scholarly journals Efficient marker free CRISPR/Cas9 genome editing for functional analysis of gene families in filamentous fungi

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Tim M. van Leeuwe ◽  
Mark Arentshorst ◽  
Tim Ernst ◽  
Ebru Alazi ◽  
Peter J. Punt ◽  
...  
Keyword(s):  
Genome Editing ◽  
Filamentous Fungi ◽  
Marker Gene ◽  
Gene Families ◽  
Selection Marker ◽  
Dna Fragments ◽  
Additional Advantage ◽  
Multiple Gene ◽  
Large Gene ◽  
Marker Free

Abstract Background CRISPR/Cas9 mediated genome editing has expedited the way of constructing multiple gene alterations in filamentous fungi, whereas traditional methods are time-consuming and can be of mutagenic nature. These developments allow the study of large gene families that contain putatively redundant genes, such as the seven-membered family of crh-genes encoding putative glucan–chitin crosslinking enzymes involved in cell wall biosynthesis. Results Here, we present a CRISPR/Cas9 system for Aspergillus niger using a non-integrative plasmid, containing a selection marker, a Cas9 and a sgRNA expression cassette. Combined with selection marker free knockout repair DNA fragments, a set of the seven single knockout strains was obtained through homology directed repair (HDR) with an average efficiency of 90%. Cas9–sgRNA plasmids could effectively be cured by removing selection pressure, allowing the use of the same selection marker in successive transformations. Moreover, we show that either two or even three separate Cas9–sgRNA plasmids combined with marker-free knockout repair DNA fragments can be used in a single transformation to obtain double or triple knockouts with 89% and 38% efficiency, respectively. By employing this technique, a seven-membered crh-gene family knockout strain was acquired in a few rounds of transformation; three times faster than integrative selection marker (pyrG) recycling transformations. An additional advantage of the use of marker-free gene editing is that negative effects of selection marker gene expression are evaded, as we observed in the case of disrupting virtually silent crh family members. Conclusions Our findings advocate the use of CRISPR/Cas9 to create multiple gene deletions in both a fast and reliable way, while simultaneously omitting possible locus-dependent-side-effects of poor auxotrophic marker expression.

scholarly journals Positive, negative and marker-free strategies for transgenic plant selection

2002 ◽  
Vol 14 (1) ◽  
pp. 01-10 ◽  
Author(s):  
Francisco José Lima Aragão ◽  
Ana Cristina Miranda Brasileiro
Keyword(s):  
Negative Selection ◽  
Marker Gene ◽  
Chloroplast Transformation ◽  
Selection Marker ◽  
Marker Genes ◽  
Plant Selection ◽  
Site Specific ◽  
Selective Agents ◽  
Marker Free ◽  
Flow Through

In this review, the use of the most common selection marker genes on plant transformation and the effects of their respective selective agents are discussed. These genes could be divided in two categories according their mode of action: genes for positive and negative selection. The retention of the marker gene flow through chloroplast transformation is also discussed. Further, strategies to recover marker-free transgenic plants, involving multi-auto-transformation (MAT), co-transformation, site-specific recombination and intragenomic relocation of transgenes through transposable elements are reviewed.

scholarly journals Forced Recycling of an AMA1-Based Genome-Editing Plasmid Allows for Efficient Multiple Gene Deletion/Integration in the Industrial Filamentous Fungus Aspergillus oryzae

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Takuya Katayama ◽  
Hidetoshi Nakamura ◽  
Yue Zhang ◽  
Arnaud Pascal ◽  
Wataru Fujii ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Genetic Engineering ◽  
Genome Editing ◽  
Aspergillus Oryzae ◽  
Filamentous Fungus ◽  
Gene Deletion ◽  
Multiple Gene ◽  
Content Type ◽  
Industrial Strains ◽  
Marker Free

