ArticleMicrobial cell factories2022
Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris.
Article in Microbial cell factories, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 21 citations in OpenAlex.
- A CRISPR-Cas9 Toolkit Enabling Tunable Integration and Transient Homologous Recombination Enhancement in Yarrowia lipolytica.Biotechnology and bioengineering · 2026Article
- An advanced genome engineering platform for Komagataella phaffii enabling versatile in vivo DNA assembly and multiplex integration.Journal of biological engineering · 2026Article
- Genome-Wide Landscape of Position Effect Variegation inJournal of fungi (Basel, Switzerland) · 2026Article
- In Vivo DNA Assembly inACS synthetic biology · 2025Article
- CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies inSynthetic and systems biotechnology · 2025Article
- Applications of the Methylotrophic YeastJournal of fungi (Basel, Switzerland) · 2024Review
- Article
- Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches.International journal of molecular sciences · 2024Review
- Genetic tools for metabolic engineering ofEngineering microbiology · 2023Review
- Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production inInternational journal of molecular sciences · 2023Article
- An outlook to sophisticated technologies and novel developments for metabolic regulation in theFrontiers in bioengineering and biotechnology · 2023Review
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Authors and funding
7 authors at 4 institutions in 1 country.
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Abstract
backgroundThe 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.
resultsThe 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.
conclusionsFusion 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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.