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Soyoung A Oh, Akiko Seki, Sascha Rutz
CRISPR/Cas9 has enabled the rapid and efficient generation of gene knockouts across various cell types of several species. T cells are central players in adaptive immune responses. Gene editing in primary T cells not only represents a valuable research tool, but is also critical for next generation immunotherapies, such as CAR T cells. Broad application of CRIPSR/Cas9 for gene editing in primary T cells has been hampered by limitations in transfection efficiency and the requirement for TCR stimulation. In this article, we provide a detailed protocol for Cas9/gRNA ribonucleoprotein (RNP) transfection of primary mouse and human T cells without the need for TCR stimulation that achieves near complete loss of target gene expression at the population level...
October 18, 2018: Current Protocols in Immunology
Praveen Balabaskaran-Nina, Sanjay A Desai
Genetic manipulation of the human malaria parasite Plasmodium falciparum is needed to explore pathogen biology and evaluate antimalarial targets. It is, however, aggravated by a low transfection efficiency, a paucity of selectable markers and a biased A/T-rich genome. While various enabling technologies have been introduced over the past two decades, facile and broad-range modification of essential genes remains challenging. We recently devised a new application of the Bxb1 integrase strategy to meet this need through an intronic attB sequence within the gene of interest...
October 17, 2018: Parasites & Vectors
Tingting Sui, Yuning Song, Zhiquan Liu, Mao Chen, Jichao Deng, Yuanyuan Xu, Liangxue Lai, Zhanjun Li
In previous studies, CRISPR/Cas9 was shown to induce unexpected exon skipping; however, the mechanism by which this phenomenon is triggered is controversial. By analyzing 22 gene-edited rabbit lines generated using CRISPR/Cas9, we provide evidence of exon skipping at high frequency in premature termination codon-mutated rabbits but not in the rabbits with a premature termination codon mutation in exon 1 rabbits with non-frameshift or missense mutations. Our results suggest that CRISPR-mediated exon skipping depends on premature termination codon mutation-induced nonsense-associated altered splicing...
October 17, 2018: Genome Biology
Tao Gong, Boyu Tang, Xuedong Zhou, Jumei Zeng, Miao Lu, Xiaoxin Guo, Xian Peng, Lei Lei, Bo Gong, Yuqing Li
Streptococcus mutans is the primary aetiological agent of human dental caries. Its major virulence factors, glucosyltransferases (Gtfs), utilize sucrose to synthesize extracellular polysaccharides (EPS), leading to the formation of dental plaque biofilm. The current study was designed to develop a novel self-targeting gene editing technology that targeted gtfs to inhibit biofilms formation. The CRISPR-Cas system (i.e., clustered regularly interspaced short palindromic repeat, with CRISPR-associated proteins) provides sequence-specific protection against foreign genetic materials in archaea and bacteria,and has been widely developed for genomic engineering...
October 17, 2018: Molecular Oral Microbiology
Xingliang Guo, Hua Jiang, Bizhi Shi, Min Zhou, Honghong Zhang, Zhimin Shi, Guoxiu Du, Hong Luo, Xiuqi Wu, Yi Wang, Ruixin Sun, Zonghai Li
Cancer immunotherapy has made unprecedented breakthrough in the fields of chimeric antigen receptor-redirected T (CAR T) cell therapy and immune modulation. Combination of CAR modification and the disruption of endogenous inhibitory immune checkpoints on T cells represent a promising immunotherapeutic modality for cancer treatment. However, the potential for the treatment of hepatocellular carcinoma (HCC) has not been explored. In this study, the gene expressing the programmed death 1 receptor (PD-1) on the Glypican-3 (GPC3)-targeted second-generation CAR T cells employing CD28 as the co-stimulatory domain was disrupted using the CRISPR/Cas9 gene-editing system...
2018: Frontiers in Pharmacology
Zulema Romero, Mark DeWitt, Mark C Walters
Sickle cell anemia (SCA) is a hereditary blood disease caused by a single-gene mutation that affects millions of individuals world-wide. In this review, we focus on techniques to treat SCA by ex vivo genetic manipulation of hematopoietic stem/progenitor cells (HSPCs), emphasizing replacement gene therapy and gene editing. Areas covered: Viral transduction of an anti-sickling β-like globin gene has been tested in pre-clinical and early-phase clinical studies, and shows promising preliminary results. Targeted editing of endogenous genes by site-directed nucleases has been developed more recently, and several approaches also are nearing clinical translation...
October 16, 2018: Expert Opinion on Biological Therapy
Joanna M Feehan, Paloma Stanar, Beatrice M Tam, Colette Chiu, Orson L Moritz
Xenopus laevis have proven to be a useful system for rapid generation and analysis of transgenic models of human retinal disease. However, experimental approaches in this system were limited by lack of a robust knockdown or knockout technology. Here we describe a protocol for generation of Cas9-edited X. laevis embryos. The technique introduces point mutations into the genome of X. laevis resulting in in-frame and out-of-frame insertions and deletions that allow modeling of both dominant and recessive human diseases and efficiently generates gene knockdown and knockout...
