Part:BBa_K3841021

gRNA for CRISPR neutral insertion site 4 in Komagataella phaffii

This part is a CRISPR RNA (crRNA) targeting a neutral region in the genome of Komagataella phaffii ideal for chromosomal integration in this yeast.

Target
The crRNA is complementary to chromosome 4 (position 1633787-1633806) within the assembled genome of K. phaffii GS115 strain [1] (Accession number FN392322). This part is targeting the same neutral region as the crRNAs BBa_K3385085-BBa_K3385087.

Usage
Co-transformation with the CRISPR-Cas9 plasmid and a repair oligo will mediate homology directed repair (HDR) [2]. For insertion, flank the expression cassette of interest by homology arms BBa_K3385065 and BBa_K3385066 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

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.

Results
Below picture shows K. phaffii GS115 transformed using a CRISPR-Cas9 plasmid harboring the BBa_K3385021 targeting an intergenic region within the genome. As evident from the plates, Δku70 have remarkably fewer colonies as compared to the wildtype.

TAPE showing sgRNA efficiency for BBa_K3841021. Δku70 plates have remarkably fewer colonies as compared to the wildtype indicating efficient cutting at the target site. Negative control is no CRISPR-Cas9 plasmid and positive control is is a Cas9 plasmid w/o a sgRNA.

Bars showing sgRNA efficiency for various crRNAs including BBa_K3841016. Δku70 plates have remarkably fewer CFUs/μg plasmid colonies as compared to the wildtype indicating efficient cutting at the target site.

As evident from the data, the Δku70 transformants have remarkably fewer colonies when compared to the wildtype as a consequence of transformation with CRISPR/Cas9 plasmids harbouring the sgRNAs. The following numbers refer to fold decrease in cell viability for Δku70 compared to the wild type:
BBa_K38410_16 with -1.8
BBa_K38410_19 with -31.5
BBa_K38410_20 with -333
BBa_K38410_21 with -136

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.