scholarly journals Tailor-made exopolysaccharides—CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Marius Rütering ◽  
Brady F Cress ◽  
Martin Schilling ◽  
Broder Rühmann ◽  
Mattheos A G Koffas ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Deletion ◽  
State Of The Art ◽  
Paenibacillus Polymyxa ◽  
Vector System ◽  
Wild Type ◽  
Monomer Composition ◽  
Homology Directed Repair ◽  
Highly Efficient ◽  
Micro Organisms

Abstract Application of state-of-the-art genome editing tools like CRISPR-Cas9 drastically increase the number of undomesticated micro-organisms amenable to highly efficient and rapid genetic engineering. Adaptation of these tools to new bacterial families can open up entirely new possibilities for these organisms to accelerate as biotechnologically relevant microbial factories, also making new products economically competitive. Here, we report the implementation of a CRISPR-Cas9 based vector system in Paenibacillus polymyxa, enabling fast and reliable genome editing in this host. Homology directed repair allows for highly efficient deletions of single genes and large regions as well as insertions. We used the system to investigate the yet undescribed biosynthesis machinery for exopolysaccharide (EPS) production in P. polymyxa DSM 365, enabling assignment of putative roles to several genes involved in EPS biosynthesis. Using this simple gene deletion strategy, we generated EPS variants that differ from the wild-type polymer not only in terms of monomer composition, but also in terms of their rheological behavior. The developed CRISPR-Cas9 mediated engineering approach will significantly contribute to the understanding and utilization of socially and economically relevant Paenibacillus species and extend the polymer portfolio.

scholarly journals Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

2018 ◽  
Author(s):  
Wannaporn Ittiprasert ◽  
Victoria H. Mann ◽  
Shannon E. Karinshak ◽  
Avril Coghlan ◽  
Gabriel Rinaldi ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Genome Editing ◽  
Human Macrophage ◽  
Wild Type ◽  
End Joining ◽  
Chromosomal Breakage ◽  
Knock Out ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

scholarly journals CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing

2021 ◽  
Vol 22 (7) ◽  
pp. 3741
Author(s):  
Nina Reuven ◽  
Julia Adler ◽  
Nadav Myers ◽  
Yosef Shaul
Keyword(s):  
Genome Editing ◽  
Selectable Marker ◽  
Small Subset ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Biologically Relevant ◽  
Homology Directed Repair ◽  
Endogenous Genes ◽  
Short Palindromic Repeat ◽  
Single Stranded Oligonucleotide

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is “scarless” since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential TAF1 gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system’s utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR.

scholarly journals Efficient and Modular CRISPR-Cas9 Vector System for Physcomitrella patens

2019 ◽  
Author(s):  
Darren R. Mallett ◽  
Mingqin Chang ◽  
Xiaohang Cheng ◽  
Magdalena Bezanilla
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Dna Sequences ◽  
Physcomitrella Patens ◽  
Fluorescent Proteins ◽  
Stop Codon ◽  
Vector System ◽  
Null Alleles ◽  
Homology Directed Repair ◽  
Multiple Loci

ABSTRACTCRISPR-Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA-guided nuclease to initiate double-strand breaks. Until recently, classical RAD51-mediated homologous recombination has been a powerful tool for gene targeting in the moss Physcomitrella patens. However, CRISPR-Cas9 mediated genome editing in P. patens was shown to be more efficient than traditional homologous recombination (Collonnier et al. 2017). CRISPR-Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR-Cas9 genome editing in P. patens, here we describe the generation and use of a flexible and modular CRISPR-Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology-directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5’ and 3’ ends of gene coding regions. With regards to homology-directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with down-stream near-effortless genotyping.

scholarly journals Highly efficient homology-directed repair using Cas9 protein in Ceratitis capitata

2018 ◽  
Author(s):  
Roswitha A. Aumann ◽  
Marc F. Schetelig ◽  
Irina Häecker
Keyword(s):  
Control Systems ◽  
Genome Editing ◽  
Pest Control ◽  
Genetically Modified Organisms ◽  
Ceratitis Capitata ◽  
Fluorescent Protein ◽  
Great Promise ◽  
Homology Directed Repair ◽  
Highly Efficient ◽  
Ribonucleoprotein Complexes

AbstractBackgroundThe Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing vast economical damage in horticultural systems. A currently used control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. Transgenic approaches hold great promise to improve key aspects of a successful SIT program. However, there is strict or even prohibitive legislation regarding the release of genetically modified organisms (GMO), while novel CRISPR-Cas technologies might allow to develop genetically enhanced strains for SIT programs classified as non-transgenic.ResultsHere we describe highly efficient homology-directed repair genome editing in C. capitata by injecting pre-assembled CRISPR-Cas9 ribonucleoprotein complexes using different guide RNAs and a short single-stranded oligodeoxynucleotide donor to convert an enhanced green fluorescent protein in C. capitata into a blue fluorescent protein. Six out of seven fertile and individually backcrossed G0 individuals generated 57-90% knock-in rate within their total offspring and 70-96% knock-in rate within their phenotypically mutant offspring.ConclusionConsidering the possibility that CRISPR-induced alterations in organisms could be classified as a non-GMO in the US and Europe, our approach to homology-directed repair genome editing can be used to genetically improve strains for pest control systems like SIT without the need to struggle with GMO directives. Furthermore, it can be used to recreate and use mutations, found in classical mutagenesis screens, for pest control systems.