2019: Methods in Molecular Biology
Daniela Benati, Valeria Marigo, Alessandra Recchia
CRISPR/Cas9 is an efficient tool to knock down specific genes in various organisms. In this chapter, we describe how to assess knockdown of human rhodopsin (RHO) gene carrying the P23H mutation in vitro, in engineered HeLa cells, and in vivo, in P23H RHO transgenic mice. To this aim, we report two molecular assays: site-specific PCR on P23H RHO cells treated with CRISPR/Cas9 and Western blotting analysis on retinal cells prepared from P23H RHO transgenic mice electroporated with CRISPR/Cas9 and GFP plasmids...
2019: Methods in Molecular Biology
Priyanka Maiti, Hyun-Jung Kim, Ya-Ting Tu, Antoni Barrientos
Most steps on the biogenesis of the mitochondrial ribosome (mitoribosome) occur near the mitochondrial DNA nucleoid, in RNA granules, which contain dedicated RNA metabolism and mitoribosome assembly factors. Here, analysis of the RNA granule proteome identified the presence of a set of small GTPases that belong to conserved families of ribosome assembly factors. We show that GTPBP10, a member of the conserved Obg family of P-loop small G proteins, is a mitochondrial protein and have used gene-editing technologies to create a HEK293T cell line KO for GTPBP10...
October 13, 2018: Nucleic Acids Research
Andrey D Volgin, Oleg A Yakovlev, Konstantin A Demin, Murilo S de Abreu, Polina A Alekseeva, Ashton J Friend, Anton M Lakstygal, Tamara G Amstislavskaya, Wandong Bao, Cai Song, Allan V Kalueff
Currently becoming widely recognized, personalized psychiatry focuses on unique physiological and genetic profiles of patients to best tailor their therapy. However, the role of individual differences, as well as genetic and environmental factors, in human psychiatric disorders remains poorly understood. Animal experimental models are a valuable tool to improve our understanding of disease pathophysiology and its molecular mechanisms. Due to high reproduction capability, fully sequenced genome, easy gene editing, and high genetic and physiological homology with humans, zebrafish (Danio rerio) are emerging as a novel powerful model in biomedicine...
October 15, 2018: Journal of Neuroscience Research
Andrew M Tidball, Preethi Swaminathan, Louis T Dang, Jack M Parent
For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector concurrently with episomal reprogramming plasmids into fibroblasts. Our experiments have produced nearly even numbers of all 3 genotypes in autosomal genes. In addition, we provide a detailed approach for maintaining and genotyping 96-well plates of iPSC clones...
April 5, 2018: Bio-protocol
Wanwan Liang, Solveig van Wersch, Meixuezi Tong, Xin Li
Intracellular nucleotide-binding (NB) and leucine-rich repeat (NLR) proteins function as immune receptors to recognize effectors from pathogens. They often guard host proteins that are the direct targets of those effectors. Recent findings revealed that a typical NLR sometimes cooperates with another atypical NLR for effector recognition. Here, by using CRISPR/Cas9 gene editing method, knockout analysis and biochemical assays, we uncovered differential pairings of typical Toll Interleukin1 receptor (TIR) type NLR (TNL) receptor SOC3 with atypical truncated TIR-NB (TN) proteins CHS1 or TN2 to guard the homeostasis of the E3 ligase SAUL1...
October 14, 2018: New Phytologist
Tiffany A Peterson, Andrew G MacLean
Macrophages, one of the most abundant populations of leukocytes in the body, function as the first line of defense against pathogen invaders. Human Immunodeficiency virus 1 (HIV-1) remains to date one of the most extensively studied viral infections. Naturally occurring lentiviruses in domestic and primate species serve as valuable models to investigate lentiviral pathogenesis and novel therapeutics. Better understanding of the role macrophages play in HIV pathogenesis will aid in the advancement towards a cure...
October 13, 2018: Journal of Neuroimmune Pharmacology: the Official Journal of the Society on NeuroImmune Pharmacology
Yvonne Knopp, Franziska K Geis, Dirk Heckl, Stefan Horn, Thomas Neumann, Johannes Kuehle, Janine Meyer, Boris Fehse, Christopher Baum, Michael Morgan, Johann Meyer, Axel Schambach, Melanie Galla
The recently discovered CRISPR/Cas9 system is widely used in basic research and is a useful tool for disease modeling and gene editing therapies. However, long-term expression of DNA-modifying enzymes can be associated with cytotoxicity and is particularly unwanted in clinical gene editing strategies. Because current transient expression methods may still suffer from cytotoxicity and/or low efficiency, we developed non-integrating retrovirus-based CRISPR/Cas9 all-in-one particles for targeted gene knockout...