ABSTRACT Filamentous fungi are used for food fermentation and industrial production of recombinant proteins. They also serve as a source of secondary metabolites and are recently expected as hosts for heterologous production of useful secondary metabolites. Multiple-step genetic engineering is required to enhance industrial production involving these fungi, but traditional sequential modification of multiple genes using a limited number of selection markers is laborious. Moreover, efficient genetic engineering techniques for industrial strains have not yet been established. We have previously developed a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9-based mutagenesis technique for the industrial filamentous fungus Aspergillus oryzae, enabling mutation efficiency of 10 to 20%. Here, we improved the CRISPR/Cas9 approach by including an AMA1-based autonomously replicating plasmid harboring the drug resistance marker ptrA. By using the improved mutagenesis technique, we successfully modified A. oryzae wild and industrial strains, with a mutation efficiency of 50 to 100%. Conditional expression of the Aoace2 gene from the AMA1-based plasmid severely inhibited fungal growth. This enabled forced recycling of the plasmid, allowing repeated genome editing. Further, double mutant strains were successfully obtained with high efficiency by expressing two guide RNA molecules from the genome-editing plasmid. Cotransformation of fungal cells with the genome-editing plasmid together with a circular donor DNA enabled marker-free multiplex gene deletion/integration in A. oryzae. The presented repeatable marker-free genetic engineering approach for mutagenesis and gene deletion/integration will allow for efficient modification of multiple genes in industrial fungal strains, increasing their applicability. IMPORTANCE Multiple gene modifications of specific fungal strains are required for achieving industrial-scale production of enzymes and secondary metabolites. In the present study, we developed an efficient multiple genetic engineering technique for the filamentous fungus Aspergillus oryzae. The approach is based on a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system and recycling of an AMA1-based autonomous replicating plasmid. Because the plasmid harbors a drug resistance marker (ptrA), the approach does not require the construction of auxotrophic industrial strains prior to genome editing and allows for forced recycling of the gene-editing plasmid. The established plasmid-recycling technique involves an Aoace2-conditional expression cassette, whose induction severely impairs fungal growth. We used the developed genetic engineering techniques for highly efficient marker-free multiple gene deletion/integration in A. oryzae. The genome-editing approaches established in the present study, which enable unlimited repeatable genetic engineering, will facilitate multiple gene modification of industrially important fungal strains.

scholarly journals Ultra Large Gene Families: A Matter of Adaptation or Genomic Parasites?

Life ◽  
2016 ◽  
Vol 6 (3) ◽  
pp. 32 ◽  
Author(s):  
Philipp Schiffer ◽  
Jan Gravemeyer ◽  
Martina Rauscher ◽  
Thomas Wiehe
Keyword(s):  
Gene Families ◽  
Large Gene

scholarly journals Development of Beet necrotic yellow vein virus ‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

2019 ◽  
Vol 17 (7) ◽  
pp. 1302-1315 ◽  
Author(s):  
Ning Jiang ◽  
Chao Zhang ◽  
Jun‐Ying Liu ◽  
Zhi‐Hong Guo ◽  
Zong‐Ying Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genome Editing ◽  
Plant Genome ◽  
Yellow Vein ◽  
Multiple Gene ◽  
Guide Rna

scholarly journals 230 Genome editing applications in plants: high-throughput CRISPR/Cas editing for crop improvement

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 56-56
Author(s):  
Michael Thomson
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Positional Cloning ◽  
Gene Editing ◽  
Crop Improvement ◽  
Cost Effective ◽  
Gene Families ◽  
Ease Of Use ◽  
Plant Genome ◽  
Wide Range