SEM-Based Nanoprobing on 40, 32 and 28 nm CMOS Devices Challenges for Semiconductor Failure Analysis

2013 ◽  
Author(s):  
Erik Paul ◽  
Holger Herzog ◽  
Sören Jansen ◽  
Christian Hobert ◽  
Eckhard Langer
Keyword(s):  
Electron Microscope ◽  
High Resolution ◽  
Scanning Electron Microscope ◽  
Failure Analysis ◽  
Case Studies ◽  
State Of The Art ◽  
Root Cause ◽  
Highly Efficient ◽  
Technology Nodes ◽  
Scanning Electron

Abstract This paper presents an effective device-level failure analysis (FA) method which uses a high-resolution low-kV Scanning Electron Microscope (SEM) in combination with an integrated state-of-the-art nanomanipulator to locate and characterize single defects in failing CMOS devices. The presented case studies utilize several FA-techniques in combination with SEM-based nanoprobing for nanometer node technologies and demonstrate how these methods are used to investigate the root cause of IC device failures. The methodology represents a highly-efficient physical failure analysis flow for 28nm and larger technology nodes.

scholarly journals First genome edited poinsettias: targeted mutagenesis of flavonoid 3′-hydroxylase using CRISPR/Cas9 results in a colour shift

2021 ◽  
Author(s):  
Daria Nitarska ◽  
Robert Boehm ◽  
Thomas Debener ◽  
Rares Calin Lucaciu ◽  
Heidi Halbwirth
Keyword(s):  
Transgenic Plants ◽  
Genome Editing ◽  
Ornamental Plants ◽  
Targeted Mutagenesis ◽  
Cloning And Expression ◽  
Euphorbia Pulcherrima ◽  
Loss Of Function ◽  
Wild Type ◽  
Promising Tool ◽  
Reddish Orange

AbstractThe CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3′-hydroxylase (F3′H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar ‘Christmas Eve’, in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3′H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3′H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3′H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3′H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia.

scholarly journals A secure and highly efficient first-order masking scheme for AES linear operations

Cybersecurity ◽  
2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jingdian Ming ◽  
Yongbin Zhou ◽  
Huizhong Li ◽  
Qian Zhang
Keyword(s):  
Cost Reduction ◽  
Provable Security ◽  
State Of The Art ◽  
Side Channel ◽  
First Order ◽  
Highly Efficient ◽  
Non Linear ◽  
Device Independence ◽  
Practical Implications ◽  
Provably Secure

AbstractDue to its provable security and remarkable device-independence, masking has been widely accepted as a noteworthy algorithmic-level countermeasure against side-channel attacks. However, relatively high cost of masking severely limits its applicability. Considering the high tackling complexity of non-linear operations, most masked AES implementations focus on the security and cost reduction of masked S-boxes. In this paper, we focus on linear operations, which seems to be underestimated, on the contrary. Specifically, we discover some security flaws and redundant processes in popular first-order masked AES linear operations, and pinpoint the underlying root causes. Then we propose a provably secure and highly efficient masking scheme for AES linear operations. In order to show its practical implications, we replace the linear operations of state-of-the-art first-order AES masking schemes with our proposal, while keeping their original non-linear operations unchanged. We implement four newly combined masking schemes on an Intel Core i7-4790 CPU, and the results show they are roughly 20% faster than those original ones. Then we select one masked implementation named RSMv2 due to its popularity, and investigate its security and efficiency on an AVR ATMega163 processor and four different FPGA devices. The results show that no exploitable first-order side-channel leakages are detected. Moreover, compared with original masked AES implementations, our combined approach is nearly 25% faster on the AVR processor, and at least 70% more efficient on four FPGA devices.

scholarly journals Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

2021 ◽  
Vol 7 (2) ◽  
pp. 149
Author(s):  
Sarah-Maria Wege ◽  
Katharina Gejer ◽  
Fabienne Becker ◽  
Michael Bölker ◽  
Johannes Freitag ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Biology ◽  
Gene Deletion ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Model Organism ◽  
Ustilago Maydis ◽  
Genomic Locus ◽  
Targeted Gene Deletion ◽  
Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

scholarly journals Normal peripheral blood neutrophil numbers accompanying ELANE whole gene deletion mutation

2019 ◽  
Vol 3 (16) ◽  
pp. 2470-2473 ◽  
Author(s):  
Marshall S. Horwitz ◽  
Mercy Y. Laurino ◽  
Siobán B. Keel
Keyword(s):  
Genome Editing ◽  
Peripheral Blood ◽  
Gene Deletion ◽  
Deletion Mutation ◽  
Blood Neutrophil ◽  
Normal Peripheral Blood ◽  
Peripheral Blood Neutrophil ◽  
Key Points ◽  
Patient Reported

Key Points The patient reported here, along with collective observations in the literature, suggest that ELANE deletion does not cause neutropenia. Potential therapeutic genome editing involving knockout of the mutant ELANE allele is therefore not expected to produce neutropenia.

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