scholarly journals Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

2022 ◽  
Author(s):  
Thomas Swartjes ◽  
Peng Shang ◽  
Dennis van den Berg ◽  
Tim A. Kunne ◽  
Niels Geijsen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Genetic Disorders ◽  
Complementary Dna ◽  
Dna Oligonucleotides ◽  
Ribonucleoprotein Complexes ◽  
Two Factors ◽  
Complementary Region ◽  
Human Genetic Disorders

CRISPR-Cas has revolutionized genome editing and has a great potential for applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. Here we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides down-regulate Cas9 activity in human cells, reducing both on and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region, and the presence of a PAM-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

scholarly journals Human USP18 deficiency underlies type 1 interferonopathy leading to severe pseudo-TORCH syndrome

2016 ◽  
Vol 213 (7) ◽  
pp. 1163-1174 ◽  
Author(s):  
Marije E.C. Meuwissen ◽  
Rachel Schot ◽  
Sofija Buta ◽  
Grétel Oudesluijs ◽  
Sigrid Tinschert ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Genetic Disorders ◽  
Genetic Disorder ◽  
Congenital Infection ◽  
Infectious Agent ◽  
Negative Regulator ◽  
Type I ◽  
Loss Of Function

Pseudo-TORCH syndrome (PTS) is characterized by microcephaly, enlarged ventricles, cerebral calcification, and, occasionally, by systemic features at birth resembling the sequelae of congenital infection but in the absence of an infectious agent. Genetic defects resulting in activation of type 1 interferon (IFN) responses have been documented to cause Aicardi-Goutières syndrome, which is a cause of PTS. Ubiquitin-specific peptidase 18 (USP18) is a key negative regulator of type I IFN signaling. In this study, we identified loss-of-function recessive mutations of USP18 in five PTS patients from two unrelated families. Ex vivo brain autopsy material demonstrated innate immune inflammation with calcification and polymicrogyria. In vitro, patient fibroblasts displayed severely enhanced IFN-induced inflammation, which was completely rescued by lentiviral transduction of USP18. These findings add USP18 deficiency to the list of genetic disorders collectively termed type I interferonopathies. Moreover, USP18 deficiency represents the first genetic disorder of PTS caused by dysregulation of the response to type I IFNs. Therapeutically, this places USP18 as a promising target not only for genetic but also acquired IFN-mediated CNS disorders.

scholarly journals Discovery of tumoricidal DNA oligonucleotides by effect-directedin-vitroevolution

2019 ◽  
Author(s):  
Noam Mamet ◽  
Yaniv Amir ◽  
Erez Lavi ◽  
Liron Bassali ◽  
Gil Harari ◽  
...  
Keyword(s):  
Drug Discovery ◽  
De Novo ◽  
Ex Vivo ◽  
Binding Capacity ◽  
Biological Effects ◽  
Current Model ◽  
Target Cells ◽  
Dna Oligonucleotides

AbstractOur current model of drug discovery is challenged by the relative ineffectiveness of drugs against highly variable and rapidly evolving diseases and their relatively high incidence of adverse effects due to poor selectivity. Here we describe a robust and reproducible platform which could potentially address these limitations. The platform enables rapid,de-novodiscovery of DNA oligonucleotides evolvedin-vitroto exert specific biological effects on target cells. Unlike aptamers, which are selected by their ligand binding capacity, this platform is driven directly by therapeutic effect and selectivity towards target vs negative target cells. The process could, therefore, operate without anya-prioriknowledge (e.g. mutations, biomarker expression, or known drug resistance) of the target. We report the discovery of DNA oligonucleotides with direct and selective cytotoxicity towards several tumor cell lines as well as primary, patient-derived solid and hematological tumors, some with chemotherapy resistance. Oligonucleotides discovered by this platform exhibited favorable biodistribution in animals, persistence in target tumors up to 48 hours after injection, and safety in human blood. These oligonucleotides showed remarkable efficacyin-vivoas well asex-vivoin freshly obtained, 3D cultured human tumors resistant to multiple chemotherapies. With further improvement, these findings could lead to a drug discovery model which is target-tailored, mechanism-flexible, and nearly on-demand.

scholarly journals Successful i-GONAD in Brown Norway Rats by Modification of in vivo Electroporation Conditions

