Part:BBa_K3385026

CRISPR_arfA_KO

Theoretical explanation: arfA encodes the Putitative ADP ribosylation factor involved in hyphal growth and secretion. It's an essential gene and therefore the fungus should become unviable when knocking it out.

Full guide construct for knockout of the Putative ADP-ribosylation factor gene (arfA) 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_arfA_KO and the repair oligo for arfA. It shows efficient gene deletion when it's transformed with a repair oligo.

TAPE showing sgRNA efficiency. As ΔarfA is unvialbe, nothing grew on either of the transformations - with or without the repair oligo.

Summary: The fungi transformed with this part became unviable and did not grow, which was expected as it encodes an essential gene.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 489
  Illegal EcoRI site found at 660
  Illegal EcoRI site found at 831
  Illegal SpeI site found at 978
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 489
  Illegal EcoRI site found at 660
  Illegal EcoRI site found at 831
  Illegal NheI site found at 333
  Illegal SpeI site found at 978
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 489
  Illegal EcoRI site found at 660
  Illegal EcoRI site found at 831
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 489
  Illegal EcoRI site found at 660
  Illegal EcoRI site found at 831
  Illegal SpeI site found at 978
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 489
  Illegal EcoRI site found at 660
  Illegal EcoRI site found at 831
  Illegal SpeI site found at 978
  Illegal AgeI site found at 204
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 162
  

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