Abstract The precision and ease of use of CRISPR nucleases, such as Cas9 and Cpf1, for plant genome editing has the potential to accelerate a wide range of applications for crop improvement. For upstream research on gene discovery and validation, rapid gene knock-outs can enable testing of single genes and multi-gene families for functional effects. Large chromosomal deletions can test the function of tandem gene arrays and assist with positional cloning of QTLs by helping to narrow down the target region. Nuclease-deactivated Cas9 fusion proteins with transcriptional activators and repressors can be used to up and down-regulate gene expression. Even more promising, gene insertions and allele replacements can provide the opportunity to rapidly test the effects of different alleles at key loci in the same genetic background, providing a more precise alternative to marker-assisted backcrossing. Recently, Texas A&M AgriLife Research has supported the development of a Crop Genome Editing Lab at Texas A&M working towards optimizing a high-throughput gene editing pipeline and providing an efficient and cost-effective gene editing service for research and breeding groups. The lab is using rice as a model to test and optimize new approaches aimed towards overcoming current bottlenecks. For example, a wealth of genomics data from the rice community enables the development of novel approaches to predict which genes and target modifications may be most beneficial for crop improvement, taking advantage of known major genes, high-resolution GWAS data, multiple high-quality reference genomes, transcriptomics data, and resequencing data from the 3,000 Rice Genomes Project. Current projects have now expanded to work across multiple crops to provide breeding and research groups with a rapid gene editing pipeline to test candidate genes in their programs, with the ultimate goal of developing nutritious, high-yielding, stress-tolerant crops for the future.

scholarly journals A Genome-Wide Analysis of the Pentatricopeptide Repeat (PPR) Gene Family and PPR-Derived Markers for Flesh Color in Watermelon (Citrullus lanatus)

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1125
Author(s):  
Saminathan Subburaj ◽  
Luhua Tu ◽  
Kayoun Lee ◽  
Gwang-Soo Park ◽  
Hyunbae Lee ◽  
...  
Keyword(s):  
Gene Family ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Citrullus Lanatus ◽  
Gene Families ◽  
Nucleotide Polymorphisms ◽  
Pentatricopeptide Repeat ◽  
Flesh Color ◽  
Large Gene ◽  
A Genome

Watermelon (Citrullus lanatus) is an economically important fruit crop grown for consumption of its large edible fruit flesh. Pentatricopeptide-repeat (PPR) encoding genes, one of the large gene families in plants, are important RNA-binding proteins involved in the regulation of plant growth and development by influencing the expression of organellar mRNA transcripts. However, systematic information regarding the PPR gene family in watermelon remains largely unknown. In this comprehensive study, we identified and characterized a total of 422 C. lanatus PPR (ClaPPR) genes in the watermelon genome. Most ClaPPRs were intronless and were mapped across 12 chromosomes. Phylogenetic analysis showed that ClaPPR proteins could be divided into P and PLS subfamilies. Gene duplication analysis suggested that 11 pairs of segmentally duplicated genes existed. In-silico expression pattern analysis demonstrated that ClaPPRs may participate in the regulation of fruit development and ripening processes. Genotyping of 70 lines using 4 single nucleotide polymorphisms (SNPs) from 4 ClaPPRs resulted in match rates of over 0.87 for each validated SNPs in correlation with the unique phenotypes of flesh color, and could be used in differentiating red, yellow, or orange watermelons in breeding programs. Our results provide significant insights for a comprehensive understanding of PPR genes and recommend further studies on their roles in watermelon fruit growth and ripening, which could be utilized for cultivar development of watermelon.

scholarly journals Exploration of the Germline Genome of the Ciliate Chilodonella uncinata through Single-Cell Omics (Transcriptomics and Genomics)

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Xyrus X. Maurer-Alcalá ◽  
Rob Knight ◽  
Laura A. Katz
Keyword(s):  
Single Cell ◽  
Large Scale ◽  
Gc Content ◽  
Gene Families ◽  
Genome Rearrangements ◽  
Paramecium Tetraurelia ◽  
Protein Coding ◽  
Genome Data ◽  
Large Gene ◽  
Disproportionate Number

