Part:BBa_K3841034

Repair template for AOX2 knockout

This repair template is used in combination with BBa_K38410229 or BBa_K3841030 to knock out the aox2 gene in Komagataella phaffii GS115 (previously denoted Pichia pastoris GS115).

Target
The DNA fusion construct is complementary to the upstream region of the aox2 gene on chromosome 4 (position 301757- 301801) and the downstream region (position 303794-303838) within the assembled genome of K. phaffii GS115 strain [1] (Accession number FN392322).

Usage
Co-transformation with a CRISPR-Cas9 plasmid and this repair oligo will mediate homology directed repair (HDR) [2]. For scarless deletion of the aox2 gene, co-transform the repair template with a CRISPR-Cas9 plasmid with crRNA BBa_K3841029 or BBa_K3841030 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.

Conceptual map of CRISPR-Cas9 plasmid system assembled using USER cloning [3]. The crRNA should be placed in front of the gRNA backbone to make the complete sgRNA. The plasmid contains features for being replicated in both bacteria and yeast and appropriate resistance markers. For more information of the assembly of the plasmid, visit 2021 DTU-Denmark’s experimental page

Theoretical expectation
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.

Functionality
The sgRNA and repair template efficiency was examined using the technique to assess protospacer efficiency (TAPE) [4] in a GS115 Δku70 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 Δku70 strain provided with a repair template.

Results
Below is a picture showing K. phaffii GS115 Δku70 transformed with pDIV153_aox2_KO and this repair oligo.

Transformed K. phaffii GS115 Δku70 with respective gRNAs and repair templates, if provided. Each gRNA had two candidates (C1 & C2). C1 seems to more efficient than C2. The negative control consists of K. phaffii GS115 Δku70 electroporated with water to check for contamination. The other control was K. phaffii GS115 Δku70 strain transformed without a repair template (RT)(BBa K3841033). The Triple knockout refers to an experiment were all three knockout plasmids were co-electroporated into K. phaffii GS115 Δku70 with respective repair templates. Since it only relies on taking up one plasmid to obtain resistance to NTC, a different approach is recommended to do multiple knockouts.

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
Δaox1	500 bp	2500 bp
Δaox2	500 bp	2500 bp
Δadh2	500 bp	1550 bp

Colony PCRs performed on Δaox1, Δaox2, and Δaox2. Each band corresponds to a unique colony picked from a plate of transformed K. phaffii GS115 Δku70. 16 colony PCRs were loaded to the gel on the left, eight aox1 and eight aox2 knockout colony PCRs, respectively. Eight adh2 knockout colonies were loaded the gel on the right. The gel pictures have been cut to fit both in one figure. One band was observed for aox1 and aox2 while two bands were observed for adh2.

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.

References
[1] De Schutter, Kristof, et al. “Genome Sequence of the Recombinant Protein Production Host Pichia Pastoris.” Nature Biotechnology, vol. 27, no. 6, NATURE PUBLISHING GROUP, 2009, pp. 561–66, doi:10.1038/nbt.1544.
[2] Jakociunas, Tadas, et al. “CRISPR/Cas9 Advances Engineering of Microbial Cell Factories.” Metabolic Engineering, vol. 34, Academic Press Inc., 2016, pp. 44–59, doi:10.1016/j.ymben.2015.12.003.
[3] Geu-Flores, Fernando, et al. “USER Fusion: A Rapid and Efficient Method for Simultaneous Fusion and Cloning of Multiple PCR Products.” Nucleic Acids Research, vol. 35, no. 7, OXFORD UNIV PRESS, 2007, p. e55, doi:10.1093/nar/gkm106.

[4] Garcia Vanegas, Katherina, et al. “SWITCH: a Dynamic CRISPR Tool for Genome Engineering and Metabolic Pathway Control for Cell Factory Construction in Saccharomyces Cerevisiae.” Microbial Cell Factories, vol. 16, no. 25, BioMed Central Ltd., 2017, p. 25, doi:10.1186/s12934-017-0632-x.