Part:BBa_K3738022

Lbu-Cas13a with an N-Terminal 6xHistidine Tag and C-Terminal Anionic Tag

Lbu-crRNA (Lbu signifying Leptotrichia buccalis, the source of the Cas13a protein and crRNA) is an RNA structure containing a direct-repeat stem loop, a recognition element for binding with the Lbu-Cas13a protein (see parts BBA_K3738020 and BBA_K3738019). The crRNA also contains a downstream complementary region, designed to base-pair with a target ssRNA sequence. Complex formation of crRNA-Cas13a occurs, and when the target sequence is perfectly paired with the crRNA, activation of the enzyme occurs and subsequent non-discriminate cleavage of collateral ssRNAs (O’Connell., 2019).

This crRNA was designed to bind with an important protein in the synthesis of harmful toxins called microcystins produced by blue-green algae (cyanobacteria) blooms. McyH is a gene in the Mcy gene cluster of Microcystic Aeruginosa and codes for a transporter protein. The protein is comprised of two homodimers each with a hydrophobic N-terminus domain and C-terminus containing an ATPase domain. Pearson et al., 2004 experimentally examined the impacts of impairing expression of this gene, and combined with bioinformatic data, found that McyH is likely a vital exporter of harmful microcystins and essential in their biosynthetic pathway. For this reason, we have designed crRNA to target the 5’-end of McyH in order to inhibit production of microcystins.

O'Connell MR. Molecular Mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR–Cas Systems. Journal of Molecular Biology. 2019;431(1):66-87. doi: https://doi.org/10.1016/j.jmb.2018.06.029.

Pearson LA, Hisbergues M, Börner T, Dittmann E, Neilan BA. Inactivation of an ABC transporter gene, mcyH, results in loss of microcystin production in the cyanobacterium Microcystis aeruginosa PCC 7806. Appl Environ Microbiol. 2004;70(11):6370-8. Epub 2004/11/06. doi: 10.1128/aem.70.11.6370-6378.2004. PubMed PMID: 15528494; PubMed Central PMCID: PMCPMC525210.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 7
  Illegal EcoRI site found at 868
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 7
  Illegal EcoRI site found at 868
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 7
  Illegal EcoRI site found at 868
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 7
  Illegal EcoRI site found at 868
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 7
  Illegal EcoRI site found at 868
  
• 1000
  COMPATIBLE WITH RFC[1000]