Part:BBa_K5490003

Inducible endonuclease targets RNA via gRNA, contains an HA tag

gRNAs (guide RNAs) are relatively small RNA molecules that play a crucial role in gene editing and RNA-targeting technologies. They are typically expressed under the control of a promoter, often polymerase III, which is ideal given the high complexity of the gRNA's secondary structure. These gRNAs are composed of two main components:

The scaffold or direct repeat region: This region forms a complex secondary structure after transcription, allowing it to bind effectively to an effector molecule such as CasRx, which acts as an RNA nuclease, specifically targeting single-stranded RNA. There are two types of direct repeat sequences commonly used--one that is 30 nucleotides long and another that is 36 nucleotides long. The 36-nucleotide direct repeat has been shown to have a higher affinity for CasRx, improving the overall efficiency of the RNA-targeting system.

The spacer: This is a 23-nucleotide sequence that is complementary to the target RNA, guiding CasRx to the specific RNA region for cleavage. In this case, the target is a region within the West Nile Virus (WNV) genome. The spacer sequence was selected using an algorithm designed to optimize its binding and cutting efficiency, ensuring that the CasRx-gRNA complex can cleave the target RNA with high specificity and effectiveness.

By integrating both the scaffold and spacer, researchers can achieve precise RNA cleavage. The CasRx effector dimer, guided by the gRNA, binds to the target RNA region, enabling highly efficient RNA degradation. This optimized approach allows for better control and precision in RNA-targeting applications, such as combating viral infections like WNV or studying gene expression pathways.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 499
  Illegal EcoRI site found at 919
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 499
  Illegal EcoRI site found at 919
  Illegal NotI site found at 2908
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 499
  Illegal EcoRI site found at 919
  Illegal BamHI site found at 2899
  Illegal XhoI site found at 2374
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 499
  Illegal EcoRI site found at 919
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 499
  Illegal EcoRI site found at 919
  Illegal NgoMIV site found at 187
  
• 1000
  COMPATIBLE WITH RFC[1000]