scholarly journals Cell-specific CRISPR–Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins

2019 ◽  
Vol 47 (13) ◽  
pp. e75-e75 ◽  
Author(s):  
Mareike D Hoffmann ◽  
Sabine Aschenbrenner ◽  
Stefanie Grosse ◽  
Kleopatra Rapti ◽  
Claire Domenger ◽  
...  
Keyword(s):  
Specific Activity ◽  
Genetic Disorders ◽  
Rapid Development ◽  
Gene Activation ◽  
Target Cells ◽  
Cardiac Muscle Cells ◽  
Regulated Expression ◽  
Target Sites ◽  
A Cell ◽  
Cell Type Specific

Abstract The rapid development of CRISPR–Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3′UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR–Cas orthologue, for which a potent anti-CRISPR protein is known.

scholarly journals Cell-specific CRISPR/Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins

2018 ◽  
Author(s):  
Mareike D. Hoffmann ◽  
Sabine Aschenbrenner ◽  
Stefanie Grosse ◽  
Kleopatra Rapti ◽  
Claire Domenger ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Genetic Disorders ◽  
Rapid Development ◽  
Gene Activation ◽  
Target Cells ◽  
Cell Type ◽  
Regulated Expression ◽  
Target Sites ◽  
A Cell ◽  
Cell Type Specific

ABSTRACTThe rapid development of CRISPR/Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and muscle cells, respectively, into the 3’UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and correspondingly in Cas9 activation solely in hepatocytes or myocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activation of any CRISPR/Cas orthologue, for which a potent anti-CRISPR protein is known.

scholarly journals Secreted reporter assay enables quantitative and longitudinal monitoring of neuronal activity

2021 ◽  
Author(s):  
Ana C. Santos ◽  
Sungjin Park
Keyword(s):  
Neuronal Activity ◽  
Specific Activity ◽  
Repeated Measurements ◽  
Reporter Assay ◽  
Cell Type ◽  
Conditional Expression ◽  
Specific Manner ◽  
A Cell ◽  
Cell Type Specific ◽  
And Function

AbstractThe ability to measure changes in neuronal activity in a quantifiable and precise manner is of fundamental importance to understand neuron development and function. Repeated monitoring of neuronal activity of the same population of neurons over several days is challenging and, typically, low-throughput. Here, we describe a new biochemical reporter assay that allows for repeated measurements of neuronal activity in a cell type-specific manner. We coupled activity-dependent elements from the Arc/Arg3.1 gene with a secreted reporter, Gaussia luciferase, to quantify neuronal activity without sacrificing the neurons. The reporter predominantly senses calcium and NMDA receptor-dependent activity. By repeatedly measuring the accumulation of the reporter in cell media, we can profile the developmental dynamics of neuronal activity in cultured neurons from male and female mice. The assay also allows for longitudinal analysis of pharmacological treatments, thus distinguishing acute from delayed responses. Moreover, conditional expression of the reporter allows for monitoring cell type-specific changes. This simple, quantitative, cost-effective, automatable, and cell type-specific activity reporter is a valuable tool to study the development of neuronal activity in normal and disease-model conditions, and to identify small molecules or protein factors that selectively modulate the activity of a specific population of neurons.SignificanceNeurological and neurodevelopmental disorders are prevalent worldwide. Despite significant advances in our understanding of synapse formation and function, developing effective therapeutics remains challenging, in part due to the lack of simple and robust high-throughput screening assays of neuronal activity. Here, we describe a simple biochemical assay that allows for repeated measurements of neuronal activity in a cell type-specific manner. Thus filling the need for assays amenable to longitudinal studies, such as those related to neural development. Other advantages include its simple and quantitative nature, logitudinal profiling, cell type-specificity, and being multiplexed with other invasive techniques.

scholarly journals Deterministic splicing of Dscam2 is regulated by Muscleblind

2018 ◽  
Author(s):  
Joshua Shing Shun Li ◽  
S.Sean Millard
Keyword(s):  
Alternative Splicing ◽  
Surface Protein ◽  
Dendritic Morphology ◽  
Protein Isoforms ◽  
Splicing Factor ◽  
Regulated Expression ◽  
A Cell ◽  
Cell Type Specific ◽  
Isoform B ◽  
The Brain

