Synthetic Notch-Receptor-Mediated Transmission of a Transient Signal into Permanent Information via CRISPR/Cas9-Based Genome Editing

Synthetic receptor biology and genome editing are emerging techniques, both of which are currently beginning to be used in preclinical and clinical applications. We were interested in whether a combination of these techniques approaches would allow for the generation of a novel type of reporter cell that would recognize transient cellular events through specifically designed synthetic receptors and would permanently store information about these events via associated gene editing. Reporting cells could be used in the future to detect alterations in the cellular microenvironment, including degenerative processes or malignant transformation into cancer cells. Here, we explored synthetic Notch (synNotch) receptors expressed in human embryonic kidney cells to investigate the efficacy of antigen recognition events in a time- and dose-dependent manner. First, we evaluated the most suitable conditions for synNotch expression based on dsRed-Express fluorophore expression. Then, we used a synNotch receptor coupled to transcriptional activators to induce the expression of a Cas9 nuclease targeted to a specific genomic DNA site. Our data demonstrate that recognition of various specific antigens via synNotch receptors robustly induced Cas9 expression and resulted in an indel formation frequency of 34.5%–45.5% at the targeted CXCR4 locus. These results provide proof of concept that reporter cells can be designed to recognize a given event and to store transient information permanently in their genomes.

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A screen of FDA-approved drugs identifies inhibitors of protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3)

Scientific Reports ◽

10.1038/s41598-021-89668-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Dylan R. Rivas ◽

Mark Vincent C. Dela Cerna ◽

Caroline N. Smith ◽

Shilpa Sampathi ◽

Blaine G. Patty ◽

...

Keyword(s):

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Dependent Manner ◽

Allosteric Site ◽

Human Embryonic Kidney Cells ◽

Protein Tyrosine ◽

Tumor Cell Migration ◽

Colorectal Cancer Cells ◽

Approved Drugs ◽

Fda Approved Drugs

AbstractProtein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) is highly expressed in a variety of cancers, where it promotes tumor cell migration and metastasis leading to poor prognosis. Despite its clinical significance, small molecule inhibitors of PRL-3 are lacking. Here, we screened 1443 FDA-approved drugs for their ability to inhibit the activity of the PRL phosphatase family. We identified five specific inhibitors for PRL-3 as well as one selective inhibitor of PRL-2. Additionally, we found nine drugs that broadly and significantly suppressed PRL activity. Two of these broad-spectrum PRL inhibitors, Salirasib and Candesartan, blocked PRL-3-induced migration in human embryonic kidney cells with no impact on cell viability. Both drugs prevented migration of human colorectal cancer cells in a PRL-3 dependent manner and were selective towards PRLs over other phosphatases. In silico modeling revealed that Salirasib binds a putative allosteric site near the WPD loop of PRL-3, while Candesartan binds a potentially novel targetable site adjacent to the CX5R motif. Inhibitor binding at either of these sites is predicted to trap PRL-3 in a closed conformation, preventing substrate binding and inhibiting function.

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An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

Molecular Biology Reports ◽

10.1007/s11033-020-06125-8 ◽

2021 ◽

Author(s):

Eugene V. Gasanov ◽

Justyna Jędrychowska ◽

Michal Pastor ◽

Malgorzata Wiweger ◽

Axel Methner ◽

...

Keyword(s):

Genome Editing ◽

High Efficiency ◽

Target Site ◽

Proof Of Concept ◽

Intervention Site ◽

End Joining ◽

Guide Rna ◽

Guide Rnas ◽

Cas9 Nuclease ◽

Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

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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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The retromer complex safeguards against neural progenitor-derived tumorigenesis by regulating Notch receptor trafficking

eLife ◽

10.7554/elife.38181 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 5

Author(s):

Bo Li ◽

Chouin Wong ◽

Shihong Max Gao ◽

Rulan Zhang ◽

Rongbo Sun ◽

...

