scholarly journals Golden Gate vectors for efficient gene fusion and gene deletion in diverse filamentous fungi

2020 ◽  
Author(s):  
Tim A. Dahlmann ◽  
Dominik Terfehr ◽  
Kordula Becker ◽  
Ines Teichert
Keyword(s):  
Resistance Genes ◽  
Gene Fusion ◽  
Gene Deletion ◽  
Developmental Model ◽  
Selection Marker ◽  
Hygromycin B ◽  
Golden Gate ◽  
One Pot ◽  
Marker Recycling ◽  
Efficient Gene

AbstractThe cloning of plasmids can be time-consuming or expensive. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including the generation of deletion mutants and the localization of gene products. Here, we provide Golden Gate vectors for fast and easy cloning of gene fusion as well as gene deletion vectors applicable to diverse fungi. In Golden Gate cloning, restriction and ligation occur simultaneously in a one-pot reaction. Our vector set contains recognition sites for the commonly used type IIS restriction endonuclease BsaI. We generated plasmids for C- as well as N-terminal tagging with GFP, mRFP and 3xFLAG. For gene deletion, we provide five different donor vectors for selection marker cassettes. These include standard cassettes for hygromycin B, nourseothricin and phleomycin resistance genes as well as FLP/FRT-based marker recycling cassettes for hygromycin B and nourseothricin resistance genes. To make cloning most feasible, we provide robust protocols, namely (1) an overview of cloning procedures described in this paper, (2) specific Golden Gate reaction protocols and (3) standard primers for cloning and sequencing of plasmids and generation of deletion cassettes by PCR and split-marker PCR. We show that our vector set is applicable for the biotechnologically relevant Penicillium chrysogenum and the developmental model system Sordaria macrospora. We thus expect these vectors to be beneficial for other fungi as well. Finally, the vectors can easily be adapted to organisms beyond the kingdom fungi.

Rapid and Efficient Gene Deletion by CRISPR/Cas9

2019 ◽  
pp. 233-247 ◽  
Author(s):  
Signe Neldeborg ◽  
Lin Lin ◽  
Magnus Stougaard ◽  
Yonglun Luo
Keyword(s):  
Gene Deletion ◽  
Efficient Gene

scholarly journals Robust inducible Cre recombinase activity in the human malaria parasite P lasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle

2013 ◽  
Vol 88 (4) ◽  
pp. 687-701 ◽  
Author(s):  
Christine R. Collins ◽  
Sujaan Das ◽  
Eleanor H. Wong ◽  
Nicole Andenmatten ◽  
Robert Stallmach ◽  
...  
Keyword(s):  
Malaria Parasite ◽  
Gene Deletion ◽  
Growth Cycle ◽  
Cre Recombinase ◽  
Human Malaria Parasite ◽  
Human Malaria ◽  
Efficient Gene

scholarly journals Nucleotide Sequence of Plasmid pCNB1 from Comamonas Strain CNB-1 Reveals Novel Genetic Organization and Evolution for 4-Chloronitrobenzene Degradation

2007 ◽  
Vol 73 (14) ◽  
pp. 4477-4483 ◽  
Author(s):  
Ying-Fei Ma ◽  
Jian-Feng Wu ◽  
Sheng-Yue Wang ◽  
Cheng-Ying Jiang ◽  
Yun Zhang ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Resistance Genes ◽  
Gene Deletion ◽  
Degradation Pathway ◽  
Open Reading Frames ◽  
Mercury Resistance ◽  
Dna Molecules ◽  
Chromate Resistance ◽  
Reverse Transcription Pcr ◽  
Reading Frames

ABSTRACT The nucleotide sequence of a new plasmid pCNB1 from Comamonas sp. strain CNB-1 that degrades 4-chloronitrobenzene (4CNB) was determined. pCNB1 belongs to the IncP-1β group and is 91,181 bp in length. A total of 95 open reading frames appear to be involved in (i) the replication, maintenance, and transfer of pCNB1; (ii) resistance to arsenate and chromate; and (iii) the degradation of 4CNB. The 4CNB degradative genes and arsenate resistance genes were located on an extraordinarily large transposon (44.5 kb), proposed as TnCNB1. TnCNB1 was flanked by two IS1071 elements and represents a new member of the composite I transposon family. The 4CNB degradative genes within TnCNB1 were separated by various truncated genes and genetic homologs from other DNA molecules. Genes for chromate resistance were located on another transposon that was similar to the Tn21 transposon of the class II replicative family that is frequently responsible for the mobilization of mercury resistance genes. Resistance to arsenate and chromate were experimentally confirmed, and transcriptions of arsenate and chromate resistance genes were demonstrated by reverse transcription-PCR. These results described a new member of the IncP-1β plasmid family, and the findings suggest that gene deletion and acquisition as well as genetic rearrangement of DNA molecules happened during the evolution of the 4CNB degradation pathway on pCNB1.

scholarly journals Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Jara Radeck ◽  
Daniel Meyer ◽  
Nina Lautenschläger ◽  
Thorsten Mascher
Keyword(s):  
Bacillus Subtilis ◽  
Homologous Recombination ◽  
Free Choice ◽  
Fixed Combination ◽  
Golden Gate ◽  
One Pot ◽  
Natural Competence ◽  
As Species ◽  
Resistance Markers ◽  
Integrative Vectors

