Part:BBa_K3385003

gRNA backbone

About the part: The part was obtained as part of the CRISPR cloning vector pFC902[1]. The gRNA backbone forms a stem-loop structure that attaches and activates Cas9 to mediate double-stranded breaks at the crRNA target.

Plasmid map of pFC902[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 the transformed A. niger. It shows efficient gene deletion when it's 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. 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
ΔspaA	672 bp	3528 bp
Δgul-1	545 bp	5022 bp
ΔpkaR	370 bp	1661 bp

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

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

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

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

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