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CRISPR AND animal Models

Zhuchi Tu, Hui Zhao, Bang Li, Sen Yan, Lu Wang, Yongjin Tang, Zhujun Li, Dazhang Bai, Caijuan Li, Yinqi Lin, Yuefeng Li, Jianrong Liu, Hao Xu, Xiangyu Guo, Yong-Hui Jiang, Yong Q Zhang, Xiao-Jiang Li
Monogenic mutations in the SHANK3 gene, which encodes a postsynaptic scaffold protein, play a causative role in autism spectrum disorder (ASD). Although a number of mouse models with Shank3 mutations have been valuable for investigating the pathogenesis of ASD, species-dependent differences in behaviors and brain structures post considerable challenges to use small animals to model ASD and to translate experimental therapeutics to the clinic. We have used CRISPR/Cas9 to generate a cynomolgus monkey model by disrupting SHANK3 at exon 6 and 12...
October 16, 2018: Human Molecular Genetics
Aikaterini N Tsouka, Constantinos C Tellis, Alexandros D Tselepis
Protein Convertase Subtilisin/Kexin type 9 (PCSK9) is a serine protease primarily expressed in the liver, which represents the main source of the plasma enzyme. The best characterized function of PCSK9 relates to the binding to low-density lipoprotein receptor (LDL-R) in hepatocytes, increasing its endosomal and lysosomal degradation. This results in the inhibition of LDL-R recycling to the cell surface and therefore the reduction of the hepatic uptake of LDL, leading to the increase in plasma levels of LDL-cholesterol, a major risk factor of cardiovascular diseases (CVD)...
October 10, 2018: Current Pharmaceutical Design
Robert C Orchard, Meagan E Sullender, Bria F Dunlap, Dale R Balce, John G Doench, Herbert W Virgin
Noroviruses (NoVs) are a leading cause of gastroenteritis world-wide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation (CRISPRa) genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in human cells. Our screens identified with high confidence 49 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprising, the majority of the genes identified are not associated with innate or adaptive immunity nor with any antiviral activity...
October 10, 2018: Journal of Virology
Bin Fang, Xueyang Ren, Ying Wang, Ze Li, Lihua Zhao, Manling Zhang, Chu Li, Zhengwei Zhang, Lei Chen, Xiaoxue Li, Jiying Liu, Qiang Xiong, Lining Zhang, Yong Jin, Xiaorui Liu, Lin Li, Hong Wei, Haiyuan Yang, Rongfeng Li, Yifan Dai
Miniature pigs have advantages over rodents in modeling atherosclerosis because their cardiovascular system and physiology are similar to that of humans. Apolipoprotein E (ApoE) deficiency has long been implicated in cardiovascular disease in humans. To establish an improved large animal model of familial hypercholesterolemia and atherosclerosis, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 system (CRISPR/Cas9) was used to disrupt the ApoE gene in Bama miniature pigs...
October 10, 2018: Disease Models & Mechanisms
Tiffany Leidy-Davis, Kai Cheng, Leslie O Goodwin, Judith L Morgan, Wen Chun Juan, Xavier Roca, S Tiong Ong, David E Bergstrom
Here, we describe an expansion of the typical DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus by zygotic injection. Driving our efforts was the desire to create a whole animal model that would replace 17 kilobase pairs (kbp) of the mouse Bcl2l11 gene with the corresponding 25-kbp segment of human BCL2L11, including a conditionally removable segment (2...
October 9, 2018: Scientific Reports
Saba N Baskoylu, Jill Yersak, Patrick O'Hern, Sarah Grosser, Jonah Simon, Sarah Kim, Kelsey Schuch, Maria Dimitriadi, Katherine S Yanagi, Jeremy Lins, Anne C Hart
Mutations in Cu/Zn superoxide dismutase 1 (SOD1) lead to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that disproportionately affects glutamatergic and cholinergic motor neurons. Previous work with SOD1 overexpression models supports a role for SOD1 toxic gain of function in ALS pathogenesis. However, the impact of SOD1 loss of function in ALS cannot be directly examined in overexpression models. In addition, overexpression may obscure the contribution of SOD1 loss of function in the degeneration of different neuronal populations...
