scholarly journals Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation

2018 ◽  
Author(s):  
Jana Ordon ◽  
Mauro Bressan ◽  
Carola Kretschmer ◽  
Luca Dall’Osto ◽  
Sylvestre Marillonnet ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Frequency ◽  
Single Gene ◽  
Arabidopsis Genome ◽  
Regulatory Sequences ◽  
Expression Systems ◽  
Ubiquitin Promoter ◽  
Single Generation ◽  
Gene Redundancy ◽  
Mutant Lines

AbstractGenetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.

scholarly journals Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation

2019 ◽  
Vol 20 (1) ◽  
pp. 151-162 ◽  
Author(s):  
Jana Ordon ◽  
Mauro Bressan ◽  
Carola Kretschmer ◽  
Luca Dall’Osto ◽  
Sylvestre Marillonnet ◽  
...  
Keyword(s):  
Genome Editing ◽  
Arabidopsis Genome ◽  
Expression Systems ◽  
Single Generation ◽  
Highly Efficient

Kv3.1–Kv3.2 Channels Underlie a High-Voltage–Activating Component of the Delayed Rectifier K+ Current in Projecting Neurons From the Globus Pallidus

1999 ◽  
Vol 82 (3) ◽  
pp. 1512-1528 ◽  
Author(s):  
R. Hernández-Pineda ◽  
A. Chow ◽  
Y. Amarillo ◽  
H. Moreno ◽  
M. Saganich ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Globus Pallidus ◽  
High Frequency ◽  
Calcium Binding ◽  
Repetitive Firing ◽  
Delayed Rectifier ◽  
Expression Systems ◽  
Electrophysiological Properties ◽  
Channel Proteins ◽  
Heterologous Expression Systems

The globus pallidus plays central roles in the basal ganglia circuitry involved in movement control as well as in cognitive and emotional functions. There is therefore great interest in the anatomic and electrophysiological characterization of this nucleus. Most pallidal neurons are GABAergic projecting cells, a large fraction of which express the calcium binding protein parvalbumin (PV). Here we show that PV-containing pallidal neurons coexpress Kv3.1 and Kv3.2 K+ channel proteins and that both Kv3.1 and Kv3.2 antibodies coprecipitate both channel proteins from pallidal membrane extracts solubilized with nondenaturing detergents, suggesting that the two channel subunits are forming heteromeric channels. Kv3.1 and Kv3.2 channels have several unusual electrophysiological properties when expressed in heterologous expression systems and are thought to play special roles in neuronal excitability including facilitating sustained high-frequency firing in fast-spiking neurons such as interneurons in the cortex and the hippocampus. Electrophysiological analysis of freshly dissociated pallidal neurons demonstrates that these cells have a current that is nearly identical to the currents expressed by Kv3.1 and Kv3.2 proteins in heterologous expression systems, including activation at very depolarized membrane potentials (more positive than −10 mV) and very fast deactivation rates. These results suggest that the electrophysiological properties of native channels containing Kv3.1 and Kv3.2 proteins in pallidal neurons are not significantly affected by factors such as associated subunits or postranslational modifications that result in channels having different properties in heterologous expression systems and native neurons. Most neurons in the globus pallidus have been reported to fire sustained trains of action potentials at high-frequency. Kv3.1–Kv3.2 voltage-gated K+channels may play a role in helping maintain sustained high-frequency repetitive firing as they probably do in other neurons.

scholarly journals Progress in Gene Therapy to Prevent Retinal Ganglion Cell Loss in Glaucoma and Leber’s Hereditary Optic Neuropathy

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Sara E. Ratican ◽  
Andrew Osborne ◽  
Keith R. Martin
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Optic Nerve ◽  
Genome Editing ◽  
Optic Neuropathy ◽  
Single Gene ◽  
Leber’S Hereditary Optic Neuropathy ◽  
Leber's Hereditary Optic Neuropathy ◽  
Potential Use ◽  
Hereditary Optic Neuropathy

