Programmable DNA cleavage in vitro by Cas9

2013 ◽  
Vol 41 (6) ◽  
pp. 1401-1406 ◽  
Author(s):  
Tautvydas Karvelis ◽  
Giedrius Gasiunas ◽  
Virginijus Siksnys
Keyword(s):  
Dna Cleavage ◽  
Gene Editing ◽  
Streptococcus Thermophilus ◽  
Cell Types ◽  
Strand Break ◽  
Eukaryotic Cell ◽  
Modular Organization ◽  
Cleavage Reaction

The ternary Cas9–crRNA–tracrRNA complex (Cas9t) of the Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) system functions as an Mg2+-dependent RNA-directed DNA endonuclease that locates its DNA target guided by the crRNA (CRISPR RNA) in the tracrRNA–crRNA structure and introduces a double-strand break at a specific site in DNA. The simple modular organization of Cas9t, where specificity for the DNA target is encoded by a small crRNA and the cleavage reaction is executed by the Cas9 endonuclease, provides a versatile platform for the engineering of universal RNA-directed DNA endonucleases. By altering the crRNA sequence within the Cas9t complex, programmable endonucleases can be designed for both in vitro and in vivo applications. Cas9t has been recently employed as a gene-editing tool in various eukaryotic cell types. Using Streptococcus thermophilus Cas9t as a model system, we demonstrate the feasibility of Cas9t as a programmable molecular tool for in vitro DNA manipulations.

scholarly journals CBIO-19. LOW GLOBAL HISTONE H4 ACETYLATION LEVELS REVEAL CANDIDATE QUIESCENT CELL POPULATIONS IN GLIOMA AND DISTINGUISH TUMORS BY GRADE

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii19-ii19
Author(s):  
Anca Mihalas ◽  
Heather Feldman ◽  
Anoop Patel ◽  
Patrick Paddison
Keyword(s):  
Cell Types ◽  
Strand Break ◽  
Cell Cycle Gene ◽  
Low Grade ◽  
Histone H4 ◽  
Current Standard ◽  
A Genome ◽  
Histone H4 Acetylation

Abstract Current standard of care therapy for glioblastoma (GBM) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, the presence of quiescent-like/G0 states, therefore, represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. To gain insight into how glioblastoma cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) harboring a G0-reporter to identify genes that when inhibited trap GSCs in G0-like states. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, which targets both histones (e.g., H4) and non-histone proteins for acetylation. NuA4 functions as a transcriptional activator, whose activities are coordinated with MYC in certain contexts, and also participates in DNA double-strand break repair by facilitating chromatin opening. However, currently little is known about the roles for NuA4 complex in GBM biology. Through modeling KAT5 function in GSC in vitro cultures and in vivo tumors, we find that KAT5 inhibition causes cells to arrest in a G0-like state with high p27 levels, G1-phase DNA content, low protein synthesis rates, low rRNA rates, lower metabolic rate, suppression of cell cycle gene expression, and low histone H4 acetylation. Interestingly, partial inhibition of KAT5 activity slows highly aggressive tumor growth, while increasing p27hi H4-aclow populations. Remarkably, we that low grade gliomas have significantly higher H4-aclow subpopulations and generally lower H4-ac levels than aggressive grade IV tumors. Taken together, our results suggest that NuA4/KAT5 activity may play a key role in quiescence ingress/egress in glioma and that targeting its activity in high grade tumors may effectively “down grade” them, thus, increase patient survival.

scholarly journals Chimeric RNA:DNA Donorguide Improves HDR in vitro and in vivo

2021 ◽  
Author(s):  
Brandon W Simone ◽  
Han B Lee ◽  
Camden L Daby ◽  
Santiago Restrepo-Castillo ◽  
Hirotaka Ata ◽  
...  
Keyword(s):  
Cell Types ◽  
Repair Process ◽  
Strand Break ◽  
Genetic Alterations ◽  
Precise Integration ◽  
Genetic Changes ◽  
Area Of Interest ◽  
Temporal Localization