Abstract Bacillus subtilis combines natural competence for genetic transformation with highly efficient homologous recombination. These features allow using vectors that integrate into the genome via double homologous recombination. So far, their utilization is restricted by the fixed combination of resistance markers and integration loci, as well as species- or strain-specific regions of homology. To overcome these limitations, we developed a toolbox for the creation of personalized Bacillus vectors in a standardized manner with a focus on fast and easy adaptation of the sequences specifying the integration loci. We based our vector toolkit on the Standard European Vector Architecture (SEVA) to allow the usage of their vector parts. The Bacillus SEVA siblings are assembled via efficient one-pot Golden Gate reactions from four entry parts with the choice of four different enzymes. The toolbox contains seven Bacillus resistance markers, two Escherichia coli origins of replication, and a free choice of integration loci. Vectors can be customized with a cargo, before or after vector assembly, and could be used in different B. subtilis strains and potentially beyond. Our adaptation of the SEVA-standard provides a powerful and standardized toolkit for the convenient creation of personalized Bacillus vectors.

scholarly journals A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Qusai Al Abdallah ◽  
Wenbo Ge ◽  
Jarrod R. Fortwendel
Keyword(s):  
Drug Resistance ◽  
Gene Deletion ◽  
Gene Replacement ◽  
Antifungal Drug ◽  
Wild Type ◽  
Gene Manipulation ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
Efficient Gene

ABSTRACT Tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A. fumigatus. We anticipate that our system will simplify genome editing in A. fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A. fumigatus. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a novel genome-editing system that has been successfully established in Aspergillus fumigatus. However, the current state of the technology relies heavily on DNA-based expression cassettes for delivering Cas9 and the guide RNA (gRNA) to the cell. Therefore, the power of the technology is limited to strains that are engineered to express Cas9 and gRNA. To overcome such limitations, we developed a simple and universal CRISPR-Cas9 system for gene deletion that works across different genetic backgrounds of A. fumigatus. The system employs in vitro assembly of dual Cas9 ribonucleoproteins (RNPs) for targeted gene deletion. Additionally, our CRISPR-Cas9 system utilizes 35 to 50 bp of flanking regions for mediating homologous recombination at Cas9 double-strand breaks (DSBs). As a proof of concept, we first tested our system in the ΔakuB (ΔakuB ku80 ) laboratory strain and generated high rates (97%) of gene deletion using 2 µg of the repair template flanked by homology regions as short as 35 bp. Next, we inspected the portability of our system across other genetic backgrounds of A. fumigatus, namely, the wild-type strain Af293 and a clinical isolate, A. fumigatus DI15-102. In the Af293 strain, 2 µg of the repair template flanked by 35 and 50 bp of homology resulted in highly efficient gene deletion (46% and 74%, respectively) in comparison to classical gene replacement systems. Similar deletion efficiencies were also obtained in the clinical isolate DI15-102. Taken together, our data show that in vitro-assembled Cas9 RNPs coupled with microhomology repair templates are an efficient and universal system for gene manipulation in A. fumigatus. IMPORTANCE Tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A. fumigatus. We anticipate that our system will simplify genome editing in A. fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A. fumigatus.

scholarly journals Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity and bias profiling

2018 ◽  
Author(s):  
Potapov Vladimir ◽  
Jennifer L. Ong ◽  
Rebecca B. Kucera ◽  
Bradley W. Langhorst ◽  
Katharina Bilotti ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Dna Assembly ◽  
Golden Gate ◽  
End Joining ◽  
One Pot ◽  
Data Set ◽  
Flexible Assembly ◽  
Comprehensive Method ◽  
Sequence Dependent ◽  
Single Reaction

ABSTRACTModern synthetic biology depends on the manufacture of large DNA constructs from libraries of genes, regulatory elements or other genetic parts. Type IIS restriction enzyme-dependent DNA assembly methods (e.g., Golden Gate) enable rapid one-pot, ordered, multi-fragment DNA assembly, facilitating the generation of high-complexity constructs. The order of assembly of genetic parts is determined by the ligation of flanking Watson-Crick base-paired overhangs. The ligation of mismatched overhangs leads to erroneous assembly, and the need to avoid such pairings has typically been accomplished by using small sets of empirically vetted junction pairs, limiting the number of parts that can be joined in a single reaction. Here, we report the use of a comprehensive method for profiling end-joining ligation fidelity and bias to predict highly accurate sets of connections for ligation-based DNA assembly methods. This data set allows quantification of sequence-dependent ligation efficiency and identification of mismatch-prone pairings. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions, and enabled efficient assembly of a lac cassette from up to 24-fragments in a single reaction. Application of the ligation fidelity profile to inform choice of junctions thus enables highly flexible assembly design, with >20 fragments in a single reaction.

scholarly journals Validating molecular markers for barley leaf rust resistance genes Rph20 and Rph24

2020 ◽  
Author(s):  
PM Dracatos ◽  
RF Park ◽  
D Singh
Keyword(s):  
Molecular Markers ◽  
Leaf Rust ◽  
Resistance Genes ◽  
Leaf Rust Resistance ◽  
Puccinia Hordei ◽  
Adult Plant ◽  
Barley Leaf Rust ◽  
Efficient Gene ◽  
Barley Leaf ◽  
Barley Germplasm

Improving resistance to barley leaf rust (caused by Puccinia hordei) is an important breeding objective in most barley growing regions worldwide. The development and subsequent utilisation of high-throughput PCR-based co-dominant molecular markers remains an effective approach to select genotypes with multiple effective resistance genes, permitting efficient gene deployment and stewardship. The genes Rph20 and Rph24 confer widely effective adult plant resistance (APR) to leaf rust, are common in European and Australian barley germplasm (often in combination), and act interactively to confer high levels of resistance (Dracatos et al. 2015; Zeims et al. 2017; Singh et al. 2018). Here we report on the development and validation of co-dominant insertion-deletion (indel) based PCR markers that are highly predictive for the Rph20 and Rph24 resistances.

scholarly journals Efficient Gene Editing Through an Intronic Selection Marker in Cells

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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