The eye is at the forefront of the application of gene therapy techniques to medicine. In the United States, a gene therapy treatment for Leber’s congenital amaurosis, a rare inherited retinal disease, recently became the first gene therapy to be approved by the FDA for the treatment of disease caused by mutations in a specific gene. Phase III clinical trials of gene therapy for other single-gene defect diseases of the retina and optic nerve are also currently underway. However, for optic nerve diseases not caused by single-gene defects, gene therapy strategies are likely to focus on slowing or preventing neuronal death through the expression of neuroprotective agents. In addition to these strategies, there has also been recent interest in the potential use of precise genome editing techniques to treat ocular disease. This review focuses on recent developments in gene therapy techniques for the treatment of glaucoma and Leber’s hereditary optic neuropathy (LHON). We discuss recent successes in clinical trials for the treatment of LHON using gene supplementation therapy, promising neuroprotective strategies that have been employed in animal models of glaucoma and the potential use of genome editing techniques in treating optic nerve disease.

scholarly journals Genome Editing Human Pluripotent Stem Cells to Model β-Cell Disease and Unmask Novel Genetic Modifiers

2021 ◽  
Vol 12 ◽  
Author(s):  
Matthew N. George ◽  
Karla F. Leavens ◽  
Paul Gadue
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Genetic Disease ◽  
Single Gene ◽  
Monogenic Diabetes ◽  
Human Pluripotent Stem Cells ◽  
Genetic Modifiers ◽  
Β Cell ◽  
Protein Coding

A mechanistic understanding of the genetic basis of complex diseases such as diabetes mellitus remain elusive due in large part to the activity of genetic disease modifiers that impact the penetrance and/or presentation of disease phenotypes. In the face of such complexity, rare forms of diabetes that result from single-gene mutations (monogenic diabetes) can be used to model the contribution of individual genetic factors to pancreatic β-cell dysfunction and the breakdown of glucose homeostasis. Here we review the contribution of protein coding and non-protein coding genetic disease modifiers to the pathogenesis of diabetes subtypes, as well as how recent technological advances in the generation, differentiation, and genome editing of human pluripotent stem cells (hPSC) enable the development of cell-based disease models. Finally, we describe a disease modifier discovery platform that utilizes these technologies to identify novel genetic modifiers using induced pluripotent stem cells (iPSC) derived from patients with monogenic diabetes caused by heterozygous mutations.

scholarly journals Translation elongation factor 1A2 is encoded by one of four closely related eef1a genes and is dispensable for survival in zebrafish

2020 ◽  
Vol 40 (1) ◽  
Author(s):  
Nwamaka J. Idigo ◽  
Dinesh C. Soares ◽  
Catherine M. Abbott
Keyword(s):  
De Novo ◽  
Single Gene ◽  
Expression Patterns ◽  
Elongation Factor ◽  
Model Organisms ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Tissue Specific ◽  
Elongation Factor 1A2 ◽  
Mutant Lines

Abstract Zebrafish are valuable model organisms for the study of human single-gene disorders: they are genetically manipulable, their development is well understood, and mutant lines with measurable, disease-appropriate phenotypic abnormalities can be used for high throughput drug screening approaches. However, gene duplication events in zebrafish can result in redundancy of gene function, masking loss-of-function phenotypes and thus confounding this approach to disease modelling. Furthermore, recent studies have yielded contrasting results depending on whether specific genes are targeted using genome editing to make mutant lines, or whether morpholinos are used (morphants). De novo missense mutations in the human gene EEF1A2, encoding a tissue-specific translation elongation factor, cause severe neurodevelopmental disorders; there is a real need for a model system to study these disorders and we wanted to explore the possibility of a zebrafish model. We identified four eef1a genes and examined their developmental and tissue-specific expression patterns: eef1a1l1 is first to be expressed while eef1a2 is only detected later during development. We then determined the effects of introducing null mutations into translation elongation factor 1A2 (eEF1A2) in zebrafish using CRISPR/Cas9 gene editing, in order to compare the results with previously described morphants, and with severe neurodegenerative lethal phenotype of eEF1A2-null mice. In contrast with both earlier analyses in zebrafish using morpholinos and with the mouse eEF1A2-null mice, disruption of the eef1a2 gene in zebrafish is compatible with normal lifespan. The resulting lines, however, may provide a valuable platform for studying the effects of expression of mutant human eEF1A2 mRNA.