Introducing small genetic changes to study specific mutations or reverting clinical mutations to wild type has been an area of interest in precision genomics for several years. In fact, it has been found that nearly 90% of all human pathogenic mutations are caused by small genetic variations, and the methods to efficiently and precisely correct these errors are critically important. One common way to make these small DNA changes is to provide a single stranded DNA (ssDNA) donor containing the desired alteration together with a targeted double-strand break (DSB) at the genomic target. The donor is typically flanked by regions of homology that are often ~30-100bp in length to leverage the homology directed repair (HDR) pathway. Coupling a ssDNA donor with a CRISPR-Cas9 to produce a targeted DSB is one of the most streamlined approaches to introduce small changes. However, in many cell types this approach results in a low rate of incorporation of the desired alteration and has undesired imprecise repair at the 5' or 3' junction due to artifacts of the DNA repair process. We herein report a technology that couples the spatial temporal localization of an ssDNA repair template and leverages the nucleic acid components of the CRISPR-Cas9 system. We show that by direct fusion of an ssDNA template to the trans activating RNA (tracrRNA) to generate an RNA-DNA chimera, termed Donorguide, we recover precise integration of genetic alterations, with both increased integration rates and decreased imprecision at the 5' or 3' junctions relative to an ssODN donor in vitro in HEK293T cells as well as in vivo in zebrafish. Further, we show that this technology can be used to enhance gene conversion with other gene editing tools such as TALENs.

scholarly journals Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

2018 ◽  
Vol 115 (19) ◽  
pp. 4903-4908 ◽  
Author(s):  
Hong-Xia Wang ◽  
Ziyuan Song ◽  
Yeh-Hsing Lao ◽  
Xin Xu ◽  
Jing Gong ◽  
...  
Keyword(s):  
Gene Editing ◽  
Transfection Efficiency ◽  
Gene Activation ◽  
Cell Types ◽  
Biological Research ◽  
Knock Out ◽  
Safe Delivery ◽  
Pegylated Nanoparticles

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

scholarly journals Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing

2019 ◽  
Vol 5 (12) ◽  
pp. eaay3255 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
John Choi ◽  
Mahita Varanasi ◽  
Katie Sanders ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Gene Editing ◽  
Gene Knockout ◽  
Cell Types ◽  
Endosomal Escape ◽  
Biological Research ◽  
Amino Ester ◽  
Cytosolic Protein

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

scholarly journals SCIDOT-40. THERAPEUTIC GENOME EDITING AS A NOVEL TREATMENT FOR GBM

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi280-vi280
Author(s):  
Daniel Rosenblum ◽  
Anna Gutkin ◽  
Dan Peer
Keyword(s):  
Gene Editing ◽  
Unmet Need ◽  
Gene Mutations ◽  
Infiltration Rate ◽  
Strand Break ◽  
Therapeutic Modality ◽  
Chromosomal Dna ◽  
Genomics And Proteomics

Abstract Glioblastoma multiforme (GBM), is the most aggressive form of glioma, a brain tumor that arises from glial cells. GBM is considered a complex malignancy with multiple gene mutations, aberrations, and overexpression together with high infiltration rate and resistance to apoptosis. The current treatment consists of surgical resection, aggressive radiation and chemotherapy regimen. This therapeutic strategy had not changed since 2005 when the chemotherapy Temozolomide was approved. This therapeutic approach extended GBM patients’ life expectancy to approximately 15 months since diagnosis. Although recent progress in genomics and proteomics has paved the way for identifying potential therapeutic targets for treating GBM, the majority of these leading drug candidates remain ineffective. Therefore, novel and effective treatment to GBM presents an unmet need. Lipid nanoparticles (LNPs) are the most clinically advanced delivery platform to date for systemic administration of RNA therapeutics, with the recent approval of Patisiran, siRNA encapsulating LNPs. In recent years, several gene editing technologies have been discovered in bacteria, enabling precise and permanent manipulations at the DNA level. The most advanced and versatile system is based on the CRISPR nuclease Cas9. The recognition of its target chromosomal DNA by Cas9 results in a site-specific double-strand break (DSB), that eventually results in gene disruption. This approach opens multiple venues for research and treatment of diseases including cancer. We have utilized CRISPR encapsulating LNPs to promote a therapeutic gene editing by disrupting key GBM survival genes in vitro in murine and human GBM cell lines as well as in vivo in an aggressive syngeneic GBM mouse model. Our results suggest that treatment with our LNPs based system can specifically and efficiently target GBM cells in vitro and in vivo. Our CRISPR-LNPs based platform could potentially mature to a clinical trial, and ultimately might become a new therapeutic modality in GBM.

