Part:BBa_K3385034

CRISPR_aplD_KO

Theoretical expectation: aplD encodes a gamma-adaptin involved in endosomal cargo transport and affects filamentous growth and pellet formation. Deletion of the gene was expected to result in a hyperbranched morphology.

Full guide construct for knockout of the transport involved protein aplD in A. niger. This BioBlock has to be cloned into the PacI/Nt.BbvCI digested pFC330 backbone.

Plasmid map of pFC330[1].

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_aplD_KO and the repair oligo for aplD. 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.

Summary: From the different experiments it can be assumed that ΔaplD plays an important role in proper growth. Therefore, the ΔaplD mutant is not viable.