Host-Informed Expression of CRISPR Guide RNA for Genomic Engineering in <i>Komagataella phaffii</i>

Dalvie, Neil C.; Leal, Justin; Whittaker, Charles A.; Yang, Yuchen; Brady, Joseph; Love, Kerry R.; Love, J. Christopher

doi:10.1021/acssynbio.9b00372

Cited by 43 publications

(65 citation statements)

References 48 publications

(82 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…If integration occurs with a frequency of 10%, then the probability of missing a correct clone when screening 32 colonies is 0.034. With the successful implementation of CRISPR‐Cas9 in P. pastoris it will be of interest to re‐screen the genes in question to try to conclusively determine whether they are essential or non‐essential 36‐38 …”

Section: Resultsmentioning

confidence: 99%

Knockout of RSN1, TVP18 or CSC1‐2 causes perturbation of Golgi cisternae in Pichia pastoris

Wachter

Laukens

et al. 2020

Traffic

View full text Add to dashboard Cite

The structural organization of the Golgi stacks in mammalian cells is intrinsically linked to function, including glycosylation, but the role of morphology is less clear in lower eukaryotes. Here we investigated the link between the structural organization of the Golgi and secretory pathway function using Pichia pastoris as a model system. To unstack the Golgi cisternae, we disrupted 18 genes encoding proteins in the secretory pathway without loss of viability. Using biosensors, confocal microscopy and transmission electron microscopy we identified three strains with irreversible perturbations in the stacking of the Golgi cisternae, all of which had disruption in genes that encode proteins with annotated function as or homology to calcium/calcium permeable ion channels. Despite this, no variation in the secretory pathway for ER size, whole cell glycomics or recombinant protein glycans was observed. Our investigations showed the robust nature of the secretory pathway in P. pastoris and suggest that Ca2+ concentration, homeostasis or signalling may play a significant role for Golgi stacking in this organism and should be investigated in other organisms.

show abstract

Section: Resultsmentioning

confidence: 99%

Knockout of RSN1, TVP18 or CSC1‐2 causes perturbation of Golgi cisternae in Pichia pastoris

Wachter

Laukens

et al. 2020

Traffic

View full text Add to dashboard Cite

show abstract

“…The gene containing the RBD was codon optimized, synthesized (Integrated DNA Technologies), and cloned into a custom vector. The RBD vector was transformed as described previously ( Dalvie et al, 2019 ). Transcription factors mit1 and mxr1 were integrated into the genome near genomic loci GQ67_02967 and GQ67_04576, respectively, using a markerless CRISPR-Cas9 system described previously ( Dalvie et al, 2019 ).…”

Section: Methodsmentioning

confidence: 99%

“…The RBD vector was transformed as described previously ( Dalvie et al, 2019 ). Transcription factors mit1 and mxr1 were integrated into the genome near genomic loci GQ67_02967 and GQ67_04576, respectively, using a markerless CRISPR-Cas9 system described previously ( Dalvie et al, 2019 ). Both mit1 and mxr1 were under control of the P CAT1 promoter from K. phaffii .…”

Section: Methodsmentioning

confidence: 99%

“…Several studies, however, have demonstrated that constitutive overexpression of activating transcription factors can lead to consistent activation of P AOX1 without methanol ( Shi et al, 2019 ; Vogl et al, 2018 ). To test production of RBD without methanol, we integrated additional copies of the endogenous transcription factors mit1 and mxr1 into the K. phaffii genome under a glycerol-repressible promoter ( Dalvie et al, 2019 ). We cultivated these strains for protein production by feeding with only sorbitol ( Fig.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii

Dalvie

Biedermann

Rodriguez-Aponte

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply on-going demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol-induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5x by alleviating protein folding stress. Removal of methanol from the production process enabled scale up to a 1,200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

show abstract

“… 35 This strain was further modified to express mCherry by cotransformation of a donor cassette and a circular plasmid containing Cas9 and guide RNA sequences, described previously. 36 IGR libraries were similarly created by cotransformation of a donor cassette for eGFP expression and a circular plasmid for CRISPR/Cas9-based targeting to the desired IGR. Donor cassettes contained the following (5′ to 3′): a 450 bp 5′ homology arm, the TEF1 promoter, the mCherry or eGFP gene, a transcription terminator, and a 450 bp 3′ homology arm.…”

Section: Methodsmentioning

confidence: 99%

Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq

et al. 2020

Self Cite

View full text Add to dashboard Cite

Constructing efficient cellular factories often requires integration of heterologous pathways for synthesis of novel compounds and improved cellular productivity. Few genomic sites are routinely used, however, for efficient integration and expression of heterologous genes, especially in nonmodel hosts. Here, a data-guided framework for informing suitable integration sites for heterologous genes based on ATAC-seq was developed in the nonmodel yeast Komagataella phaffii . Single-copy GFP constructs were integrated using CRISPR/Cas9 into 38 intergenic regions (IGRs) to evaluate the effects of IGR size, intensity of ATAC-seq peaks, and orientation and expression of adjacent genes. Only the intensity of accessibility peaks was observed to have a significant effect, with higher expression observed from IGRs with low- to moderate-intensity peaks than from high-intensity peaks. This effect diminished for tandem, multicopy integrations, suggesting that the additional copies of exogenous sequence buffered the transcriptional unit of the transgene against effects from endogenous sequence context. The approach developed from these results should provide a basis for nominating suitable IGRs in other eukaryotic hosts from an annotated genome and ATAC-seq data.

show abstract

Host-Informed Expression of CRISPR Guide RNA for Genomic Engineering in Komagataella phaffii

Cited by 43 publications

References 48 publications

Knockout of RSN1, TVP18 or CSC1‐2 causes perturbation of Golgi cisternae in Pichia pastoris

Knockout of RSN1, TVP18 or CSC1‐2 causes perturbation of Golgi cisternae in Pichia pastoris

Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii

Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq

Contact Info

Product

Resources

About