2020 ◽  
Vol 4 (4) ◽  
Author(s):  
Shuji Takabayashi ◽  
◽  
Takuya Aoshima ◽  
Yukari Kobayashi ◽  
Hisayoshi Takagi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Preimplantation Embryos ◽  
Brown Norway ◽  
Sprague Dawley ◽  
In Vivo Electroporation ◽  
Nucleic Acids Delivery ◽  
A Current

Improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) was developed for in situ genome editing of the preimplantation embryos present within the oviductal lumen of mice. This method is based on intra-oviductal instillation of genome editing components and subsequent in vivo electroporation (EP) in the entire oviduct. Therefore, i-GONAD differs from the previous methods (i.e., zygote microinjection and in vitro EP) in producing genome-edited mice, which relied on ex vivo handling of preimplantation embryos and egg transfer to the recipient females. We have previously demonstrated that i-GONAD can be successfully applied to produce genome-edited rats, including albino Sprague-Dawley and albino Lewis rats (however, not pigmented Brown Norway [BN] rats). We observed that the successful i-GONAD was dependent on the mouse strain used; for example, in random-bred mice, such as ICR and C3H/He × C57BL/6, it was successful under relatively stringent electrical conditions but not in the C57BL/6 strain. Under less stringent conditions, i-GONAD was successful in the C57BL/6 strain. We speculated that this would also be true for i-GONAD using BN rats. On applying a current of >500 mA, we failed to obtain rat offspring (fetuses/newborns); however, i-GONAD under a current of 100-300 mA using NEPA21 (NEPA GENE) led to the production of genome-edited BN rats with efficiencies of 75%-100%. Similarly, i-GONAD, under a current of 150-200 mA using CUY21EDIT II (BEX Co.) led to the production of genome-edited BN rats with efficiencies of 24%-55%. These experiments suggest the importance of selecting the appropriate current value, depending on the rat strain used, when performing i-GONAD.

Emerging therapeutic applications of CRISPR genome editing

2019 ◽  
Vol 3 (3) ◽  
pp. 257-260 ◽  
Author(s):  
Waseem Qasim
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Ex Vivo ◽  
Single Gene ◽  
Rapid Evolution ◽  
Small Animal ◽  
Clinical Phase ◽  
Blood Stem Cells ◽  
Novel Therapeutic Strategies

Abstract The rapid evolution of tools for genome editing has created a dizzying array of possibilities for novel therapeutic strategies, even though to date only a handful of clinical applications have been realised. Proof-of-concept demonstrations of targeted genome modification in vitro and in small animal models of inherited single gene disorders have to be translated into effective therapies. Interest has naturally gravitated towards opportunities for collection, ex vivo modification and return of blood, immune and stem cells. Initial applications designed to modify T cells to protect against HIV or to confer potent anti-leukaemic effects have reached clinical phase, and further applications to modify blood stem cells are close to being applied. There are generic considerations of safety, on- and off-target effects and possible genotoxicity as well as issues relating to more sophisticated systemic approaches where niche occupation and host immunity become relevant. Such issues will be likely addressed over time, with carefully designed clinical trials required to determine therapeutic risks and benefits.

scholarly journals CRISPR-Cas9-based gene editing of human mesenchymal stromal cells to improve the outcome of cell therapy

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
Y Schary ◽  
R.Y Brzezinski ◽  
O Teper-Shaihov ◽  
N Naftali-Shani ◽  
J Leor
Keyword(s):  
Cell Therapy ◽  
Genome Editing ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Gene Editing ◽  
Ex Vivo ◽  
Medical Center ◽  
Funding Source ◽  
Tlr4 Gene

