Part:BBa_K3385036

crRNA_pkaR_KO_up

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.

This crRNA is for upstream targeting of the regulatory subunit (repressor) of the cAPM-dependent protein kinase gene (pkaR) in A. niger. It was used in combination with BBa_K3385037, but can be used in combination with any crRNA targeting downstream this target site.

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
  COMPATIBLE WITH RFC[25]
• 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