A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts

Abstract

Methylotrophic yeasts such as Ogataea polymorpha and Komagataella phaffii (sin. Hansenula polymorpha and Pichia pastoris, respectively) are commonly used in basic research and biotechnological applications, frequently those requiring genome modifications. However, the CRISPR-Cas9 genome editing approaches reported for these species so far are relatively complex and laborious. In this work we present an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts. This includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of a sgRNA gene for a particular target sequence requires only the insertion of a 24 bp oligonucleotide duplex into the scaffold. Prior to yeast transformation, each plasmid is cleaved at two sites, one of which is located within the selectable marker, so that the functional marker can be restored only via recombination of the Cas9-containing fragment with the sgRNA gene-containing fragment. This recombination leads to the formation of an autonomously replicating plasmid, which can be lost from yeast clones after acquisition of the required genome modification. The vector set allows the use of G418-resistance and LEU2 auxotrophic selectable markers. The functionality of this setup has been demonstrated in O. polymorpha, O. parapolymorpha, O. haglerorum and Komagataella phaffii.

Keywords: CRISPR; Cas9; Komagataella; Ogataea; genome editing; genome engineering; methylotrophic yeast.

Figure 1

Genetic constructs used to introduce Cas9- and sgRNA-encoding genes into yeast cells. (A) Schemes of pKAM944 and pKAM966 plasmids. P_LAT1, Komagataella phaffii LAT1 promoter; Cas9, gene coding for Cas9 with SV40 NLS; ori, the bacterial ColE1 replication origin; 2μ, fragment of S. cerevisiae 2μ DNA bearing STB locus; HARS6, an O. parapolymorpha autonomously replicating sequence; G418/kan^R, a selectable marker providing G418 resistance in yeast and kanamycin resistance in E. coli; tY(UAC), O. polymorpha gene encoding tyrosine tRNA without the terminator sequence, mCherry, a gene coding for mCherry under control of the E. coli promoter; Scaffold, the sgRNA-encoding sequence lacking the targeting part; T_SNR52, a S. cerevisiae SNR52 terminator. (B) Scheme of insertion of the targeting sequence into the plasmid with the blank sgRNA construct. The upper and lower strands of the target sequence are shown as blue and green N characters, respectively. The PAM sequence is shown in red. (C) Scheme of the in vivo recombination (crossed dashed red lines) of plasmid fragments creating the autonomously replicating plasmid bearing Cas9 and sgRNA encoding genes.

Figure 2

O. haglerorum transformants obtained using a mix of SgfI–BcuI-digested pKAM944, the donor DNA for OhADE2 deletion, and the SalI–NruI-digested plasmid encoding the sgRNA with an inserted OhADE2F1–OhADE2R1 oligonucleotide duplex.

Figure 3

Sequences of the Cas9 target site in the O. haglerorum wild-type strain (WT) and Ade⁻ transformants possessing 1 bp deletion or insertion within this site. Sequence of the 20 bp target site and its flanking sequences are shown in black and gray, respectively; PAM sequence is shown in red; mutations are shown in blue; arrows indicate predicted Cas9 cleavage site.

Figure 4

Scheme of the in vivo recombination (crossed dashed red lines) of plasmid fragments producing the autonomously replicating plasmid bearing Cas9- and sgRNA-encoding genes and a LEU2 selectable marker. Designations as in the Figure 1.

Figure 5

Porphyrin fluorescence in K. phaffii met8 mutants obtained with the transformation of the X-33 strain with a mix of SgfI–BcuI-digested pKAM944 and the SalI–NruI-digested plasmid coding sgRNA, with the insertion of the KpMET8F–KpMET8R oligonucleotide duplex. Mutant clones (T1–T5) and untransformed strain (WT) were patched onto a YPD plate, incubated for two days at 30 °C and photographed through a yellow filter under illumination with 405 nm light emitting diode.