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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
Alewo Idoko-Akoh, Lorna Taylor, Helen M Sang, Michael J McGrew
Primordial germ cells (PGCs), the embryonic precursors of the sperm and egg, are used for the introduction of genetic modifications into avian genome. Introduction of small defined sequences using genome editing has not been demonstrated in bird species. Here, we compared oligonucleotide-mediated HDR using wild type SpCas9 (SpCas9-WT) and high fidelity SpCas9-HF1 in PGCs and show that many loci in chicken PGCs can be precise edited using donors containing CRISPR/Cas9-blocking mutations positioned in the protospacer adjacent motif (PAM)...
October 11, 2018: Scientific Reports
Yanchi Wang, Junya Zhao, Nannan Duan, Wei Liu, Yuxuan Zhang, Miaojin Zhou, Zhiqing Hu, Mai Feng, Xionghao Liu, Lingqian Wu, Zhuo Li, Desheng Liang
Hemophilia B (HB) is an X-linked recessive bleeding disorder, caused by F9 gene deficiency. Gene therapy combined with the CRISPR/Cas9 technology offers a potential cure for hemophilia B. Now the Cas9 nickase (Cas9n) shows a great advantage in reducing off-target effect compared with wild-type Cas9. In this study, we found that in the multicopy ribosomal DNA (rDNA) locus, the homology directed recombination (HDR) efficiency induced by sgRNA-Cas9n was much higher than sgRNA-Cas9, meanwhile without off-target in six predicted sites...
October 5, 2018: International Journal of Molecular Sciences
Lukas Villiger, Hiu Man Grisch-Chan, Helen Lindsay, Femke Ringnalda, Chiara B Pogliano, Gabriella Allegri, Ralph Fingerhut, Johannes Häberle, Joao Matos, Mark D Robinson, Beat Thöny, Gerald Schwank
CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1 , using recently developed CRISPR-Cas-associated base editors2-4 ...
October 2018: Nature Medicine
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
Yuning Song, Yuxin Zhang, Mao Chen, Jichao Deng, Tingting Sui, Liangxue Lai, Zhanjun Li
BACKGROUND: Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by reduced melanin that are caused by mutations in the gene encoding tyrosinase (TYR), which is the rate-limiting enzyme in the production of the pigment melanin. Many studies or meta-analyses have suggested an association between the TYR T373K SNP and OCA1, but there is limited biochemical and genetic evidence to support this association. METHODS: We overexpressed TYR-WT and TYR-T373K mutants on HK293T cells and tested the changes of melanin production and tyrosinase activity...
September 28, 2018: EBioMedicine
Eric J Aird, Klaus N Lovendahl, Amber St Martin, Reuben S Harris, Wendy R Gordon
The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome, where the Cas9 nuclease then induces a double-stranded break (DSB). The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded oligodeoxynucleotide (ssODN) to the Cas9-guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease, thus spatially and temporally co-localizing the DSB machinery and donor DNA...
2018: Communications biology
Cristina Parola, Derek M Mason, Andreas Zingg, Daniel Neumeier, Sai T Reddy
From the perspective of academic and small research laboratories, the most common and practical strategy for recombinant expression of full-length monoclonal antibodies is to perform transient plasmid transfection of mammalian cells, resulting in small-scale and limited protein production. The generation of stable antibody producing mammalian cell lines enables larger-scale and consistent expression, however this approach is rarely pursued due to the time-consuming and expensive process of single colony screening and characterization...
2018: Methods in Molecular Biology
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
Nicholas D Bonawitz, W Michael Ainley, Asuka Itaya, Sivarama R Chennareddy, Tobias Cicak, Katherine Effinger, Ke Jiang, Tejinder Kumar Mall, Pradeep Reddy Marri, J Pon Samuel, Nagesh Sardesai, Matthew Simpson, Otto Folkerts, Rodrigo Sarria, Steven R Webb, Delkin O Gonzalez, Daina H Simmonds, Dayakar R Pareddy
Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence-specific endonucleases to generate DNA double strand breaks (DSBs) at user-specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non-homologous end joining (NHEJ)-mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology-directed repair (HDR)-mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9...
September 16, 2018: Plant Biotechnology Journal
Amanda Haupt, Tanya Grancharova, Joy Arakaki, Margaret A Fuqua, Brock Roberts, Ruwanthi N Gunawardane
A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation...
August 25, 2018: Journal of Visualized Experiments: JoVE
Huibin Tian, Jun Luo, Zhifei Zhang, Jiao Wu, Tianying Zhang, Sebastiano Busato, Lian Huang, Ning Song, Massimo Bionaz
Stearoyl-CoA desaturase 1 (SCD1) is a fatty acid desaturase catalyzing cis-double-bond formation in the Δ9 position to produce monounsaturated fatty acids essential for the synthesis of milk fat. Previous studies using RNAi methods have provided support for a role of SCD1 in goat mammary epithelial cells (GMEC); however, RNAi presents several limitations that might preclude a truthful understanding of the biological function of SCD1. To explore the function of SCD1 on fatty acid metabolism in GMEC, we used CRISPR-Cas9-mediated SCD1 knockout through non-homologous end-joining (NHEJ) and homology-directed repair (HDR) pathways in GMEC...
