Part:BBa_K3841017

gRNA for CRISPR neutral insertion site 2 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 2 (position 666701-666720) within the assembled genome of K. phaffii GS115 strain [1] (Accession number FN392320). This part is targeting the same neutral region as the crRNAs BBa_K3385018, BBa_K3385081-BBa_K3385082.

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_K3385045 and BBa_K3385046 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.

Results
Below picture shows K. phaffii GS115 Δku70 transformed using a CRISPR-Cas9 plasmid harboring the BBa_K3385017 targeting an intergenic region within the genome. As evident from the plates, when treated with BBa_K3385017 no colonies appeared.

TAPE showing sgRNA efficiency for BBa_K3385017. Positive control is a Cas9 plasmid w/o a sgRNA. No colonies appeared in the strain transformed with the CRISPR-Cas9 plasmid with BBa_K3385017 indicating an extremely efficient cutting at the sgRNA.

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.