Part:BBa_K1699002

gRNA for SaCas9 targeting human ubiquitin B gene

Ribozyme flanked gRNA compatible for SaCas9. gRNA sequence complements 3 different loci in the second exon of human UBB gene. It has a hammerhead ribozyme on its 5' and an HDV ribozyme on its 3' end. Upon transcription the ribozymes should cleave the mRNA at specific locations to release the gRNA (6).

Usage and Biology

guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds Cas9 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. gRNA scaffold sequence for SaCas9 was used (1). In order to utilize the cancer specific promoter hyperactivation we used an RGR (Ribozyme gRNA Ribozyme). This design allows for gRNAs to be transcribed and processed using RNA polymerase II promoters, since these are the main promoters controlling gene activation (2).

Characterization

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 146
  Illegal NgoMIV site found at 175
  
• 1000
  COMPATIBLE WITH RFC[1000]