Part:BBa_K3841030

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).

Target
The crRNA is complementary to the aox2 gene in chromosome 4 (position 302054-302076) within the assembled genome of K. phaffii GS115 strain [1] (Accession number FN392322). This part is targeting the same gene as the crRNA BBa_K3385029.

Usage
Co-transformation with the CRISPR-Cas9 plasmid and a repair oligo will mediate homology directed repair (HDR) [2]. 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.

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 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 the repair oligo for aox2 BBa_K3841034.

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, we performed colony PCRs on a subset of the gRNAs. The technique to assess if a knock out is successful can be carried out in a similar fashion as BBa_K3841027.

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.