A repackaged CRISPR platform increases homology-directed repair for yeast engineering

Ploessl, Deon; Zhao, Yuxin; Cao, Mingfeng; Ghosh, Saptarshi; López, Carmen Trinidad; Sayadi, Maryam; Chudalayandi, Siva; Severin, Andrew J.; Huang, Lei; Gustafson, Marissa; Shao, Zengyi

doi:10.1038/s41589-021-00893-5

Cited by 25 publications

(30 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has been reported that knockout of KU70 or other genes involved in the NHEJ pathway can hinder the fitness of a strain due to their shared role in telomere maintenance ( Liti and Louis, 2003 ; Polotnianka et al, 1998 ). The deleterious effects of NHEJ gene knockouts on strain fitness has been demonstrated in some yeasts including S. cerevisiae ( Palmbos et al, 2005 ) and S. stipitis ( Ploessl et al, 2021 ) . However, comparison of the growth rate of CBS 712Δ U and CBS 712Δ U Δ K grown in YPD at 37 °C revealed no significant differences between the two strains ( Fig.…”

Section: Resultsmentioning

confidence: 99%

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

Bever

Wheeldon

Silva

2022

Metabolic Engineering Communications

View full text Add to dashboard Cite

Section: Resultsmentioning

confidence: 99%

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

Bever

Wheeldon

Silva

2022

Metabolic Engineering Communications

View full text Add to dashboard Cite

“…This transient CRISPR−Cas9 system has been applied to other yeast as well. 33,39 In Vivo Assembly of Multi-DNA Fragments. The tactic of one-step assembly of DNA fragments has been used for multiple gene integration by the CRISPR−Cas9 system in S. cerevisiae 37,40 and C. tropicalis.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…In general, the transient CRISPR–Cas9 system could also highly improve the efficiency of the target gene disruption. This transient CRISPR–Cas9 system has been applied to other yeast as well. , …”

Section: Resultsmentioning

confidence: 99%

A CRISPR–Cas9 System-Mediated Genetic Disruption and Multi-fragment Assembly in Starmerella bombicola

Shi

Zhang

et al. 2022

ACS Synth. Biol.

View full text Add to dashboard Cite

Gene editing technology plays an extremely significant role in synthetic biology and metabolic engineering. Traditional genetic manipulation methods, such as homologous recombination, however, are inefficient, timeconsuming, and barely feasible when disrupting multiple genes simultaneously. Starmerella bombicola, a nonconventional yeast that overproduces sophorolipids, lacks convenient genetic tools for engineering strains. Here, we developed an efficient CRISPR−Cas9 genome editing technology by combining molecular element mining and expression system optimization for S. bombicola. This CRISPR−Cas9 system improved the efficiency of gene-integration/target geneintroducing disruption by homology-directed repair and realized the multi-gene simultaneous disruptions. Based on this CRISPR− Cas9 system, we also further constructed an engineered strain via the in vivo assembly of multiple DNA fragments (10 kb) that can produce acid-type sophorolipids. These results showed that the CRISPR−Cas9 system may be an efficient and convenient strategy to perform genetic manipulation in S. bombicola.

show abstract

“…Thus, this strategy can help to enhance HR process without disrupting NHEJ procedure. Previous studies showed that interfering with the NHEJ pathway, especially deletion of KU70Δ, resulted in genetic instability of chromosomes [17], sensitive to UV rays, negative effect on the cell growth and production [23][24][25]. Furthermore, methanol biotransformation in P. pastoris will suffers the methanol toxicity that might bring chromosome damage and the NHEJ pathway can help cells restore their viability rapidly.…”

Section: Discussionmentioning

confidence: 99%

Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris

et al. 2022

View full text Add to dashboard Cite

Background The methylotrophic yeast Pichia pastoris is considered as an ideal host for the production of recombinant proteins and chemicals. However, low homologous recombination (HR) efficiency hinders its precise and extensive genetic manipulation. To enhance the homology-directed repair over non-homologous end joining (NHEJ), we expressed five exonucleases that were fused with the Cas9 for enhancing end resection of double strand breaks (DSBs) of DNA cuts. Results The endogenous exonuclease Mre11 and Exo1 showed the highest positive rates in seamless deletion of FAA1, and fusing the MRE11 to the C-terminal of CAS9 had the highest positive rate and relatively high number of clones. We observed that expression of CAS9-MRE11 significantly improved positive rates when simultaneously seamless deletion of double genes (from 76.7 to 86.7%) and three genes (from 10.8 to 16.7%) when overexpressing RAD52. Furthermore, MRE11 overexpression significantly improved the genomic integration of multi-fragments with higher positive rate and clone number. Conclusions Fusion expression of the endogenous exonuclease Mre11 with Cas9 enhances homologous recombination efficiency in P. pastoris. The strategy described here should facilitate the metabolic engineering of P. pastoris toward high-level production of value-added compounds.

show abstract

A repackaged CRISPR platform increases homology-directed repair for yeast engineering

Cited by 25 publications

References 60 publications

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

A CRISPR–Cas9 System-Mediated Genetic Disruption and Multi-fragment Assembly in Starmerella bombicola

Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris

Contact Info

Product

Resources

About