October 8, 2018: PLoS Genetics
Lijuan Du, Amy Zhou, Alex Sohr, Sougata Roy
Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression...
September 19, 2018: Journal of Visualized Experiments: JoVE
Marcela Vilarino, Fabian Patrik Suchy, Sheikh Tamir Rashid, Helen Lindsay, Juan Reyes, Bret Roberts McNabb, Talitha van der Meulen, Mark O Huising, Hiromitsu Nakauchi, Pablo Juan Ross
The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep...
October 3, 2018: Transgenic Research
Zhiqiang Fan, Iuri Viotti Perisse, Calvin U Cotton, Misha Regouski, Qinggang Meng, Chaim Domb, Arnaud J Van Wettere, Zhongde Wang, Ann Harris, Kenneth L White, Irina A Polejaeva
Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The major cause of limited life span in CF patients is progressive lung disease. CF models have been generated in 4 species (mice, rats, ferrets, and pigs) to enhance our understanding of the CF pathogenesis. Sheep may be a particularly relevant animal to model CF in humans due to the similarities in lung anatomy and development in the two species. Here, we describe the generation of a sheep model for CF using CRISPR/Cas9 genome editing and somatic cell nuclear transfer (SCNT) techniques...
October 4, 2018: JCI Insight
Tao Zhang, Xin Jiang, Min Xu, Haifang Wang, Xiao Sang, Meiling Qin, Puhua Bao, Ruiqi Wang, Chenchen Zhang, Huiping Lu, Yuzhuo Li, Jin Ren, Hung-Chun Chang, Jun Yan, Qiang Sun, Jin Xu
Mutations in fused in sarcoma (Fus) cause familial amyotrophic lateral sclerosis (ALS) and occasionally frontotemporal dementia. Here we report the establishment and characterization of a novel knockin (KI) rat model expressing a Fus point mutation (R521C) via CRISPR/Cas9. The mutant animals developed adult-onset learning and memory behavioral deficits, with reduced spine density in hippocampal neurons. Remarkably, sleep-wake cycle and circadian abnormalities preceded the onset of cognitive deficit. RNA-seq study further demonstrated altered expression of some key sleep and circadian regulators, such as orexin/hypocretin receptor type 2 and casein kinase 1 epsilon, in the mutant rats...
September 5, 2018: Neurobiology of Aging
Zhe Yang, Shihao Chen, Songlei Xue, Xinxiu Li, Zhen Sun, Yu Yang, Xuming Hu, Tuoyu Geng, Hengmi Cui
OBJECTIVES: To investigate the effect of endogenous Cas9 on genome editing efficiency in transgenic zebrafish. RESULTS: Here we have constructed a transgenic zebrafish strain that can be screened by pigment deficiency. Compared with the traditional CRISPR injection method, the transgenic zebrafish can improve the efficiency of genome editing significantly. At the same time, we first observed that the phenotype of vertebral malformation in early embryonic development of zebrafish after ZFERV knockout...
September 22, 2018: Biotechnology Letters
Rui Lu, Tingting Yuan, Yingge Wang, Ting Zhang, Yuguo Yuan, Daijin Wu, Minya Zhou, Zhengyi He, Yaoyao Lu, Yajie Chen, Jianglin Fan, Jingyan Liang, Yong Cheng
Rabbits (Oryctolagus cuniculus) have been the very frequently used as animal models in the study of human lipid metabolism and atherosclerosis, because they have similar lipoprotein metabolism to humans. Most of hyperlipidemia and atherosclerosis rabbit models are produced by feeding rabbits a high-cholesterol diet. Gene editing or knockout (KO) offered another means of producing rabbit models for study of the metabolism of lipids and lipoproteins. Even so, apolipoprotein (Apo)E KO rabbits must be fed a high-cholesterol diet to induce hyperlipidemia...
