gRNA for CRISPR mediated knockout of the aox2 gene in Komagataella phaffii
This part is a CRISPR RNA (crRNA) targeting the aox2 gene in the genome of Komagataella phaffii (previously denoted Pichia pastoris GS115).
The crRNA is complementary to the aox2 gene in chromosome 4 (position 303293-303312) within the assembled genome of K. phaffii GS115 strain  (Accession number FN392322). This part is targeting the same gene as the crRNA BBa_K3385030.
Co-transformation with the CRISPR-Cas9 plasmid and a repair oligo will mediate homology directed repair (HDR) . For scarless deletion of the aox2 gene, co-transform the fused homology arms BBa_K3841034 to aid the HDR. The crRNA should be correctly inserted into a CRISPR-Cas9 plasmid system after a gRNA backbone (we used BBa_K3841003) to induce a double-stranded DNA break. For confidential matters, the CRISPR-Cas9 plasmids sequence cannot be added to the iGEM registry before it has been published. A conceptual map of the CRISPR-Cas9 plasmid system used is seen below.
aox2 encodes an alcohol oxidase, which catalyzes the oxidation of methanol to formaldehyde and hydrogen peroxide, the first step in the methanol utilization pathway of methylotrophic yeasts. Deletion of the gene was expected to impair the growth of Komagataella phaffii GS115 on methanol.
The sgRNA efficiency was examined using the technique to assess protospacer efficiency (TAPE)  in a GS115 Îku70 strain. Highly efficient sgRNA will result in no colonies, while less efficient sgRNA will show a reduced number of colonies as compared to the wildtype or a GS115 Îku70 strain provided with a repair template.
Below is a picture showing K. phaffii GS115 Îku70 transformed with pDIV153_aox2_KO and the repair oligo for aox2 BBa_K3841034.
To see if the knockout was successful, colony 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 the gene has been knocked out the primers are going to be closer to each other resulting in a smaller band in the colony PCR. However if the gene is still present in the genome, the band will include its whole length as seen in the table below.
Expected length of the knockouts
|Targeted gene||Expected gene length after knockout||Control lenght|
|Îaox1||500 bp||2500 bp|
|Îaox2||500 bp||2500 bp|
|Îadh2||500 bp||1550 bp|
To validate the scarless deletion of aox2 we sent the colony PCRs for Sanger Sequencing. The results we received were inconclusive, and due to time constraints we did not have a chance to repeat the sequencing.
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