Part:BBa_K3385019

spaA_repair_oligo

Theoretical expectation: It has been suggested that spaA encodes a molecule which is involved in polarity maintenance and thereby polar growth. The mutant was expected to have a dense hyperbranched morphology.

Repair oligo for the genomic spaA K/O made with the pFC330 with CRISPR_spaA_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_spaA_KO and the repair oligo for spaA. 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
ΔspaA	672 bp	3528 bp
Δgul-1	545 bp	5022 bp
ΔpkaR	370 bp	1661 bp

Picture of the tissue PCRs performed on ΔspaA, Δgul-1 and ΔpkaR.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 82
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 82
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 82
  
• 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