Part:BBa_K3385074

NLS

About the part: The part was obtained as part of the CRISPR cloning vector pFC330[1]. This is a nuclear localization signal used to ensure that Cas9 is guided to the nucleus.

Plasmid map of pFC330[1].

Functionality: The sgRNA efficiency has been assessed through the technique to assess protospacer efficiency (TAPE) [1]. 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 the transformed A. niger. It shows efficient gene deletion when it is transformed with a repair oligo, while the lack of the repair oligo renders the fungus unable to repair the double-stranded break and leads to death.

TAPE showing sgRNA efficiency. As arfA is an essential gene, it did not grow.

To see if the K/O's were successful, other than looking at macromorphology, tissue PCRs were performed. Bands lower than the genomic DNA control (i.e. pos. control on the figures) shows successful K/O of the targeted gene.

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
ΔspaA	672 bp	3528 bp
Δgul-1	545 bp	5022 bp
ΔpkaR	370 bp	1661 bp

Gel picture showing that our K/O's were successful. Tissue PCRs of ΔchsC, ΔaplD, and ΔracA are shown in this picture.

Gel picture showing that our K/O's were successful. Tissue PCRs of ΔspaA, Δgul-1, and ΔpkaR are shown in this picture.

Gel picture showing that our K/O's were successful. Tissue PCRs of ΔchsC and ΔpkaR are shown in this picture.

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] A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. Nodvig CS, Nielsen JB, Kogle ME, Mortensen UH. PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. PONE-D-15-11561 [pii] PubMed 26177455