Keyword(s):

Notch Signaling ◽

Molecular Mechanisms ◽

Neural Progenitor ◽

Tumor Formation ◽

Neural Progenitors ◽

Notch Receptor ◽

Dependent Manner ◽

Cell Fate Decisions ◽

Retromer Complex ◽

Signaling Activation

The correct establishment and maintenance of unidirectional Notch signaling are critical for the homeostasis of various stem cell lineages. However, the molecular mechanisms that prevent cell-autonomous ectopic Notch signaling activation and deleterious cell fate decisions remain unclear. Here we show that the retromer complex directly and specifically regulates Notch receptor retrograde trafficking in Drosophila neuroblast lineages to ensure the unidirectional Notch signaling from neural progenitors to neuroblasts. Notch polyubiquitination mediated by E3 ubiquitin ligase Itch/Su(dx) is inherently inefficient within neural progenitors, relying on retromer-mediated trafficking to avoid aberrant endosomal accumulation of Notch and cell-autonomous signaling activation. Upon retromer dysfunction, hypo-ubiquitinated Notch accumulates in Rab7+ enlarged endosomes, where it is ectopically processed and activated in a ligand-dependent manner, causing progenitor-originated tumorigenesis. Our results therefore unveil a safeguard mechanism whereby retromer retrieves potentially harmful Notch receptors in a timely manner to prevent aberrant Notch activation-induced neural progenitor dedifferentiation and brain tumor formation.

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Effect of interferon on colony formation in culture by blast cell progenitors in acute myeloblastic leukemia

Blood ◽

10.1182/blood.v56.3.549.549 ◽

1980 ◽

Vol 56 (3) ◽

pp. 549-552 ◽

Cited By ~ 31

Author(s):

R Taetle ◽

RN Buick ◽

EA McCulloch

Keyword(s):

Colony Formation ◽

Antitumor Agents ◽

Myelogenous Leukemia ◽

Dependent Manner ◽

Self Renewal ◽

Cell Culture Techniques ◽

Culture Techniques ◽

Cellular Events ◽

Acute Myelogenous ◽

Dose Dependent

Abstract The effect of purified human fibroblast interferon on primary and secondary colony formation by blast progenitors from the peripheral blood of patients with acute myelogenous leukemia was examined. Interferon inhibited blast progenitors and normal granulocyte/macrophage progenitors (CFU-C) in a dose-dependent manner. The magnitude of this effect on blast progenitors and CFU was similar. Interferon also inhibited secondary plating of blast progenitors (self- renewal). This effect was in marked contrast to the effect of adriamycin, which reduced primary plating efficiency of blast progenitors but did not affect self-renewal. Inhibition of blast progenitor proliferation by interferon was markedly reduced when interferon was added after 24 hr of culture and was absent when added after 72 hr. Inhibition of self-renewal was observed even when interferon was added at 72 hr. We conclude that interferon inhibits both primary proliferation and self-renewal of blast progenitors and that this effect is not due to reduction in the number of primary colonies. These experiments provide an example of how cell culture techniques may be used to test antitumor agents for effects on important cellular events other than general cytotoxicity.

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Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing

Genes ◽

10.3390/genes11111311 ◽

2020 ◽

Vol 11 (11) ◽

pp. 1311

Author(s):

Liuru Su ◽

Chenggang Shi ◽

Xin Huang ◽

Yiquan Wang ◽

Guang Li

Keyword(s):

Animal Model ◽

Thermal Treatment ◽

Genome Editing ◽

Phylogenetic Position ◽

Transcription Activator ◽

Cell Stage ◽

System A ◽

Cas9 Nuclease ◽

Cas9 Protein ◽

Amphioxus Genome

The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrates due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) have been adopted in amphioxus genome editing, its labor-intensive construction of TALEN proteins limits its usage in many laboratories. Here we reported an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data showed that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs caused no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at the two-cell stage, or of Cas9 protein and sgRNAs before fertilization, can execute efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations with efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrated that monomerization of sgRNAs via thermal treatment or modifying the sgRNA structure could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.