scholarly journals Direct LC-MS/MS Analysis of Extra- and Intracellular Glycerophosphoinositol in Model Cancer Cell Lines

2021 ◽  
Vol 12 ◽  
Author(s):  
Ana Margarida Campos ◽  
Genoveffa Nuzzo ◽  
Alessia Varone ◽  
Paola Italiani ◽  
Diana Boraschi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Solid Phase ◽  
Cell Types ◽  
Eukaryotic Cell ◽  
Water Soluble ◽  
Extracellular Milieu ◽  
Cell Functions

Glycerophosphoinositols (GPIs) are water-soluble bioactive phospholipid derivatives of increasing interest as intracellular and paracrine mediators of eukaryotic cell functions. The most representative compound of the family is glycerophosphoinositol (GroPIns), an ubiquitous component of mammalian cells that participates in cell proliferation, cell survival and cell response to stimuli. Levels and activity of this compound vary among cell types and deciphering these functions requires accurate measurements in in vitro and in vivo models. The conventional approaches for the analysis of GroPIns pose several issues in terms of sensitivity and product resolution, especially when the product is in the extracellular milieu. Here we present an UPLC-MS study for the quantitative analysis of this lipid derivative in cells and, for the first time, culture supernatants. The method is based on a solid-phase extraction that allows for fast desalting and analyte concentration. The robustness of the procedure was tested on the simultaneous measurements of intra- and extracellular levels of GroPIns in a number of human cell lines where it has been shown that the non-transformed cells are characterized by high extracellular level of GroPIns, whereas the tumor cells tended to have higher intracellular levels.

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

scholarly journals Heme Oxygenase-1 Deficiency and Oxidative Stress: A Review of 9 Independent Human Cases and Animal Models

2021 ◽  
Vol 22 (4) ◽  
pp. 1514 ◽  
Author(s):  
Akihiro Yachie
Keyword(s):  
Oxidative Stress ◽  
Animal Models ◽  
Heme Oxygenase ◽  
Inflammatory Diseases ◽  
Cell Types ◽  
Pharmacological Intervention ◽  
Heme Oxygenase 1 ◽  
Inflammatory Reactions

Since Yachie et al. reported the first description of human heme oxygenase (HO)-1 deficiency more than 20 years ago, few additional human cases have been reported in the literature. A detailed analysis of the first human case of HO-1 deficiency revealed that HO-1 is involved in the protection of multiple tissues and organs from oxidative stress and excessive inflammatory reactions, through the release of multiple molecules with anti-oxidative stress and anti-inflammatory functions. HO-1 production is induced in vivo within selected cell types, including renal tubular epithelium, hepatic Kupffer cells, vascular endothelium, and monocytes/macrophages, suggesting that HO-1 plays critical roles in these cells. In vivo and in vitro studies have indicated that impaired HO-1 production results in progressive monocyte dysfunction, unregulated macrophage activation and endothelial cell dysfunction, leading to catastrophic systemic inflammatory response syndrome. Data from reported human cases of HO-1 deficiency and numerous studies using animal models suggest that HO-1 plays critical roles in various clinical settings involving excessive oxidative stress and inflammation. In this regard, therapy to induce HO-1 production by pharmacological intervention represents a promising novel strategy to control inflammatory diseases.

scholarly journals MyD88 Is Not Required for Muscle Injury-Induced Endochondral Heterotopic Ossification in a Mouse Model of Fibrodysplasia Ossificans Progressiva

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 630
Author(s):  
Huili Lyu ◽  
Cody M. Elkins ◽  
Jessica L. Pierce ◽  
C. Henrique Serezani ◽  
Daniel S. Perrien
Keyword(s):  
Heterotopic Ossification ◽  
Muscle Injury ◽  
Cell Types ◽  
Fibrodysplasia Ossificans Progressiva ◽  
Inflammatory Signaling ◽  
Conditional Deletion ◽  
Pathway Crosstalk ◽  
Multiple Cell

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1β or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

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