September 13, 2018: Molecular Therapy. Nucleic Acids
Adriana Buskin, Lili Zhu, Valeria Chichagova, Basudha Basu, Sina Mozaffari-Jovin, David Dolan, Alastair Droop, Joseph Collin, Revital Bronstein, Sudeep Mehrotra, Michael Farkas, Gerrit Hilgen, Kathryn White, Kuan-Ting Pan, Achim Treumann, Dean Hallam, Katarzyna Bialas, Git Chung, Carla Mellough, Yuchun Ding, Natalio Krasnogor, Stefan Przyborski, Simon Zwolinski, Jumana Al-Aama, Sameer Alharthi, Yaobo Xu, Gabrielle Wheway, Katarzyna Szymanska, Martin McKibbin, Chris F Inglehearn, David J Elliott, Susan Lindsay, Robin R Ali, David H Steel, Lyle Armstrong, Evelyne Sernagor, Henning Urlaub, Eric Pierce, Reinhard Lührmann, Sushma-Nagaraja Grellscheid, Colin A Johnson, Majlinda Lako
Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/- mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/- mouse retinae and RPE...
October 12, 2018: Nature Communications
Grzegorz Kreiner
Neurodegenerative diseases are currently a major challenge in elderly care due to demographic changes and dramatic increases in the aging population worldwide. This review is focused on the most promising research discovering ways to attenuate neural loss or enhance neuroprotection and unraveling the basis of neurodegeneration using transgenic mouse models. With the recent introduction of the powerful and relatively simple gene-editing tool CRISPR-Cas9, we have entered a new era in genetic engineering that will certainly lead to a variety of new transgenic models in the near future...
September 16, 2018: Pharmacological Reports: PR
Chen Zhao, Yingze Zhao, Jingfang Zhang, Jia Lu, Li Chen, Yue Zhang, Yue Ying, Junjun Xu, Shixian Wei, Yu Wang
The CRISPR/Cas9 enabled efficient gene editing in an easy and programmable manner. Controlling its activity in greater precision is desired for biomedical research and potential therapeutic translation. Here, we engrafted the CRISPR/Cas9 system with a mutated human estrogen receptor (ERT2 ), which renders it 4-hydroxytamoxifen (4-OHT) inducible for the access of genome, and a nuclear export signal (NES), which lowers the background activity. Tight and efficient drug-inducible genome editing was achieved across several human cell types, including embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), upon vigorous optimization...
September 1, 2018: Molecular Therapy. Nucleic Acids
Q Raas, F-E Saih, C Gondcaille, D Trompier, Y Hamon, V Leoni, C Caccia, B Nasser, M Jadot, F Ménétrier, G Lizard, M Cherkaoui-Malki, P Andreoletti, S Savary
Acyl-CoA oxidase (ACOX1) deficiency is a rare and severe peroxisomal leukodystrophy associated with a very long-chain fatty acid (VLCFA) β-oxidation defect. This neurodegenerative disease lacks relevant cell models to further decipher the pathomechanisms in order to identify novel therapeutic targets. Since peroxisomal defects in microglia appear to be a key component of peroxisomal leukodystrophies, we targeted the Acox1 gene in the murine microglial BV-2 cell line. Using CRISPR/Cas9 gene editing, we generated an Acox1-deficient cell line and validated the allelic mutations, which lead to the absence of ACOX1 protein and enzymatic activity...
October 9, 2018: Biochimica et biophysica acta. Molecular and cell biology of lipids
Nitasha Sehgal, M Eileen Sylves, Ansuman Sahoo, Jacky Chow, Sarah E Walker, Paul J Cullen, James O Berry
Clustered regularly interspaced short palindromic repeats (CRISPR) are a revolutionary tool based on a bacterial acquired immune response system. CRISPR has gained widespread use for gene editing in a variety of organisms and is an increasingly valuable tool for basic genetic research, with far-reaching implications for medicine, agriculture, and industry. This lab is based on the premise that upper division undergraduate students enrolled in a Life Sciences curriculum must become familiar with cutting edge advances in biotechnology that have significant impact on society...
October 12, 2018: Biochemistry and Molecular Biology Education
Yuxiao Hu, Yanrong Jia, Xiangdong Zhao, Zihao Yang, Zhimin Hao, Jingao Dong, Fanli Zeng
Technologies development for seamless gene editing and marker recycling has allowed frequent genomic engineering in Saccharomyces cerevisiae for desired laboratory strains and cell factory. Alternative new approaches are still required for complicated scenarios. In this study, we report that inducible overexpression of cell wall protein 1 (Cwp1) by galactose addition confers yeast cells a robust growth inhibition. Direct repeats flanking the Gal-CWP1:selectable marker cassette allow for its homology recombination excision and counter selection upon galactose addition, therefore enable seamless gene editing and marker recycling...
October 11, 2018: MicrobiologyOpen
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