A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9

Table 1 Plasmids constructed in the current study

Plasmid name	Clone no	Description	Selection marker
CRISPi_04576	MF8295	Shuttle vector for sgRNA/Cas9 targeting near 04576	kanMX
CRISPi_PFK1	MF8296	Shuttle vector for sgRNA/Cas9 targeting near PFK1	kanMX
CRISPi_ROX1	MF8297	Shuttle vector for sgRNA/Cas9 targeting near ROX1	kanMX
crBB3_14_04576	MF8310	Helper plasmid for cloning of one transcription unit with 04576 homology regions	cat
crBB3_14_PFK1	MF8311	Helper plasmid for cloning of one transcription unit with PFK1 homology regions	cat
crBB3_14_ROX1	MF8312	Helper plasmid for cloning of one transcription unit with ROX1 homology regions	cat
crBB3_AC_04576	MF8437	Helper plasmid for cloning of two transcription units with 04576 homology regions	cat
crBB3_AC_PFK1	MF8438	Helper plasmid for cloning of two transcription units with PFK1 homology regions	cat
crBB3_AC_ROX1	MF8439	Helper plasmid for cloning of two transcription units with ROX1 homology regions	cat
crBB3_04576_eGFP	MF8316	crBB3_14_04576 with inserted eGFP expression cassette	cat
crBB3_PFK1_eGFP	MF8317	crBB3_14_PFK1 with inserted eGFP expression cassette	cat
crBB3_ROX1_eGFP	MF8318	crBB3_14_ROX1 with inserted eGFP expression cassette	cat
pEX-A128-αMF	MF8448	Synthesized α-MF, template for PCR	bla
pTOPO-αMF	MF8970	α-MF for fusion to OVA, cloned in pCR4Blunt-TOPO	bla, kanMX
pEX-K248-OVA		Synthesized OVA gene	kanMX
crBB3_04576_OVA	MF8982	crBB3_14_04576 with inserted OVA expression cassette	cat

ISSN: 1475-2859