Part:BBa_K4054002

Cas9
Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system.

Description

The key to cleavage is the presence of a primordial spacer adjacent motif (PAM) downstream of the target region. Cas9 is a double-stranded (DS)DNA endonuclease that uses crRNA guidance to determine the cleavage site. During the processing of the crRNA precursor, acrRNA guide is loaded on Cas9, requiring the precursor (tracrRNA) and a small RNA antisense of RNAse III.

Experience

In the project ofXJTLU-CHINA this year, thwy used Cas9(Csn1) for phage edition. Two plasmid contains crRNA and Cas9 coding gene ere used respectively to improve the efficiency of CRISPR Cas9 system. They detect the cleavage efficiency by qPCR, as they insert the target gene into plasmid.By compare the expression level in the cell, it is varified that the Cas9 and our crRNA3 could work well in the E.coli.

Figure 1. The gp23 expression level compare between the control and the group contains Cas9 gene

Just as the Figure shows, it is assumed that the transcription efficiency of gp23 without cleavage of Cas9 was 1. In the engeering bacteria, the expression effiency was repressed to a low level, decreased to around 30% of the common efficiency.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1340
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1340
  Illegal NheI site found at 1099
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1340
  Illegal BamHI site found at 3378
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1340
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1340
  
• 1000
  COMPATIBLE WITH RFC[1000]