CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

Abstract Background Gene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in various filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™). Results We present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0–40% of isolates. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum. Conclusions Using an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum for use as a research tool, for understanding the controls of secondary metabolism and hyphal development and validating prototypes of strains produced using traditional methods for strain improvement.

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CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

10.1101/2021.09.06.459129 ◽

2021 ◽

Author(s):

Fiona M Wilson ◽

Richard J Harrison

Keyword(s):

Gene Expression ◽

Transient Expression ◽

Gene Editing ◽

Polyketide Synthase ◽

Marker Gene ◽

Constitutive Expression ◽

Cost Effective ◽

Editing Efficiency ◽

Base Editing ◽

Fusarium Venenatum

AbstractBackgroundGene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in a number of filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™).ResultsWe present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0-40% of isolates, with evidence of off-target editing. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum.ConclusionsUsing an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum, such as modulation of gene expression, gene insertion, base editing and prime editing. These tools will facilitate an understanding of the controls of secondary metabolism and hyphal development during fermentation of F. venenatum for mycoprotein production and may be used to validate prototypes of strains for improvement using classical means, enabling more cost-effective and sustainable production of this industrially important fungus.

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CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

10.21203/rs.3.rs-903002/v1 ◽

2021 ◽

Author(s):

Fiona Wilson ◽

Richard J Harrison

Keyword(s):

Gene Expression ◽

Transient Expression ◽

Gene Editing ◽

Polyketide Synthase ◽

Marker Gene ◽

Constitutive Expression ◽

Cost Effective ◽

Editing Efficiency ◽

Base Editing ◽

Fusarium Venenatum

Abstract Background Gene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in a number of filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™). Results We present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0–40% of isolates, with evidence of off-target editing. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum. Conclusions Using an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum, such as modulation of gene expression, gene insertion, base editing and prime editing. These tools will facilitate an understanding of the controls of secondary metabolism and hyphal development during fermentation of F. venenatum for mycoprotein production and may be used to validate prototypes of strains for improvement using classical means, enabling more cost-effective and sustainable production of this industrially important fungus.

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Efficient Gene Editing Through an Intronic Selection Marker in Cells

10.21203/rs.3.rs-739001/v1 ◽

2021 ◽

Author(s):

Shang Wang ◽

Yuqing Li ◽

Li Zhong ◽

Kai Wu ◽

Ruhua Zhang ◽

...

Keyword(s):

Gene Editing ◽

Selection Marker ◽

End Joining ◽

Dna Variation ◽

Editing Efficiency ◽

Endogenous Gene ◽

Positive Effects ◽

Efficient Gene ◽

Almost All ◽

In Cells

Abstract Background Gene editing technology has provided researchers with the ability to modify genome sequences in almost all eukaryotes. Gene-edited cell lines are being used with increasing frequency in both bench research and targeted therapy. Despite the great importance and universality of gene editing, however, precision and efficiency are hard to achieve with the prevailing editing strategies, such as homology-directed DNA repair (HDR) and the use of base editors (BEs). Results & Discussion Our group has developed a novel gene editing technology to indicate DNA variation with an independent selection marker using an HDR strategy, which we named Gene Editing through an Intronic Selection marker (GEIS). GEIS uses a simple process to avoid nonhomologous end joining (NHEJ)-mediated false-positive effects and achieves editing efficiency as high as 91% without disturbing endogenous gene splicing and expression. We re-examined the correlation of the conversion tract and editing efficiency, and our data suggest that GEIS has the potential to edit approximately 99% of gene editing targets in human and mouse cells. The results of further comprehensive analysis suggest that the strategy may be useful for introducing multiple DNA variations in cells.

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Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

Journal of Fungi ◽

10.3390/jof7070505 ◽

2021 ◽

Vol 7 (7) ◽

pp. 505

Author(s):

Ping Zhang ◽

Yu Wang ◽

Chenxi Li ◽

Xiaoyu Ma ◽

Lan Ma ◽

...

Keyword(s):

Genome Editing ◽

Fungal Pathogens ◽

Gene Editing ◽

Recombination Frequency ◽

Target Sequence ◽

Widespread Application ◽

Genetic Studies ◽

Efficient Gene ◽

Cryptococcus Species ◽

Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

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Validation of gene editing efficiency with CRISPR-Cas9 system directly in rat zygotes using electroporationmediated delivery and embryo culture

MethodsX ◽

10.1016/j.mex.2021.101419 ◽

2021 ◽

pp. 101419

Author(s):

Anil K Challa ◽

Denise Stanford ◽

Antonio Allen ◽

Lawrence Rasmussen ◽

Ferdinand K Amanor ◽

...

Keyword(s):

Embryo Culture ◽

Gene Editing ◽

Editing Efficiency

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Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

International Journal of Molecular Sciences ◽

10.3390/ijms20153623 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3623 ◽

Cited By ~ 5

Author(s):

Tobias Bruegmann ◽

Khira Deecke ◽

Matthias Fladung

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Constitutive Expression ◽

Gc Content ◽

Seed Region ◽

Research Topics ◽

Target Region ◽

Cas9 Nuclease ◽

Different Types ◽

Poplar Hybrid

CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

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Design of time-delayed safety switches for CRISPR gene therapy

10.1101/2021.04.17.440285 ◽

2021 ◽

Author(s):

Dashan Sun

Keyword(s):

Gene Therapy ◽

Immune Response ◽

Time Delay ◽

Cell Lines ◽

Gene Editing ◽

Drug Accumulation ◽

Work Time ◽

Crispr System ◽

Efficient Gene ◽

Target Effect

CRISPR system is a powerful gene editing tool which has already been reported to address a variety of gene relevant diseases in different cell lines. However, off-target effect and immune response caused by Cas9 remain two fundamental problems. In our work, time-delayed safety switches are designed based on either artificial ultrasensitivity transmission module or intrinsic time delay in biomolecular activities. By addressing gene therapy efficiency, off-target effect, immune response and drug accumulation, we hope our safety switches may offer inspiration in realizing safe and efficient gene therapy in humans.

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