CRISPR-Cas9 Genome Editing Tool for the Production of Industrial Biopharmaceuticals

Khan, Amjad Hayat; Tye, Gee Jun; Noordin, Rahmah

doi:10.1007/s12033-020-00265-9

Cited by 5 publications

(2 citation statements)

References 109 publications

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“…A major bottleneck in strain engineering and obtaining desired genetic modifications in P. pastoris is the low frequency of homologous recombination. Lacking an efficient method for gene modification and/or gene integration cassettes has remained a challenge for many scientists 86 . To address these issues, many researchers have conducted various studies on P. pastoris based on the CRISPR/Cas9 method to enhance P. pastoris recombination machinery for recombinant heterologous cellulase expression.…”

Section: Genetic Engineering Strategiesmentioning

confidence: 99%

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

Ullah,

Madadi,

Asadollahi

et al. 2023

J of Chemical Tech & Biotech

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The yeast expression system, Pichia pastoris, is one of the most robust and versatile expression systems in biotechnology and molecular biology, generally considered as a safe host for heterologous protein expression, especially for producing cellulases and xylanases at the industrial level. Despite the high recombinant protein expression rate, the potential of the P. pastoris expression system has still not been fully explored. Cultivation of P. pastoris under optimized conditions greatly relies on the strain and is associated with certain problems such as promoter strength, sensitivity to methanol, and oxygen demand. To address these issues, different genetic engineering strategies have been employed. Advancements in promoter engineering, optimization of gene dosage and codon usage, recombinant plasmid engineering using CRISPR/Cas9 system, and directed evolution strategies have proven beneficial to the yield of cellulase expression levels. This study will systemically review recent progress in various genetic engineering strategies to enhance cellulase and xylanase expression in the P. pastoris expression system. The utilization of alcohol oxidase 1 promoter (pAOX1), methanol‐free system, and recombinant plasmid engineering for improved production of these enzymes are highlighted. Additionally, we discuss the recent advancements in the P. pastoris expression system toolbox for improved cellulase and xylanase production, thus providing a deep insight into how P. pastoris is becoming the indispensable platform for heterologous protein production. © 2023 Society of Chemical Industry (SCI).

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Section: Genetic Engineering Strategiesmentioning

confidence: 99%

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

Ullah,

Madadi,

Asadollahi

et al. 2023

J of Chemical Tech & Biotech

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show abstract

“…The clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system (Jinek et al 2012) has been used as an excellent genome editing tool in a broad range of elds and species (Banan 2020; Khan et al 2020). The CRISPR-Cas9 system consists of two components: Cas9 endonuclease and guide RNA (gRNA).…”

Section: Introductionmentioning

confidence: 99%

Applying the CRISPR-Cas9 system to targeted transgene integration in the development of recombinant lepidopteran insect cells

Matsuda

Togami

Masumi-Koizumi

et al. 2022

Preprint

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The clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system is a widely used genome editing tool, but its application for the development of stably transformed lepidopteran insect cells for recombinant protein production has been limited. In the present study, we applied the CRISPR-Cas9 system to the targeted transgene knock-in to efficiently develop recombinant insect cells. A double-strand break (DSB) in a target site of the genome of Trichoplusia ni BTI-TN-5B1-4 (High Five) cells was detected by T7 endonuclease assay after the cells were transfected with a plasmid for genome editing by employing a U6-4 promoter derived from T. ni to drive guide RNA (gRNA). Genomic DNA analysis via PCR confirmed the integration of a transgene into a target site of the High Five cell genome. The length of the homology arm affected the genome editing efficiency, and the use of a 500-bp version proved to be optimal. Via the co-transfection of High Five cells with gRNA-Cas9 plasmid and a donor vector harboring a reporter gene and the neomycin resistance gene, 21 clones of stably transformed cells were established. In 19 of those clones a site-specific knock-in of the reporter gene at the target site was confirmed, which indicates that an exogenous gene can be efficiently integrated into a target site of the High Five cell genome via the CRISPR-Cas9 system, though transcriptional hot spots in the genome should be identified to establish highly productive recombinant cells.

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Metabolic Engineering and the Synthetic Biology Toolbox forClostridium

Joseph

Kelley

Kim

et al. 2021

Metabolic Engineering

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CRISPR-Cas9 Genome Editing Tool for the Production of Industrial Biopharmaceuticals

Cited by 5 publications

References 109 publications

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

Applying the CRISPR-Cas9 system to targeted transgene integration in the development of recombinant lepidopteran insect cells

Metabolic Engineering and the Synthetic Biology Toolbox forClostridium

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