Abstract Background The environment of the failing and infarcted myocardium drives resident and transplanted mesenchymal stromal cells (MSCs) toward a pro-inflammatory phenotype, thus restricting their survival and their reparative effects in a mechanism mediated by toll-like receptor 4 (TLR4). Hence, new approaches are needed to improve the efficacy of cell therapy for heart failure. CRISPR is a promising tool for genome editing, which raises the hope for therapeutic genome editing in the clinic. Purpose We aimed to provide a new strategy to enhance MSC-based cell therapy to improve cardiac remodeling and function. We hypothesized that ex vivo knockout (KO) of the human TLR4 gene by CRISPR would switch human-cardiac MSCs (hMSCs) to an anti-inflammatory, reparative phenotype that could prevent remodeling of the left ventricle after myocardial infarction (Fig. 1A). Methods and results We achieved up to 68% (out of 4x105 cells, R2=0.93) success rate in editing the genome of primary cardiac hMSCs taken from patients with ischemic heart disease. The deletion of TLR4 in hMSCs significantly reduced the secretion of inflammatory and extracellular-matrix (ECM) proteins, compared with unedited hMSCs, by protein mass spectrometry (Fig. 1B) and by multiplex ELISA (Fig. 1C). Additionally, edited cells secreted significantly more extracellular vesicles (EVs) than unedited hMScs (Fig. 1D, p<0.001). These EVs from edited hMSCs stimulated faster migration of hMSCs in a “wound healing” assay (p<0.001). Conclusions We show, for the first time, that CRISPR-based deletion of the TLR4 gene in hMSCs inhibits inflammatory and ECM protein secretion and facilitates a reparative response by hMSCs in vitro. This precise and efficient ex vivo gene editing could provide a newly engineered cell line to improve the outcome of hMSC-based cell therapy. Figure 1 Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): The Foundation in Memory of Seymour Fefer, Sheba Medical Center

scholarly journals Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences

Molecules ◽  
2019 ◽  
Vol 25 (1) ◽  
pp. 52 ◽  
Author(s):  
Taegeun Bae ◽  
Hanseop Kim ◽  
Jeong Hee Kim ◽  
Yong Jun Kim ◽  
Seung Hwan Lee ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genetic Disorders ◽  
Point Mutations ◽  
Growth Factor Receptor ◽  
High Specificity ◽  
Complete Sequence ◽  
Single Base ◽  
Safety Issues ◽  
Ribonucleoprotein Complexes ◽  
Sequence Complementarity

In CRISPR genome editing, CRISPR proteins form ribonucleoprotein complexes with guide RNAs to bind and cleave the target DNAs with complete sequence complementarity. CRISPR genome editing has a high potential for use in precision gene therapy for various diseases, including cancer and genetic disorders, which are caused by DNA mutations within the genome. However, several studies have shown that targeting the DNA via sequence complementarity is imperfect and subject to unintended genome editing of other genomic loci with similar sequences. These off-target problems pose critical safety issues in the therapeutic applications of CRISPR technology, with particular concerns in terms of the genome editing of pathogenic point mutations, where non-mutant alleles can become an off-target with only a one-base difference. In this study, we sought to assess a novel CRISPR genome editing technique that has been proposed to achieve a high specificity by positioning the mismatches within the protospacer adjacent motif (PAM) sequence. To this end, we compared the genome editing specificities of the PAM-based and conventional methods on an oncogenic single-base mutation in the endothelial growth factor receptor (EGFR). The results indicated that the PAM-based method provided a significantly increased genome editing specificity for pathogenic mutant alleles with single-base precision.

scholarly journals Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 546 ◽  
Author(s):  
Masahiro Sato ◽  
Rico Miyagasako ◽  
Shuji Takabayashi ◽  
Masato Ohtsuka ◽  
Izuho Hatada ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Preimplantation Embryos ◽  
Nucleic Acid Delivery ◽  
Cell Stage ◽  
Initial Development ◽  
Genetic Manipulations ◽  
Target Sites

Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. During the initial development of this technique, we performed i-GONAD on Days 1.4–1.5 (corresponding to the 2-cell stage). Theoretically, this means that at least two GONAD steps (on Day 0.7 and Day 1.4–1.5) must be performed. If this is practically demonstrated, it provides additional options for various clustered regularly interspaced palindrome repeats (CRISPR)/Caspase 9 (Cas9)-based genetic manipulations. For example, it is usually difficult to induce two independent indels at the target sites, which are located very close to each other, by simultaneous transfection of two guide RNAs and Cas9 protein. However, the sequential induction of indels at a target site may be possible when repeated i-GONAD is performed on different days. Furthermore, simultaneous introduction of two mutated lox sites (to which Cre recombinase bind) for making a floxed allele is reported to be difficult, as it often causes deletion of a sequence between the two gRNA target sites. However, differential KI of lox sites may be possible when repeated i-GONAD is performed on different days. In this study, we performed proof-of-principle experiments to demonstrate the feasibility of the proposed approach called “sequential i-GONAD (si-GONAD).”

scholarly journals Quality by Design for Optimizing a Novel Liposomal Jojoba Oil-Based Emulgel to Ameliorate the Anti-Inflammatory Effect of Brucine

