CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris

Gassler, Thomas; Heistinger, Lina; Mattanovich, Diethard; Gasser, Brigitte; Prielhofer, Roland

doi:10.1007/978-1-4939-9024-5_9

Cited by 60 publications

(68 citation statements)

References 36 publications

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“…2d), the other targets also demonstrated good efficiency (Additional file 1: Table S1) and could be used for single-locus and multiloci integrations. The lower integration efficiency of the gRNA targets upstream of P GAP and downstream of AOXTT may be attributed to the fact that the eGFP cassette and its homologous arms are flanked by P GAP or AOXTT , thereby influencing donor DNA stability [30]. When two or three sites are simultaneously targeted in the Δ ku70 strain, cells with unrepaired DSB at any site may die because of its lethality.…”

Section: Discussionmentioning

confidence: 99%

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CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris

et al. 2019

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Background Pichia pastoris (syn. Komagataella phaffii ) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris . Results A non-homologous-end-joining defective strain (Δ ku70 ) was first constructed using the CRISPR–Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100 bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (P AOX1 UP-g2, P TEF1 UP-g1, and P FLD1 UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR–Cas9-mediated gene co-integration method in P. pastoris . Conclusions A CRISPR–Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris . Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.

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Section: Discussionmentioning

confidence: 99%

“…This enhanced the homology directed repair (HDR) efficiency of gene deletion and replacement [28]. These reports demonstrated the suitability of marker-free donor DNA fragments in facilitating marker-free gene integration in P. pastoris , with the loss of plasmids containing Cas9 and gRNA by continuous streaking [27, 30].…”

Section: Introductionmentioning

confidence: 99%

CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris

et al. 2019

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“…To create the FLO11-eGFP , FLO400-eGFP and FLO5-1-eGFP strains, CRISPR/Cas9 mediated homology-directed repair was employed (Gassler et al . 2019 ). Since all the three proteins contain a signal peptide pre-sequence and a signal peptidase cleavage site, the enhanced green fluorescent protein (eGFP) sequence was added internally after the pre-sequence at the amino-terminal as described for S. cerevisiae Flo11 (Lo and Dranginis 1998 ).…”

Section: Methodsmentioning

confidence: 99%

“…Human codon-optimized Cas9 under the control of K. phaffii LAT1 promoter and a guide RNA targeting the region directly upstream of the start codon under the control of the pGAP were expressed from an episomal plasmid vector (Gassler et al . 2019 ) and 500 ng of this vector was transformed along with the donor DNA for integration of the eGFP sequence. Correct integration was checked by PCR using primers binding outside the homologous regions and sequencing of the locus.…”

Section: Methodsmentioning

confidence: 99%

Pseudohyphal differentiation inKomagataella phaffii: investigating theFLOgene family

Rebnegger

Moser

et al. 2020

FEMS Yeast Research

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Many yeasts differentiate into multicellular phenotypes in adverse environmental conditions. Here we investigate pseudohyphal growth in Komagataella phaffii and the involvement of the flocculin (FLO) gene family in its regulation. The K. phaffii FLO family consists of 13 members, and the conditions inducing pseudohyphal growth are different from Saccharomyces cerevisiae. So far, this phenotype was only observed when K. phaffii was cultivated at slow growth rates in glucose-limited chemostats, but not upon nitrogen starvation or the presence of fusel alcohols. Transcriptional analysis identified that FLO11, FLO400 and FLO5–1 are involved in the phenotype, all being controlled by the transcriptional regulator Flo8. The three genes exhibit a complex mechanism of expression and repression during transition from yeast to pseudohyphal form. Unlike in S. cerevisiae, deletion of FLO11 does not completely prevent the phenotype. In contrast, deletion of FLO400 or FLO5–1 prevents pseudohyphae formation, and hampers FLO11 expression. FAIRE-Seq data shows that the expression and repression of FLO400 and FLO5–1 are correlated to open or closed chromatin regions upstream of these genes, respectively. Our findings indicate that K. phaffii Flo400 and/or Flo5–1 act as upstream signals that lead to the induction of FLO11 upon glucose limitation in chemostats at slow growth and chromatin modulation is involved in the regulation of their expression.

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“…Modern state-of-the-art genome editing technologies allow for unprecedented large-scale intervention into the host genome previously unattainable with other approaches. The heterologous pathway expression is assisted with such genome editing tools as RNAi [ 97 ], zinc-finger nucleases [ 98 ], DNA editing at replication forks [ 99 ], and the eminent CRISPR-Cas9 technology [ 10 , 100 , 101 ]. Genome editing also enables host genome stabilization by reducing the well-known problem of inactivation or recombination excision of heterologous genes [ 102 ].…”

Section: Heterologous Pathway Optimizationmentioning

confidence: 99%

Heterologous Metabolic Pathways: Strategies for Optimal Expression in Eukaryotic Hosts

2020

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Heterologous pathways are linked series of biochemical reactions occurring in a host organism after the introduction of foreign genes. Incorporation of metabolic pathways into host organisms is a major strategy used to increase the production of valuable secondary metabolites. Unfortunately, simple introduction of the pathway genes into the heterologous host in most cases does not result in successful heterologous expression. Extensive modification of heterologous genes and the corresponding enzymes on many different levels is required to achieve high target metabolite production rates. This review summarizes the essential techniques used tocreate heterologous biochemical pathways, with a focus on the key challenges arising in the process and the major strategies for overcoming them.

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CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris

Cited by 60 publications

References 36 publications

CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris

CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris

Pseudohyphal differentiation inKomagataella phaffii: investigating theFLOgene family

Heterologous Metabolic Pathways: Strategies for Optimal Expression in Eukaryotic Hosts

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