Part:BBa_K3385023

gul1_repair_oligo

Theoretical expectation: gul-1 was found in literature about the fungi Neurospora crassa where it encodes an mRNA binding protein involved in remodeling of the cell wall. A homolog was found in A. niger. The expected morphology of the Δgul-1 mutant was hyperbranched pellets.

Repair oligo for the genomic gul-1 K/O made with the pFC330 with CRISPR_glaA_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_gul-1_KO and the repair oligo for gul-1. 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.

As seen from the results above this part has been shown to be successful and can be used for creating the gul-1 knock-out.

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