Part:BBa_K3402056

Single-gene editing cassette

This device is composed of 50bp-upPXA1(BBa_K3402037), hph(BBa_K3402012), 50bp-doPXA1(BBa_K3402038), Ptef1(BBa_K3402007), sgPXA1(BBa_K3402039), Tsyn7(BBa_K3402001).

Usage and Biology

We achieve the knockout of PXA1 gene and insert the hygromycin resistant gene as a selection marker. Two parts of PXA1 are homologous arms at the ends. The strongest promoter Ptef1 is used to express sgRNA to guide Cas9 protein to edit PXA1 site.
The transformants were cultured on solid YPD medium with hygromycin. Then the genomes of positive transformants extracted for PCR and gel electrophoresis analysis. As a result, all of the amplified fragments displayed the correct stripe. So all of the target genes were verified to be inserted into the PXA1 site.

Fig. 1 Comparison of the number of transformants using CRISPR/Cas9 and traditional homologous recombination

Fig. 2 Gel electrophoresis analysis of positive transformants

The single gene-editing efficiency was 100%.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 2171
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]