Part:BBa_K3385010

CRISPR_glaA_KO

Theoretical expectation: glaA encodes a glucoamylase. This part works with the CRISPR pFC330 vector to mediate double stranded break. Full guide construct for knockout of the glucoamylase gene (glaA) in A. niger. This BioBlock has to be cloned into the PacI/Nt.BbvCI digested pFC330 backbone.

Plasmid map of pFC330[1].

Results: To confirm that the genes were correctly deleted, we performed tissue PCRs. The results are as shown on the gel below. Bands lower than the genomic DNA control (i.e. pos. control on the figures), at around 100 bp shows successful K/O of the targeted gene.

TAPE showing sgRNA efficiency.

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
  

[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