TABLE 1.

Genetic engineering tools for Pichia pastoris through HR,¹ adapted from Cai et al. (2021).

Strain engineering	Cas 9 expression	sgRNA expression	Genome cutting efficiency	Seamless deletion	Genome integration	Marker needed	Minimal HA2 length	References
/	PHTA1, episomal	PHTB1, episomal	43–95%	2.4%	Single gene (24%)	Yes	1000 bp	Weninger et al., 2016
kuΔ70 for blocking NHEJ	PHTA1, episomal	PHTB1, episomal	94%	gut1Δ (100%)	/	No	1000 bp	Weninger et al., 2018
kuΔ70 for blocking NHEJ	PENO1, episomal	PtRNA3, episomal	93%	/	Three genes (20%)	No	500 bp	Dalvie et al., 2020
kuΔ70 for blocking NHEJ	PHTA1, episomal	PFLD1, PAOX1 or PGAP, episomal	75–98%	/	Two genes (58–70%)	No	1000 bp	Liu et al., 2019
					Three genes (13–32%)
Overexpressing RAD52 and/or deleting MPH1	PHTA1, episomal	PHTB1, episomal	93%	gut1Δ (90%)	Single gene (43–70%)	No	50 bp	Cai et al., 2021
				faa1Δ (88%)	Three genes at one site (67.5%)
				faa2Δ (88%)	Three genes at three site (25%)
/	hCas9, Intergrated	PHZP–gRNA, episomal	/	/	Single loci (100%)	No	40 bp	Gao et al., 2022b
					Two loci (98%)
					Three loci (81%)
kuΔ70 for blocking NHEJ	PENO1, episomal	PtRNA3, episomal	100%	/	Single gene (89.7%)	No	50–500 bp	Dalvie et al., 2022

¹HR, homologous recombination.

²HA, homologous arms.