Part:BBa_K4335012

pCrU6.3

pCrU6.3 is a previously characterized RNA polymerase III promoter from the chromosome 8 of Chlamydomonas reinhardtii

The first prerequisite for CRISPR/Cas9 experiments is sgRNA transcription under the control of the RNA polymerase III promoter (RNAPII) . In general, the RNAPII transcription factor binding site is intrinsic to the transcriptional sequence and therefore not amenable to sgRNA transcription. U6snRNA is one of the few examples of sgRNAs with proximal RNAPII promoter elements. Therefore, we used previously characterized U6snRNA sequences from chromosome 8.

Usage

pCrU6.3 was constructed into plasmid pTX2038 and pTX2040 as the promoter of gRNA, and was successfully introduced into Chlamydomonas reinhardtii to obtain the positive transformant.

Result

We selected PSY1 gene as the target gene to detect the sgRNA transcription driven by U6.3 promoter according to the method of { ref. 2} . The plant endo synthase gene (PSY1) is involved in chlorophyll synthesis, and disruption of PSY1 produces white colonies that are easy to detect and count.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 48
  Illegal PstI site found at 121
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 48
  Illegal PstI site found at 121
  Illegal NotI site found at 453
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 48
  Illegal PstI site found at 121
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 48
  Illegal PstI site found at 121
  
• 1000
  COMPATIBLE WITH RFC[1000]