Part:BBa_K3402059
Cas9 expression cassette
This device is composed of upSBLE (BBa_K3402030), Ptef1 (BBa_K3402007), Cas9 (BBa_K3402023), NLS (BBa_K3402008), Tsyn7 (BBa_K3402001) and doSBLE (BBa_K3402031).
Usage and Biology
The homologous arms were designed for integrating the Cas9 gene into the genome of S. Bombicola. The use of the strongest promoter Ptef1 to express Cas9 protein and sgRNA could increase the gene-editing efficiency. As the Cas9 protein was too big to transport into the nucleus, a nuclear localization sequence (NLS) was needed to help the transportation of Cas9 protein.
The Cas9 expression cassette was fused to the self-excising hygromycin marker cassette to obtain the recombinant vector. After linearization, it was electroporated into wild-type Starmerella bombicola. The positive transformants were induced by galactose to eject hygromycin resistance gene. Then the recombinant strain with Cas9 gene and hygromycin resistance gene deletion was carried on the verification experiment of single, double and triple gene-editing efficiency.
We have built the Single-gene editing cassette (BBa_K3402056), Double-gene editing cassette (BBa_K3402057) and Triple-gene editing cassette (BBa_K3402058).
The single gene-editing efficiency is 100%. The double gene-editing efficiency is 99%. The triple gene-editing efficiency is 30%.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2078
Illegal BglII site found at 5642
Illegal XhoI site found at 1399
Illegal XhoI site found at 2128
Illegal XhoI site found at 2491
Illegal XhoI site found at 5443 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2905
Illegal NgoMIV site found at 4009 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1849
Illegal BsaI site found at 2566
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