September 26, 2018: Journal of Agricultural and Food Chemistry
Daniela Benati, Francesca Miselli, Fabienne Cocchiarella, Clarissa Patrizi, Marta Carretero, Samantha Baldassarri, Virginia Ammendola, Cristina Has, Stefano Colloca, Marcela Del Rio, Fernando Larcher, Alessandra Recchia
Deficiency of basement membrane heterotrimeric laminin 332 component, coded by LAMA3, LAMB3, and LAMC2 genes, causes junctional epidermolysis bullosa (JEB), a severe skin adhesion defect. Herein, we report the first application of CRISPR/Cas9-mediated homology direct repair (HDR) to in situ restore LAMB3 expression in JEB keratinocytes in vitro and in immunodeficient mice transplanted with genetically corrected skin equivalents. We packaged an adenovector carrying Cas9/guide RNA (gRNA) tailored to the intron 2 of LAMB3 gene and an integration defective lentiviral vector bearing a promoterless quasi-complete LAMB3 cDNA downstream a splice acceptor site and flanked by homology arms...
August 4, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
Sushil Devkota
Non-homologous end joining (NHEJ), and to a lesser extent, the error-free pathway known as homology-directed repair (HDR) are cellular mechanisms for recovery from double-strand DNA breaks (DSB) induced by RNA-guided programmable nuclease CRISPR/Cas. Since NHEJ is equivalent to using a duck tape to stick two pieces of metals together, the outcome of this repair mechanism is prone to error. Any out-of-frame mutations or premature stop codons resulting from NHEJ repair mechanism are extremely handy for loss-of-function studies...
September 2018: BMB Reports
Anna Buchman, Omar S Akbari
Despite the importance of Y-chromosomes in evolution and sex determination, their heterochromatic, repeat-rich nature makes them difficult to sequence (due, in part, to ambiguities in sequence alignment and assembly) and to genetically manipulate, and therefore they generally remain poorly understood. For example, the D. melanogaster Y-chromosome, one of the most extensively studied Y-chromosomes, is widely heterochromatic and composed mainly of highly repetitive sequences, with only a handful of expressed genes scattered throughout its length...
August 6, 2018: Insect Molecular Biology
Xiaoyun Xu, Dongbing Gao, Ping Wang, Jian Chen, Jinxue Ruan, Jie Xu, Xiaofeng Xia
CRISPR/Cas9 efficiently generates gene knock-out via nonhomologous end joining (NHEJ), but the efficiency of precise homology-directed repair (HDR) is substantially lower, especially in the hard-to-transfect human stem cells and primary cells. Herein we report a tube electroporation method that can effectively transfect human stem cells and primary cells with minimal cytotoxicity. When applied to genome editing using CRISPR/Cas9 along with single stranded DNA oligonucleotide (ssODN) template in human induced pluripotent stem cells (iPSCs), up to 42...
August 3, 2018: Scientific Reports
Lupeng Ye, Chengkun Wang, Lingjuan Hong, Ninghe Sun, Danyang Chen, Sidi Chen, Feng Han
CRISPR systems have been proven as versatile tools for site-specific genome engineering in mammalian species. During the gene editing processes, these RNA-guide nucleases introduce DNA double strand breaks (DSBs), in which non-homologous DNA end joining (NHEJ) dominates the DNA repair pathway, limiting the efficiency of homology-directed repair (HDR), the alternative pathway essential for precise gene targeting. Multiple approaches have been developed to enhance HDR, including chemical compound or RNA interference-mediated inhibition of NHEJ factors, small molecule activation of HDR enzymes, or cell cycle timed delivery of CRISPR complex...
2018: Cell Discovery
Lei Li, Keke Wei, Guosong Zheng, Xiaocao Liu, Shaoxin Chen, Weihong Jiang, Yinhua Lu
Streptomyces has a strong capability for producing a large number of bioactive natural products and remains invaluable as a source for the discovery of novel drug leads. Although the Streptococcus pyogenes CRISPR-Cas9-assisted genome editing tool has been developed for rapid genetic engineering in Streptomyces , it has a number of limitations, including the toxicity of Sp Cas9 expression in some important industrial Streptomyces strains and the need for complex expression constructs when targeting multiple genomic loci...
September 15, 2018: Applied and Environmental Microbiology
Jun Bo Tang, Hao Wei Cao, Rui Xu, Dan Dan Zhang, Juan Huang
Multiple genes work together to maintain the normal functions of the reproductive system. However, for many of these genes, little is known about their specific functions and mechanisms. In the present study, eight Drosophila genes, including CG4161, CG11475, CG2921, CG10541, CG7276, CG3800, CG8117 and CG16779, were selected for detailed studies based on their testis expression, undefined functions, and having highly homologous and conserved genes in humans (Homo sapiens) and mouse (Mus musculus). We analyzed their expression levels in different tissues, and determined their probably functions in male reproduction...
June 20, 2018: Yi Chuan, Hereditas
Shaoya Li, Jingying Li, Jiahui Zhang, Wenming Du, Jindong Fu, Suhas Sutar, Yunde Zhao, Lanqin Xia
The recently developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system expands the range of genome editing and is emerging as an alternative powerful tool for both plant functional genomics and crop improvement. Cpf1-CRISPR RNA (crRNA) produces double strand DNA breaks (DSBs) with long 5'-protruding ends, which may facilitate the pairing and insertion of repair templates through homology-directed repair (HDR) for targeted gene replacement and introduction of the desired DNA elements at specific gene loci for crop improvement...
September 14, 2018: Journal of Experimental Botany
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