 domain eliminate activity completely. The Rec1 domain is likely essential for Cas9 activity because it binds the repeat/anti-repeat duplex. The Protospacer Adjacent Motif (PAM) Interacting (PI) domain and RuvC nuclease domain bind the stem loops on the guide RNA. Overall, the exact process of guide RNA binding by the nuclease is unknown. However, a dramatic conformational change upon binding has been shown to produce an activated Cas9 nuclease capable of binding and cleaving specific sequences of double-stranded DNA (Nishimasu et al. 2014).
for gRNA scafold binding just REC1,REC2 and PI domain is required ?
the absence of other domain like RUVC or HNH or Helix can influence on binding of gRNA and CAS9?
As for Cas9 protein binding the guide RNA, does that mean that this complementary sequence is found in all gRNA in order for it to be recognized and bound? How does does Cas9 recognize and bind the gRNA rather than than an oligonucleotide strand floating around?
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