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

Cells ◽

10.3390/cells9020467 ◽

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.

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Microhomologies are prevalent at Cas9-induced larger deletions

Nucleic Acids Research ◽

10.1093/nar/gkz459 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7402-7417 ◽

Cited By ~ 20

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.

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Genome-scale CRISPR screens are efficient in non-homologous end-joining deficient cells

Scientific Reports ◽

10.1038/s41598-019-52078-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Joana Ferreira da Silva ◽

Sejla Salic ◽

Marc Wiedner ◽

Paul Datlinger ◽

Patrick Essletzbichler ◽

...

Keyword(s):

Genome Editing ◽

Large Scale ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

End Joining ◽

Knock Out ◽

Genetic Ablation ◽

Alternative End Joining ◽

Non Homologous End Joining ◽

Genome Scale

Abstract The mutagenic repair of Cas9 generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). In this study, by taking focused as well as genome-wide approaches, we show that this pathway is dispensable for the repair of such lesions. Genetic ablation of NHEJ is fully compensated for by alternative end joining (alt-EJ), in a POLQ-dependent manner, resulting in a distinct repair signature with larger deletions that may be exploited for large-scale genome editing. Moreover, we show that cells deficient for both NHEJ and alt-EJ were still able to repair CRISPR-mediated DNA double-strand breaks, highlighting how little is yet known about the mechanisms of CRISPR-based genome editing.

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Ketamine Inhibits Monoamine Transporters Expressed in Human Embryonic Kidney 293 Cells

Anesthesiology ◽

10.1097/00000542-199803000-00029 ◽

1998 ◽

Vol 88 (3) ◽

pp. 768-774 ◽

Cited By ~ 76

Author(s):

Mitsuhiro Nishimura ◽

Kohji Sato ◽

Tomoya Okada ◽

Ikuto Yoshiya ◽

Patrick Schloss ◽

...

Keyword(s):

Eukaryotic Expression ◽

Psychotic Symptoms ◽

Kidney Cells ◽

Dependent Manner ◽

Monoamine Transporters ◽

Human Embryonic Kidney ◽

General Anesthetic ◽

Serotonin Transporters ◽

Human Embryonic Kidney Cells ◽

Dose Dependent

Background Ketamine has been characterized as having psychotomimetic and sympathomimetic effects. These symptoms have raised the possibility that ketamine affects monoaminergic neurotransmission. To elucidate the relation between ketamine and monoamine transporters, the authors constructed three cell lines that stably express the norepinephrine, dopamine, and serotonin transporters and investigated the effects of ketamine on these transporters. Methods Human embryonic kidney cells were transfected using the Chen-Okayama method with the human norepinephrine, rat dopamine, and rat serotonin transporter cDNA subcloned into the eukaryotic expression vector. Using cells stably expressing these transporters, the authors investigated the effects of ketamine on the uptake of these compounds and compared them with those of pentobarbital. Results Inhibition analysis showed that ketamine significantly inhibited the uptake of all three monoamine transporters in a dose-dependent manner. The Ki (inhibition constant) values of ketamine on the norepinephrine, dopamine, and serotonin transporters were 66.8 microM, 62.9 microM, and 162 microM, respectively. Pentobarbital, a typical general anesthetic agent with no psychotic symptoms, did not affect the uptake of monoamines, however. Further, neither the glycine transporter 1 nor the glutamate/aspartate transporter was affected by ketamine, indicating that ketamine preferentially inhibits monoamine transporters. Conclusions Ketamine inhibited monoamine transporters expressed in human embryonic kidney cells in a dose-dependent manner. This result suggests that the ketamine-induced inhibition of monoamine transporters might contribute to its psychotomimetic and sympathomimetic effects through potentiating monoaminergic neurotransmission.

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