ABSTRACTSeparate germline and somatic genomes are found in numerous lineages across the eukaryotic tree of life, often separated into distinct tissues (e.g., in plants, animals, and fungi) or distinct nuclei sharing a common cytoplasm (e.g., in ciliates and some foraminifera). In ciliates, germline-limited (i.e., micronuclear-specific) DNA is eliminated during the development of a new somatic (i.e., macronuclear) genome in a process that is tightly linked to large-scale genome rearrangements, such as deletions and reordering of protein-coding sequences. Most studies of germline genome architecture in ciliates have focused on the model ciliatesOxytricha trifallax,Paramecium tetraurelia, andTetrahymena thermophila, for which the complete germline genome sequences are known. Outside of these model taxa, only a few dozen germline loci have been characterized from a limited number of cultivable species, which is likely due to difficulties in obtaining sufficient quantities of “purified” germline DNA in these taxa. Combining single-cell transcriptomics and genomics, we have overcome these limitations and provide the first insights into the structure of the germline genome of the ciliateChilodonella uncinata, a member of the understudied classPhyllopharyngea. Our analyses reveal the following: (i) large gene families contain a disproportionate number of genes from scrambled germline loci; (ii) germline-soma boundaries in the germline genome are demarcated by substantial shifts in GC content; (iii) single-cell omics techniques provide large-scale quality germline genome data with limited effort, at least for ciliates with extensively fragmented somatic genomes. Our approach provides an efficient means to understand better the evolution of genome rearrangements between germline and soma in ciliates.IMPORTANCEOur understanding of the distinctions between germline and somatic genomes in ciliates has largely relied on studies of a few model genera (e.g.,Oxytricha,Paramecium,Tetrahymena). We have used single-cell omics to explore germline-soma distinctions in the ciliateChilodonella uncinata, which likely diverged from the better-studied ciliates ~700 million years ago. The analyses presented here indicate that developmentally regulated genome rearrangements between germline and soma are demarcated by rapid transitions in local GC composition and lead to diversification of protein families. The approaches used here provide the basis for future work aimed at discerning the evolutionary impacts of germline-soma distinctions among diverse ciliates.

scholarly journals Phenylpropanoid Pathway Engineering: An Emerging Approach towards Plant Defense

Pathogens ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 312 ◽  
Author(s):  
Vivek Yadav ◽  
Zhongyuan Wang ◽  
Chunhua Wei ◽  
Aduragbemi Amo ◽  
Bilal Ahmed ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Defense ◽  
Plant Cell Wall ◽  
Shikimate Pathway ◽  
Gene Families ◽  
Phenylpropanoid Pathway ◽  
Pathway Engineering ◽  
Multiple Gene ◽  
Regulator Genes ◽  
Microbial Attack

Pathogens hitting the plant cell wall is the first impetus that triggers the phenylpropanoid pathway for plant defense. The phenylpropanoid pathway bifurcates into the production of an enormous array of compounds based on the few intermediates of the shikimate pathway in response to cell wall breaches by pathogens. The whole metabolomic pathway is a complex network regulated by multiple gene families and it exhibits refined regulatory mechanisms at the transcriptional, post-transcriptional, and post-translational levels. The pathway genes are involved in the production of anti-microbial compounds as well as signaling molecules. The engineering in the metabolic pathway has led to a new plant defense system of which various mechanisms have been proposed including salicylic acid and antimicrobial mediated compounds. In recent years, some key players like phenylalanine ammonia lyases (PALs) from the phenylpropanoid pathway are proposed to have broad spectrum disease resistance (BSR) without yield penalties. Now we have more evidence than ever, yet little understanding about the pathway-based genes that orchestrate rapid, coordinated induction of phenylpropanoid defenses in response to microbial attack. It is not astonishing that mutants of pathway regulator genes can show conflicting results. Therefore, precise engineering of the pathway is an interesting strategy to aim at profitably tailored plants. Here, this review portrays the current progress and challenges for phenylpropanoid pathway-based resistance from the current prospective to provide a deeper understanding.

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