SummaryAlternative splicing of genes increases the number of distinct proteins in a cell. In the brain it is highly prevalent, presumably because proteome diversity is crucial for establishing the complex circuitry between trillions of neurons. To provide individual cells with different repertoires of protein isoforms, however, this process must be regulated. Previously, we found that the mutually exclusive alternative splicing of a cell surface protein, Dscam2 produces two isoforms (exon 10A and 10B) with unique binding properties. This splicing event is tightly regulated and crucial for maintaining axon terminal size, dendritic morphology and synaptic numbers. Here, we show that Drosophila Muscleblind (Mbl), a conserved splicing factor implicated in myotonic dystrophy, controls Dscam2 alternative splicing. Removing mbl from cells that normally express isoform B induces the expression of isoform A and eliminates the expression of B, demonstrating that Mbl represses one alternative exon and selects the other. Mbl mutants exhibit phenotypes that are also observed in flies engineered to express a single isoform. Consistent with these observations, mbl expression is cell-type-specific and correlates with the expression of isoform B. Our study demonstrates how the regulated expression of a splicing factor is sufficient to provide neurons with unique protein isoforms crucial for development.

IFIH1-deficiency results in a cell-type specific alteration of autophagic activity in response to S. typhimurium

2017 ◽  
Vol 55 (05) ◽  
pp. e28-e56
Author(s):  
S Macheiner ◽  
R Gerner ◽  
A Pfister ◽  
A Moschen ◽  
H Tilg
Keyword(s):  
Cell Type ◽  
A Cell ◽  
Cell Type Specific ◽  
Autophagic Activity ◽  
Specific Alteration

Design and Delivery of Therapeutic siRNAs: Application to MERS-Coronavirus

2018 ◽  
Vol 24 (1) ◽  
pp. 62-77 ◽  
Author(s):  
Sayed Sartaj Sohrab ◽  
Sherif Aly El-Kafrawy ◽  
Zeenat Mirza ◽  
Mohammad Amjad Kamal ◽  
Esam Ibraheem Azhar
Keyword(s):  
Enzymatic Degradation ◽  
Polymeric Nanoparticles ◽  
Genetic Disorders ◽  
Inorganic Nanoparticles ◽  
Innovative Technology ◽  
Viral Diseases ◽  
Viral Gene ◽  
Efficient Delivery ◽  
Disease Therapy ◽  
Target Sites

Background: The MERS-CoV is a novel human coronavirus causing respiratory syndrome since April 2012. The replication of MERS-CoV is mediated by ORF 1ab and viral gene activity can be modulated by RNAi approach. The inhibition of virus replication has been documented in cell culture against multiple viruses by RNAi approach. Currently, very few siRNA against MERS-CoV have been computationally designed and published. Methods: In this review, we have discussed the computational designing and delivery of potential siRNAs. Potential siRNA can be designed to silence a desired gene by considering many factors like target site, specificity, length and nucleotide content of siRNA, removal of potential off-target sites, toxicity and immunogenic responses. The efficient delivery of siRNAs into targeted cells faces many challenges like enzymatic degradation and quick clearance through renal system. The siRNA can be delivered using transfection, electroporation and viral gene transfer. Currently, siRNAs delivery has been improved by using advanced nanotechnology like lipid nanoparticles, inorganic nanoparticles and polymeric nanoparticles. Conclusion: The efficacy of siRNA-based therapeutics has been used not only against many viral diseases but also against non-viral diseases, cancer, dominant genetic disorders, and autoimmune disease. This innovative technology has attracted researchers, academia and pharmaceuticals industries towards designing and development of highly effective and targeted disease therapy. By using this technology, effective and potential siRNAs can be designed, delivered and their efficacy with toxic effects and immunogenic responses can be tested against MERS-CoV.

scholarly journals A cell type‐specific expression map of NCoR1 and SMRT transcriptional co‐repressors in the mouse brain

2020 ◽  
Vol 528 (13) ◽  
pp. 2218-2238 ◽  
Author(s):  
Attilio Iemolo ◽  
Patricia Montilla‐Perez ◽  
I‐Chi Lai ◽  
Yinuo Meng ◽  
Syreeta Nolan ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Cell Type ◽  
Specific Expression ◽  
Cell Type Specific Expression ◽  
A Cell ◽  
Cell Type Specific

scholarly journals Diminished LcrV Secretion Attenuates Yersinia pseudotuberculosis Virulence

2007 ◽  
Vol 189 (23) ◽  
pp. 8417-8429 ◽  
Author(s):  
Jeanette E. Bröms ◽  
Matthew S. Francis ◽  
Åke Forsberg
Keyword(s):  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Signal Sequence ◽  
Infection Model ◽  
Target Cells ◽  
Cell Infection ◽  
Host Cell Membrane ◽  
Type Iii ◽  
A Cell

