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CRISPR AND animal Models

Shan Jiang, Natalie Wen, Zeran Li, Umber Dube, Jorge Del Aguila, John Budde, Rita Martinez, Simon Hsu, Maria V Fernandez, Nigel J Cairns, Oscar Harari, Carlos Cruchaga, Celeste M Karch
Mutations in the microtubule-associated protein tau (MAPT) gene cause autosomal dominant frontotemporal lobar degeneration with tau inclusions (FTLD-tau). MAPT p.R406W carriers present clinically with progressive memory loss and neuropathologically with neuronal and glial tauopathy. However, the pathogenic events triggered by the expression of the mutant tau protein remain poorly understood. To identify the genes and pathways that are dysregulated in FTLD-tau, we performed transcriptomic analyses in induced pluripotent stem cell (iPSC)-derived neurons carrying MAPT p...
December 13, 2018: Translational Psychiatry
Rui Zheng, Xiaoliang Fang, Lei He, Yanjiao Shao, Nana Guo, Liren Wang, Mingyao Liu, Dali Li, Hongquan Geng
BACKGROUND: Primary hyperoxaluria type 1 (PH1) is an inherited disease caused by mutations in alanine-glyoxylate aminotransferase (AGXT). It is characterized by abnormal metabolism of glyoxylic acid in liver leading to endogenous oxalate overproduction and deposition of oxalate in multiple organs, mainly the kidney. Patients of PH1 often suffer from recurrent urinary tract stones, and finally renal failure. There is no effective treatment other than combined liver-kidney transplantation...
December 11, 2018: Current Molecular Medicine
Su Yang, Shihua Li, Xiao-Jiang Li
Virus-mediated expression of CRISPR/Cas9 is commonly used for genome editing in animal brains to model or treat neurological diseases, but the potential neurotoxicity of overexpressing bacterial Cas9 in the mammalian brain remains unknown. Through RNA sequencing (RNA-seq) analysis, we find that virus-mediated expression of Cas9 influences the expression of genes involved in neuronal functions. Reducing the half-life of Cas9 by tagging with geminin, whose expression is regulated by the cell cycle, maintains the genome editing capacity of Cas9 but significantly alleviates neurotoxicity...
December 4, 2018: Cell Reports
Duško Lainšček, Lucija Kadunc, Mateja Manček-Keber, Iva Hafner-Bratkovič, Rok Romih, Roman Jerala
The CRISPR/Cas system has been developed as a potent tool for genome engineering and transcription regulation. However, the efficiency of its delivery into cells, particularly for therapeutic in vivo applications, remains a major bottleneck. Extracellular vesicles (EVs), released by eukaryotic cells, can mediate the transfer of different molecules, including nucleic acids and proteins. Here we show packaging and delivery of the CRISPR/Cas system via EVs to target cells, combining the advantages of both technological platforms...
December 4, 2018: ACS Synthetic Biology
Wan Zhu, Daniel Saw, Miriam Weiss, Zhengda Sun, Meng Wei, Sonali Shaligram, Sen Wang, Hua Su
Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. The pathogenesis of bAVM has not been fully understood. Animal models are important tools for dissecting bAVM pathogenesis and testing new therapies. We have developed several mouse bAVM models using genetically modified mice. However, due to the body size, mouse bAVM models have some limitations. Recent studies identified somatic mutations in sporadic human bAVM. To develop a feasible tool to create sporadic bAVM in rodent and animals larger than rodent, we made tests using the CRISPR/Cas9 technique to induce somatic gene mutations in mouse brain in situ...
December 3, 2018: Translational Stroke Research
Behnom Farboud, Aaron F Severson, Barbara J Meyer
The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and non-model organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes for both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-stranded breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans We also devised strategies to insert long (10 kb) exogenous sequences and incorporate multiple nucleotide substitutions at considerable distance from DSBs...
November 30, 2018: Genetics
Neda Nejati Moharrami, Erlend Bjørkøy Tande, Liv Ryan, Terje Espevik, Victor Boyartchuk
ROR family of nuclear receptor transcription factors forms nodes connecting metabolic and inflammatory signaling pathways. The RORα members of the family have intrinsic transcriptional activity and they are involved in both activation and repression of a wide range of genes. The role of RORα in control of inflammation has been extensively studied using animal models but its function in human cells is not as well understood. To address this shortcoming, we analyzed how RORα is shaping the inflammatory state of human macrophages...
