Part:BBa_K3385039

pkaR_repair_oligo

Theoretical expectation: pkaR encodes a regulatory subunit in Protein Kinase A (PKA) which is involved in stress response and regulation of morphology and growth. The gene has been characterized in Neurospora crassa and a homolog was found in A. niger.

Repair oligo for the pkaR K/O made with the pFC330 with CRISPR_pkaR_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_pkaR_KO and the repair oligo for pkaR. It shows efficient gene deletion when it's transformed with a repair oligo.

TAPE showing sgRNA efficiency.

To see if the K/O’s were successful, other than looking at macromorphology, tissue PCRs were performed. By the amplification of specific primers, upstream and downstream of the gene, it can be verified if the gene has successfully been knocked out. If it has been knocked out the primers are gonna be closer to each other resulting in a smaller band in the Tissue PCR. However if the gene is still present in the genome, the band size will be the same as the target gene as seen in the table below.



Expected length of each K/O

Targeted gene	Expected gene length after K/O	Control lenght
ΔchsC	704 bp	1867 bp
ΔpkaR	370 bp	1661 bp

Picture of the tissue PCRs performed on ΔchsC and ΔpkaR.

The results from the Tissue PCR showed that we successfully integrated the part into A. niger.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 9
  
• 1000
  COMPATIBLE 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