September 19, 2018: EBioMedicine
Xiao-Lan Li, Guo-Hua Li, Juan Fu, Ya-Wen Fu, Lu Zhang, Wanqiu Chen, Cameron Arakaki, Jian-Ping Zhang, Wei Wen, Mei Zhao, Weisheng V Chen, Gary D Botimer, David Baylink, Leslie Aranda, Hannah Choi, Rachel Bechar, Prue Talbot, Chang-Kai Sun, Tao Cheng, Xiao-Bing Zhang
Genome editing of human induced pluripotent stem cells (iPSCs) is instrumental for functional genomics, disease modeling, and regenerative medicine. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockin (KI) or knockout (KO) iPSC lines, which is largely due to massive cell death after electroporation with editing plasmids. Here, we report that the transient delivery of BCL-XL increases iPSC survival by ∼10-fold after plasmid transfection, leading to a 20- to 100-fold increase in homology-directed repair (HDR) KI efficiency and a 5-fold increase in non-homologous end joining (NHEJ) KO efficiency...
September 20, 2018: Nucleic Acids Research
Huaqiang Yang, Zhenfang Wu
Pigs serve as an important agricultural resource and animal model in biomedical studies. Efficient and precise modification of pig genome by using recently developed gene editing tools has significantly broadened the application of pig models in various research areas. The three types of site-specific nucleases, namely, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein, are the main gene editing tools that can efficiently introduce predetermined modifications, including knockouts and knockins, into the pig genome...
2018: Frontiers in Genetics
Yichi Zhang, Aaron MacCosham
Inherited cardiomyopathies are cardiovascular disorders that are one of the leading causes of death and are strongly associated with genetic mutations. These include hypertrophic, dilated, restrictive, as well as arrhythmogenic right ventricular cardiomyopathies. Among the patients presenting with these specific forms of cardiomyopathies, there is significant phenotypic, genotypic, and environmental heterogeneity. Over the years, the identification of the underlying mutations common to specific forms of cardiomyopathies have facilitated clinic diagnosis...
September 19, 2018: Lifestyle genomics
Han Wang, Heenam Park, Jonathan Liu, Paul W Sternberg
Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons...
September 17, 2018: G3: Genes—Genomes—Genetics
Arife Unal Eroglu, Timothy S Mulligan, Liyun Zhang, David T White, Sumitra Sengupta, Cathy Nie, Noela Y Lu, Jiang Qian, Lisha Xu, Wuhong Pei, Shawn M Burgess, Meera T Saxena, Jeff S Mumm
Thousands of genes have been implicated in retinal regeneration, but only a few have been shown to impact the regenerative capacity of Müller glia-an adult retinal stem cell with untapped therapeutic potential. Similarly, among nearly 300 genetic loci associated with human retinal disease, the majority remain untested in animal models. To address the large-scale nature of these problems, we are applying CRISPR/Cas9-based genome modification strategies in zebrafish to target over 300 genes implicated in retinal regeneration or degeneration...
2018: Frontiers in Cell and Developmental Biology
Andrew J Waters, Paolo Capriotti, David C A Gaboriau, Philippos Aris Papathanos, Nikolai Windbichler
The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci...
September 3, 2018: Scientific Reports
Leonela Amoasii, John C W Hildyard, Hui Li, Efrain Sanchez-Ortiz, Alex Mireault, Daniel Caballero, Rachel Harron, Thaleia-Rengina Stathopoulou, Claire Massey, John M Shelton, Rhonda Bassel-Duby, Richard J Piercy, Eric N Olson
Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational "hotspot" in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery ( n = 2) or 8 weeks after systemic delivery ( n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type...
October 5, 2018: Science
Thomas Naert, Kris Vleminckx
The recent advent of CRISPR/Cas9 as a straightforward genome editing tool has allowed the establishment of the first bona fide genetic cancer models within the diploid aquatic model organism Xenopus tropicalis (X. tropicalis). Within this chapter, we demonstrate the methods for targeting tumor suppressors with the CRISPR/Cas9 system in the developing X. tropicalis embryo. We further illustrate genotyping and phenotyping of the resulting tumor-bearing F0 mosaic mutant animals (crispants). We focus in detail on the histopathological analysis of cancer neoplasms, the methodology to illustrate high proliferative index by proliferation marker immunofluorescence and how to isolate specific (tumor) cell populations by laser capture microdissection...
2018: Methods in Molecular Biology
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