scholarly journals Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 467
Author(s):  
Min Hao ◽  
Zhaoguan Wang ◽  
Hongyan Qiao ◽  
Peng Yin ◽  
Jianjun Qiao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
Genome Engineering ◽  
Single Gene ◽  
Rolling Circle Replication ◽  
Rolling Circle ◽  
Cas9 Nuclease ◽  
Dynamic Genome ◽  
Circular Ssdna

As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.

scholarly journals Microhomologies are prevalent at Cas9-induced larger deletions

2019 ◽  
Vol 47 (14) ◽  
pp. 7402-7417 ◽  
Author(s):  
Dominic D G Owens ◽  
Adam Caulder ◽  
Vincent Frontera ◽  
Joe R Harman ◽  
Alasdair J Allan ◽  
...  
Keyword(s):  
Genome Editing ◽  
Biomedical Research ◽  
High Frequency ◽  
Cultured Cells ◽  
Mouse Embryos ◽  
Deletion Breakpoint ◽  
End Joining ◽  
Crispr System ◽  
Cas9 Nuclease ◽  
Target Sites

Abstract The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.

scholarly journals High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

2011 ◽  
Vol 8 (9) ◽  
pp. 753-755 ◽  
Author(s):  
Fuqiang Chen ◽  
Shondra M Pruett-Miller ◽  
Yuping Huang ◽  
Monika Gjoka ◽  
Katarzyna Duda ◽  
...  
Keyword(s):  
Genome Editing ◽  
Zinc Finger ◽  
High Frequency ◽  
Zinc Finger Nucleases

scholarly journals Arabidopsis Ca2+-ATPases 1, 2, and 7 in the endoplasmic reticulum contribute to growth and pollen fitness

2021 ◽  
Author(s):  
Maryam Rahmati Ishka ◽  
Elizabeth Brown ◽  
Alexa Rosenberg ◽  
Shawn Romanowsky ◽  
James A Davis ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Endoplasmic Reticulum ◽  
Salicylic Acid ◽  
High Frequency ◽  
Male Fertility ◽  
Single Gene ◽  
Transmission Efficiency ◽  
Marker Genes ◽  
Pathogenesis Related ◽  
Gene Knockouts

Abstract Generating cellular Ca2+ signals requires coordinated transport activities from both Ca2+ influx and efflux pathways. In Arabidopsis (Arabidopsis thaliana), multiple efflux pathways exist, some of which involve Ca2+-pumps belonging to the Autoinhibited Ca2+-ATPase (ACA) family. Here we show that ACA1, 2, and 7 localize to the endoplasmic reticulum (ER) and are important for plant growth and pollen fertility. While phenotypes for plants harboring single gene knockouts (KOs) were weak or undetected, a triple KO of aca1/2/7 displayed a 2.6-fold decrease in pollen transmission efficiency, whereas inheritance through female gametes was normal. The triple KO also resulted in smaller rosettes showing a high frequency of lesions. Both vegetative and reproductive phenotypes were rescued by transgenes encoding either ACA1, 2, or 7, suggesting that all three isoforms are biochemically redundant. Lesions were suppressed by expression of a transgene encoding NahG, an enzyme that degrades salicylic acid (SA). Triple KO mutants showed elevated mRNA expression for two SA-inducible marker genes, PR1 (Pathogenesis-related 1) and PR2. The aca1/2/7 lesion phenotype was similar but less severe than SA-dependent lesions associated with a double KO of vacuolar pumps aca4 and 11. Imaging of Ca2+ dynamics triggered by blue light or the pathogen elicitor flg22 revealed that aca1/2/7 mutants display Ca2+ transients with increased magnitudes and durations. Together, these results indicate that ER-localized ACAs play important roles in regulating Ca2+ signals, and that the loss of these pumps results in male fertility and vegetative growth deficiencies.

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