ABSTRACT Many gram-negative bacterial pathogenicity factors that function beyond the outer membrane are secreted via a contact-dependent type III secretion system. Two types of substrates are predestined for this mode of secretion, namely, antihost effectors that are translocated directly into target cells and the translocators required for targeting of the effectors across the host cell membrane. N-terminal secretion signals are important for recognition of the protein cargo by the type III secretion machinery. Even though such signals are known for several effectors, a consensus signal sequence is not obvious. One of the translocators, LcrV, has been attributed other functions in addition to its role in translocation. These functions include regulation, presumably via interaction with LcrG inside bacteria, and immunomodulation via interaction with Toll-like receptor 2. Here we wanted to address the significance of the specific targeting of LcrV to the exterior for its function in regulation, effector targeting, and virulence. The results, highlighting key N-terminal amino acids important for LcrV secretion, allowed us to dissect the role of LcrV in regulation from that in effector targeting/virulence. While only low levels of exported LcrV were required for in vitro effector translocation, as deduced by a cell infection assay, fully functional export of LcrV was found to be a prerequisite for its role in virulence in the systemic murine infection model.

scholarly journals Development of COVID-19 vaccines utilizing gene therapy technology

2021 ◽  
Author(s):  
Hironori Nakagami
Keyword(s):  
Viral Vectors ◽  
High Efficiency ◽  
Genetic Disorders ◽  
Rapid Development ◽  
Adenovirus Vector ◽  
Phase 3 ◽  
Double Blind ◽  
Time Period ◽  
Short Period ◽  
Long Time

Abstract There is currently an outbreak of respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus disease 2019 (COVID-19) is caused by infection with SARS-CoV-2. Individuals with COVID-19 have symptoms that are usually asymptomatic or mild in most initial cases. However, in some cases, moderate and severe symptoms have been observed with pneumonia. Many companies are developing COVID-19 vaccine candidates using different technologies that are classified into four groups (intact target viruses, proteins, viral vectors and nucleic acids). For rapid development, RNA vaccines and adenovirus vector vaccines have been urgently approved, and their injection has already started across the world. These types of vaccine technologies have been developed over more than 20 years using translational research for use against cancer or diseases caused by genetic disorders but the COVID-19 vaccines are the first licensed drugs to prevent infectious diseases using RNA vaccine technology. Although these vaccines are highly effective in preventing COVID-19 for a short period, safety and efficiency evaluations should be continuously monitored over a long time period. As the time of writing, more than 10 projects are now in phase 3 to evaluate the prevention of infection in double-blind studies. Hopefully, several projects may be approved to ensure high-efficiency and safe vaccines.

Gene therapy can target mutations such as BRAF, which have been shown to make tumors more susceptible to autophagy suppression

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Signaling Pathways ◽  
Chemotherapy Resistance ◽  
Effective Strategy ◽  
Cell Type ◽  
Normal Cells ◽  
A Cell ◽  
Cell Type Specific ◽  
Catabolic Process ◽  
Specific Impact

Autophagy is a double-edged sword in cancer, and numerous aspects should be taken into account before deciding on the most effective strategy to target the process. The fact that several clinical studies are now ongoing does not mean that the patient group that may benefit from autophagy-targeting medicines has been identified. Autophagy inhibitors that are more potent and specialized, as well as autophagy indicators, are also desperately required. The fact that these inhibitors only work against tumors that rely on autophagy for survival (RAS mutants) makes it difficult to distinguish them from tumors that continue to develop even when autophagy is absent. Furthermore, mutations such as BRAF have been shown to make tumors more susceptible to autophagy suppression, suggesting that targeting such tumours may be a viable strategy for overcoming their chemotherapy resistance. In the meantime, we are unable to identify if autophagy regulation works in vivo or whether it selectively targets a disease while inflicting injury to other healthy organs and tissues. A cell-type-specific impact appears to be observed with such therapy. As a result, it is just as important to consider the differences between tumors that originate in different organs as it is to consider the signaling pathways that are similar across them. For a therapy or cure to be effective, the proposed intervention must be tailored to the specific needs of each patient.Over the last several years, a growing amount of data has implicated autophagy in a variety of disorders, including cancer. In normal cells, this catabolic process is also required for cell survival and homeostasis. Despite the fact that medications targeting intermediates in the autophagy signaling pathway are being created and evaluated at both the preclinical and clinical levels, given the complicated function of autophagy in cancer, we still have a long way to go in terms of establishing an effective therapeutic approach. This article discusses current tactics for exploiting cancer cells' autophagy dependency, as well as obstacles in the area. We believe that the unanswered concerns raised in this work will stimulate researchers to investigate previously unknown connections between autophagy and other signaling pathways, which might lead to the development of novel, highly specialized autophagy therapies.

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