2018: PloS One
Mignhui Li, Xingyong Liu, Shengfei Dai, Hesheng Xiao, Deshou Wang
The CRISPR/Cas9 has been successfully applied for disruption of protein coding sequences in a variety of organisms. The majority of the animal genome is actually non-coding sequences, which are key regulators associated with various biological process. In this study, to understand the biological signifi-cance of these sequences, we used one or dual gRNA guided Cas9 nuclease to achieve specific deletion of non-coding sequences including microRNA and 3' untranslated region (UTR) in tilapia, which is an important fish for studying sex determination and evolution...
November 27, 2018: G3: Genes—Genomes—Genetics
Hirohito Ishigaki, Takashi Shiina, Kazumasa Ogasawara
Cynomolgus macaques are useful experimental animals that are physiologically and genetically close to humans. We have developed two kinds of experimental usage of cynomolgus macaque: transplantation and disease models. First, we identified certain major histocompatibility complex (MHC) haplotypes including homozygotes and heterozygotes in cynomolgus macaques native to the Philippines, because they have less polymorphism in the MHC than that in other origins such as Vietnam and Indonesia. As a preclinical model of the induced pluripotent stem cell (iPSC) stock project, we established iPSCs from various types of MHC homozygous macaques, which were transplanted into compatible MHC heterozygous macaques, the iPSC stock project was experimentally shown to be effective...
2018: Inflammation and Regeneration
Kizuki Yuza, Masato Nakajima, Masayuki Nagahashi, Junko Tsuchida, Yuki Hirose, Kohei Miura, Yosuke Tajima, Manabu Abe, Kenji Sakimura, Kazuaki Takabe, Toshifumi Wakai
BACKGROUND: Pancreatic cancer is a disease with poor prognosis, and development of new treatments is necessary. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays a critical role in progression of many types of cancer. However, little is known about the role of sphingosine kinases in pancreatic cancer. This study investigated the roles of sphingosine kinases in pancreatic cancer progression. MATERIALS AND METHODS: S1P levels in pancreatic cancer and noncancerous pancreatic tissue were measured in 10 patients...
December 2018: Journal of Surgical Research
Carmen Hurtado Del Pozo, Elena Garreta, Juan Carlos Izpisúa Belmonte, Nuria Montserrat
Understanding epigenetic mechanisms is crucial to our comprehension of gene regulation in development and disease. In the past decades, different studies have shown the role of epigenetic modifications and modifiers in renal disease, especially during its progression towards chronic and end-stage renal disease. Thus, the identification of genetic variation associated with chronic kidney disease has resulted in better clinical management of patients. Despite the importance of these findings, the translation of genotype-phenotype data into gene-based medicine in chronic kidney disease populations still lacks faithful cellular or animal models that recapitulate the key aspects of the human kidney...
November 20, 2018: Disease Models & Mechanisms
Diarra K Williams, Carlos Pinzón, Shannon Huggins, Jane H Pryor, Alyssa Falck, Forrest Herman, James Oldeschulte, Michael B Chavez, Brian L Foster, Sarah H White, Mark E Westhusin, Larry J Suva, Charles R Long, Dana Gaddy
The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness...
November 16, 2018: Scientific Reports
Richard Whitley, Joel Baines
Infection with herpes simplex virus (HSV) types 1 and 2 is ubiquitous in the human population. Most commonly, virus replication is limited to the epithelia and establishes latency in enervating sensory neurons, reactivating periodically to produce localized recurrent lesions. However, these viruses can also cause severe disease such as recurrent keratitis leading potentially to blindness, as well as encephalitis, and systemic disease in neonates and immunocompromised patients. Although antiviral therapy has allowed continual and substantial improvement in the management of both primary and recurrent infections, resistance to currently available drugs and long-term toxicity pose a current and future threat that should be addressed through the development of new antiviral compounds directed against new targets...
