Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.

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An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽

10.1128/mspheredirect.00149-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 33

Author(s):

Namkha Nguyen ◽

Morgan M. F. Quail ◽

Aaron D. Hernday

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Fungal Pathogen ◽

Gene Knockout ◽

Strain Engineering ◽

Molecular Genetic Analysis ◽

Content Type ◽

Highly Efficient ◽

Discovery Research ◽

Gene Knockouts

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

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A genome-editing off switch

Nature Reviews Genetics ◽

10.1038/nrg.2016.166 ◽

2016 ◽

Vol 18 (2) ◽

pp. 69-69

Author(s):

Ross Cloney

Keyword(s):

Genome Editing ◽

A Genome

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Constraint-based metabolic control analysis for rational strain engineering

10.1101/2020.11.26.399576 ◽

2020 ◽

Author(s):

Sophia Tsouka ◽

Meric Ataman ◽

Tuure Hameri ◽

Ljubisa Miskovic ◽

Vassily Hatzimanikatis

Keyword(s):

Metabolic Engineering ◽

Genome Editing ◽

Metabolic Control ◽

Metabolic Flux ◽

Strain Engineering ◽

Metabolic Control Analysis ◽

Physiological Data ◽

Response Analysis ◽

Control Analysis ◽

Wide Range

AbstractThe advancements in genome editing techniques over the past years have rekindled interest in rational metabolic engineering strategies. While Metabolic Control Analysis (MCA) is a well-established method for quantifying the effects of metabolic engineering interventions on flows in metabolic networks and metabolic concentrations, it fails to account for the physiological limitations of the cellular environment and metabolic engineering design constraints. We report here a constraint-based framework based on MCA, Network Response Analysis (NRA), for the rational genetic strain design that incorporates biologically relevant constraints, as well as genome editing restrictions. The NRA core constraints being similar to the ones of Flux Balance Analysis, allow it to be used for a wide range of optimization criteria and with various physiological constraints. We show how the parametrization and introduction of biological constraints enhance the NRA formulation compared to the classical MCA approach, and we demonstrate its features and its ability to generate multiple alternative optimal strategies given several user-defined boundaries and objectives. In summary, NRA is a sophisticated alternative to classical MCA for rational metabolic engineering that accommodates the incorporation of physiological data at metabolic flux, metabolite concentration, and enzyme expression levels.

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Choosing a genome editing strategy and target site

Genome Editing ◽

10.1016/b978-0-12-823484-6.00011-6 ◽

2021 ◽

pp. 21-39

Author(s):

Kiran Musunuru

Keyword(s):

Genome Editing ◽

Target Site ◽

A Genome

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Nanopore sequencing reveals a structural alteration of mirror‐image duplicated genes in a genome‐editing mouse line

Congenital Anomalies ◽

10.1111/cga.12364 ◽

2019 ◽

Vol 60 (4) ◽

pp. 120-125 ◽

Cited By ~ 1

Author(s):

Sachiko Miyamoto ◽

Kazushi Aoto ◽

Takuya Hiraide ◽

Mitsuko Nakashima ◽

Shuji Takabayashi ◽

...

Keyword(s):

Genome Editing ◽

Mirror Image ◽

Structural Alteration ◽

Nanopore Sequencing ◽

Duplicated Genes ◽

Mouse Line ◽

A Genome

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CRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering

Chemical Society Reviews ◽

10.1039/c6cs00197a ◽

2016 ◽

Vol 45 (24) ◽

pp. 6666-6684 ◽

Cited By ~ 16

Author(s):

Amrita Singh ◽

Debojyoti Chakraborty ◽

Souvik Maiti

Keyword(s):

Genome Editing ◽

Chemical Biology ◽

Genome Engineering ◽

A Genome

The development and adaptation of CRISPR–Cas9 as a genome editing tool and chemical biology approaches for modulating its activity.

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A fast and robust iterative genome-editing method based on a Rock-Paper-Scissors strategy

Nucleic Acids Research ◽

10.1093/nar/gkaa1141 ◽

2020 ◽

Author(s):

Jichao Wang ◽

Xinyue Sui ◽

Yamei Ding ◽

Yingxin Fu ◽

Xinjun Feng ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Genome Editing ◽

The Other ◽

Plasmid Curing ◽

Gene Deletions ◽

A Genome ◽

Single Clone ◽

Previous Round ◽

Genome Modifications

Abstract The production of optimized strains of a specific phenotype requires the construction and testing of a large number of genome modifications and combinations thereof. Most bacterial iterative genome-editing methods include essential steps to eliminate selection markers, or to cure plasmids. Additionally, the presence of escapers leads to time-consuming separate single clone picking and subsequent cultivation steps. Herein, we report a genome-editing method based on a Rock-Paper-Scissors (RPS) strategy. Each of three constructed sgRNA plasmids can cure, or be cured by, the other two plasmids in the system; plasmids from a previous round of editing can be cured while the current round of editing takes place. Due to the enhanced curing efficiency and embedded double check mechanism, separate steps for plasmid curing or confirmation are not necessary, and only two times of cultivation are needed per genome-editing round. This method was successfully demonstrated in Escherichia coli and Klebsiella pneumoniae with both gene deletions and replacements. To the best of our knowledge, this is the fastest and most robust iterative genome-editing method, with the least times of cultivation decreasing the possibilities of spontaneous genome mutations.

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Progress and Challenges in the Improvement of Ornamental Plants by Genome Editing

Plants ◽

10.3390/plants9060687 ◽

2020 ◽

Vol 9 (6) ◽

pp. 687 ◽

Cited By ~ 3

Author(s):

Chang Ho Ahn ◽

Mummadireddy Ramya ◽

Hye Ryun An ◽

Pil Man Park ◽

Yae-Jin Kim ◽

...

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Genetically Modified Organisms ◽

Ornamental Plants ◽

Safety Evaluation ◽

Evaluation Process ◽

Vase Life ◽

Transcription Activator ◽

A Genome ◽

Dna Specificity

Biotechnological approaches have been used to modify the floral color, size, and fragrance of ornamental plants, as well as to increase disease resistance and vase life. Together with the advancement of whole genome sequencing technologies, new plant breeding techniques have rapidly emerged in recent years. Compared to the early versions of gene editing tools, such as meganucleases (MNs), zinc fingers (ZFNs), and transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) is capable of altering a genome more efficiently and with higher accuracy. Most recently, new CRISPR systems, including base editors and prime editors, confer reduced off-target activity with improved DNA specificity and an expanded targeting scope. However, there are still controversial issues worldwide for the recognition of genome-edited plants, including whether genome-edited plants are genetically modified organisms and require a safety evaluation process. In the current review, we briefly summarize the current progress in gene editing systems and also introduce successful/representative cases of the CRISPR system application for the improvement of ornamental plants with desirable traits. Furthermore, potential challenges and future prospects in the use of genome-editing tools for ornamental plants are also discussed.

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