Part:BBa_K3385029

crRNA_racA_KO_down

Theoretical expectation: racA encodes a Rho GTPase which controls actin dynamics and it is thereby also involved in polarity regulation. The knockout mutant was expected to have a hyperbranched morphology.

This crRNA is for downstream targeting of Rho GTPase involved in regulation of cell polarity gene (racA) in A. niger. It was used in combination with BBa_K3385028, but can be used in combination with any crRNA targeting upstream 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_racA_KO and the repair oligo for racA. 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
ΔaplD	590 bp	3807 bp
ΔracA	709 bp	1920 bp

Picture of the tissue PCRs performed on ΔchsC, ΔaplD and ΔracA.

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

As seen from the results above this part has been shown to be successful and can be used for creating a 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