2018: F1000Research
Ji-Feng Fei, Wilson Pak-Kin Lou, Dunja Knapp, Prayag Murawala, Tobias Gerber, Yuka Taniguchi, Sergej Nowoshilow, Shahryar Khattak, Elly M Tanaka
Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR-Cas9 system. The protocol, which takes 7-8 weeks to complete, describes generation of targeted gene knockouts and knock-ins and includes site-specific integration of large targeting constructs...
December 2018: Nature Protocols
Lingyu Li, Wei Li, Naifei Chen, Haixin Zhao, Guang Xu, Yijing Zhao, Xin Pan, Xiaoying Zhang, Lei Zhou, Dehai Yu, Ailing Li, Jifan Hu, Jiuwei Cui
PURPOSE: The aberrantly upregulated Friend leukemia virus integration 1 ( FLI1 ) is closely correlated with the malignant phenotype of small cell lung cancer (SCLC). This study attempts to examine if FLI1 exonic circular RNAs (FECRs) function as a new malignant driver that determines the metastatic phenotype in SCLC. EXPERIMENTAL DESIGN: The expression of FECRs was examined in SCLC tissues and serum exosomes. The oncogenic activity of FECRs was investigated in SCLC cell lines and animal xenograft studies...
November 14, 2018: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Jeffrey M Lynch, Bing Li, Parvaneh Katoli, Chuanxi Xiang, Barrett Leehy, Nalini Rangaswamy, Veronica Saenz-Vash, Y Karen Wang, Hong Lei, Thomas B Nicholson, Erik Meredith, Dennis Rice, Ganesh Prasanna, Amy Chen
Myocilin ( MYOC ) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of wild-type (wt) MYOC has remained elusive and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum (ER), and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution which is the equivalent of the pathologic human MYOC Y437H mutation...
November 2, 2018: Journal of Biological Chemistry
Shun-Wa Tsang, Yanjiang Guo, Long-Hei Chan, Yingyu Huang, King L Chow
MAB21L2(R51C) is one of the five documented MAB21L2 mutations in human patients with bilateral eye malformations identified via whole exome sequencing. In addition to the eye abnormality, patients with MAB21L2R51C/+ mutation also have skeletal dysplasia and intellectual disability. To evaluate the pathology of this mutant allele systematically in understanding the functional role of MAB21L2 in human development, we introduce the R51C mutation into the mouse genome by CRISPR/Cas9 system to generate a mouse model for detailed characterization...
October 30, 2018: Genesis: the Journal of Genetics and Development
Ryan P Liegel, Erin Finnerty, Lauren Ward, Andrew DiStasio, Robert B Hufnagel, Howard M Saal, Kristen L Sund, Cynthia A Prows, Rolf Stottmann
A recent convergence of technological innovations has re-energized the ability to apply genetics to research in human craniofacial development. Next-generation exome and whole genome sequencing have significantly dropped in price, making it relatively trivial to sequence and analyze patients and families with congenital craniofacial anomalies. A concurrent revolution in genome editing with the use of the CRISPR-Cas9 system enables the rapid generation of animal models, including mouse, which can precisely recapitulate human variants...
October 29, 2018: Genesis: the Journal of Genetics and Development
Fuminori Tanihara, Maki Hirata, Nhien T Nguyen, Quynh A Le, Takayuki Hirano, Tatsuya Takemoto, Michiko Nakai, Dai-Ichiro Fuchimoto, Takeshige Otoi
Recently, we established the GEEP ("gene editing by electroporation of Cas9 protein") method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome-editing efficiency. To produce an animal model for diabetes, we targeted PDX-1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion...
October 25, 2018: Animal Science Journal, Nihon Chikusan Gakkaihō
Sergey V Prykhozhij, Jason N Berman
The zebrafish is an increasingly popular model organism for human genetic disease research. CRISPR/Cas9-based approaches are currently used for multiple gene-editing purposes in zebrafish, but few studies have developed reliable ways to introduce precise mutations. Point mutation knock-in using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) is currently the most promising technology for this purpose. Despite some progress in applying this technique to zebrafish, there is still a great need for improvements in terms of its efficiency, optimal design of sgRNA and ssODNs and broader applicability...
October 24, 2018: Disease Models & Mechanisms
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