Part:BBa_K3385027
arfA_repair_oligo
Theoretical explanation: arfA encodes the Putitative ADP ribosylation factor involved in hyphal growth and secretion. It's an essential gene and therefore the fungus should become unviable when knocking it out.
Repair oligo for the genomic arfA K/O made with the pFC330 with CRISPR_arfA_KO integrated. This part fits the regions just outside the cutting areas, for the repair of the DNA.
Functionality: The sgRNA efficiency has been accessed through the technique to assess protospacer efficiency (TAPE) [2]. A repair oligo is used to mediate homologous recombination, where a highly efficient sgRNA will show no colonies without the repair oligo, while less efficient sgRNA will show a reduced number of colonies.
Results: Below is a picture showing A. niger transformed with CRISPR_arfA_KO and the repair oligo for arfA. It shows efficient gene deletion when it's transformed with a repair oligo.
Summary: The fungi transformed with this part became unviable and did not grow, which was expected as it encodes an essential gene.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References:
[1] Efficient Oligo nucleotide mediated CRISPR-Cas9 Gene Editing in Aspergilli. Nodvig CS, Hoof JB, Kogle ME, Jarczynska ZD, Lehmbeck J, Klitgaard DK, Mortensen UH. Fungal Genet Biol. 2018 Jan 8. pii: S1087-1845(18)30004-5. doi: 10.1016/j.fgb.2018.01.004. 10.1016/j.fgb.2018.01.004 PubMed 29325827
[2] Efficient Oligo nucleotide mediated CRISPR-Cas9 Gene Editing in Aspergilli. Nodvig CS, Hoof JB, Kogle ME, Jarczynska ZD, Lehmbeck J, Klitgaard DK, Mortensen UH. Fungal Genet Biol. 2018 Jan 8. pii: S1087-1845(18)30004-5. doi: 10.1016/j.fgb.2018.01.004. 10.1016/j.fgb.2018.01.004 PubMed 29325827
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