Part:BBa_K5096400

pCas9

The pCas9 plasmid encodes the pCas9 protein, which is used in CRISPR. Cas9 derived from Streptococcus pyogenes. Cas9 does have nuclease activity, therefore it can cut the DNA. It does this by binding to single-guide RNA (sgRNA) to form a complex, effectively knocking down the gene targeted by the sgRNA. This part was originally developed by Marshall et al. (2018).

Cas9 and dCas9 were tested to compare effectiveness in repression. Cas9 was used in CRIPSR, and dCas9 was used in CRISPR interference. Although CRISPR resulted in lower fluorescence, indicating stronger repression, both CRISPR and CRISPRi showed successful outcomes when simulated in silica. The CRISPR reaction simulation predicted around 80-90% repression of deGFP, while CRISPRi resulted in around 60% downregulation. These results demonstrate that while CRISPR achieved greater repression than CRISPRi, both surpassed the 50% threshold for effective GFP repression in the simulation. Lambert iGEM decided to run a CRISPR reaction using pCas9 protein to compare the rates of repression between gene knockdown and knockout. We tested our CRISPR vs. CRISPRi reactions by following the same protocol as our CRISPRi reaction and utilized pCas9 with sgRNA9 to repress and cut the P70a-deGFP . Although the CRISPR reaction resulted in lower fluorescence, indicating greater repression, both CRISPR and CRISPRi yielded successful results. The CRISPR reaction achieved 80.0% repression of deGFP, while CRISPRi achieved 59.8% downregulation.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3478
  Illegal EcoRI site found at 7442
  Illegal XbaI site found at 1292
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3478
  Illegal EcoRI site found at 7442
  Illegal NheI site found at 3237
  Illegal NheI site found at 6832
  Illegal NheI site found at 8024
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3478
  Illegal EcoRI site found at 7442
  Illegal BamHI site found at 5516
  Illegal BamHI site found at 6978
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3478
  Illegal EcoRI site found at 7442
  Illegal XbaI site found at 1292
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3478
  Illegal EcoRI site found at 7442
  Illegal XbaI site found at 1292
  Illegal AgeI site found at 8110
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 6571
  Illegal BsaI.rc site found at 6548