Part:BBa_K1559002

rearranged CRISPR/Cas9 system without promoter

This is an engineered version of pCas9 (available in the registry as Part:BBa_K1218011) to facilitate control of CRISPR activity while keeping all essential components of the CRISPR/Cas9 (Type II CRISPR system) together in one part.

We confirmed via email with Wenyan Jiang (lead author of the paper describing pCas9) that the original pCas9 had a bidirectional promoter that constitutively expressed both the tracrRNA and Cas9 (see design page). Since the region containing the bidirectional promoter is in the middle of the pCas9 sequence, it is difficult to swap out the promoter for a different one.

We moved the tracrRNA and the bidirectional promoter region to the end, so that the Cas9 gene is now right at the start of the biobrick. This allows Cas9 to be controlled by any promoter/rbs placed upstream of this part. The expression of the tracrRNA and crRNA array are unaffected since they have not been moved relative to their original promoters.

Usage and Biology

Add a promoter and rbs upstream of this part to express Cas9. The tracrRNA and crRNA array are constitutively active. The crRNA array contains a placeholder in the spacer insertion site. Use the [http://www.addgene.org/static/cms/files/Marraffini_pCas9_protocol.pdf Marrafini pCas9 protocol] (Golden Gate Assembly) to add your own spacer.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1099
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3378
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4320
  Illegal BsaI.rc site found at 4297