Gels ◽  
2021 ◽  
Vol 7 (4) ◽  
pp. 219
Author(s):  
Marwa H. Abdallah ◽  
Heba S. Elsewedy ◽  
Amr S. AbuLila ◽  
Khaled Almansour ◽  
Rahamat Unissa ◽  
...  
Keyword(s):  
Ex Vivo ◽  
In Vitro Release ◽  
Therapeutic Effects ◽  
Jojoba Oil ◽  
Drug Permeability ◽  
Two Factors ◽  
Natural Alkaloid ◽  
Anti Inflammatory ◽  
Inflammatory Effect

One of the recent advancements in research is the application of natural products in developing newly effective formulations that have few drawbacks and that boost therapeutic effects. The goal of the current exploration is to investigate the effect of jojoba oil in augmenting the anti-inflammatory effect of Brucine natural alkaloid. This is first development of a formulation that applies Brucine and jojoba oil int a PEGylated liposomal emulgel proposed for topical application. Initially, various PEGylated Brucine liposomal formulations were fabricated using a thin-film hydration method. (22) Factorial design was assembled using two factors (egg Phosphatidylcholine and cholesterol concentrations) and three responses (particle size, encapsulation efficiency and in vitro release). The optimized formula was incorporated within jojoba oil emulgel. The PEGylated liposomal emulgel was inspected for its characteristics, in vitro, ex vivo and anti-inflammatory behaviors. Liposomal emulgel showed a pH of 6.63, a spreadability of 48.8 mm and a viscosity of 9310 cP. As much as 40.57% of Brucine was released after 6 h, and drug permeability exhibited a flux of 0.47 µg/cm2·h. Lastly, % of inflammation was lowered to 47.7, which was significant effect compared to other formulations. In conclusion, the anti-inflammatory influence of jojoba oil and Brucine was confirmed, supporting their integration into liposomal emulgel as a potential nanocarrier.

Effects of the Tibetan herbal preparation PADMA 28 on blood lipids and lipid oxidisability in subjects with mild hypercholesterolaemia

VASA ◽  
2005 ◽  
Vol 34 (1) ◽  
pp. 11-17 ◽  
Author(s):  
Brunner-La Rocca ◽  
Schindler ◽  
Schlumpf ◽  
Saller ◽  
Suter
Keyword(s):  
Endothelial Dysfunction ◽  
Blood Lipids ◽  
Ex Vivo ◽  
Hdl Cholesterol ◽  
Blood Lipid ◽  
Tibetan Medicine ◽  
Double Blind ◽  
Intraarterial Infusion ◽  
Padma 28

Background: Previous studies showed an anti-atherosclerotic effect of PADMA 28, an herbal formula based on Tibetan medicine. As the mechanisms of action are not fully understood, we investigated whether PADMA 28 may lower blood lipids and lipid oxidisability, and affect early endothelial dysfunction. Patients and methods: Sixty otherwise healthy subjects with total cholesterol ≥5.2 mmol/l and < 8.0 mmol/l were randomly assigned to placebo or PADMA 28, 3 x 2 capsules daily, for 4 weeks (double-blind). Blood lipids (total, LDL-, and HDL-cholesterol, triglycerides, Apo-lipoprotein A1 and B) and ex vivo lipid oxidisability were measured before and after treatment. In a subset of 24 subjects, endothelial function was assessed using venous occlusion plethysmography with intraarterial infusion of acetylcholine. Isolated LDL and plasma both untreated and pre-treated with PADMA 28 extract were oxidised by the radical generator AAPH. Conjugated diene formation was measured at 245 nm. Results: Blood lipids did not change during the study in both groups. In contrast to previous reports in mild hypercholesterolaemia, no endothelial dysfunction was seen and, consequently, was not influenced by therapy. Ex vivo blood lipid oxidisability was significantly reduced with PADMA 28 (area under curve: 5.29 ± 1.62 to 4.99 ± 1.46, p = 0.01), and remained unchanged in the placebo group (5.33 ± 1.88 to 5.18 ± 1.78, p > 0.1). This effect persisted one week after cessation of medication. In vitro experiments confirmed the prevention of lipid peroxidation in the presence of PADMA 28 extracts. Persistent protection was also seen for LDL isolated from PADMA 28-pretreated blood after being subjected to rigorous purification. Conclusions: This study suggests that the inhibition of blood lipid oxidisability by PADMA 28 may play a role in its anti-atherosclerotic effect.

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