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https://www.readbyqxmd.com/read/28809467/multidimensional-control-of-cas9-by-evolved-rna-polymerase-based-biosensors
#1
Jinyue Pu, Kaitlin Kentala, Bryan C Dickinson
Systems to control Cas9 with spatial and temporal precision offer opportunities to decrease side effects, protect sensitive tissues, and create gene therapies that are only activated at defined times and places. Here, we present the design of new Cas9 controllers based on RNA polymerase (RNAP)-based biosensors that produce gRNAs, thereby regulating target knockout. After development and validation of a new abscisic acid-inducible biosensor to control Cas9, we lowered the background of the system using continuous evolution...
August 15, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28800611/using-local-chromatin-structure-to-improve-crispr-cas9-efficiency-in-zebrafish
#2
Yunru Chen, Shiyang Zeng, Ruikun Hu, Xiangxiu Wang, Weilai Huang, Jiangfang Liu, Luying Wang, Guifen Liu, Ying Cao, Yong Zhang
Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines...
2017: PloS One
https://www.readbyqxmd.com/read/28800587/highly-efficient-gene-inactivation-by-adenoviral-crispr-cas9-in-human-primary-cells
#3
Olaf Voets, Frans Tielen, Edo Elstak, Julian Benschop, Max Grimbergen, Jan Stallen, Richard Janssen, Andre van Marle, Christian Essrich
Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells...
2017: PloS One
https://www.readbyqxmd.com/read/28794219/dodging-silver-bullets-good-crispr-gene-drive-design-is-critical-for-eradicating-exotic-vertebrates
#4
Thomas A A Prowse, Phillip Cassey, Joshua V Ross, Chandran Pfitzner, Talia A Wittmann, Paul Thomas
Self-replicating gene drives that can spread deleterious alleles through animal populations have been promoted as a much needed but controversial 'silver bullet' for controlling invasive alien species. Homing-based drives comprise an endonuclease and a guide RNA (gRNA) that are replicated during meiosis via homologous recombination. However, their efficacy for controlling wild populations is threatened by inherent polymorphic resistance and the creation of resistance alleles via non-homologous end-joining (NHEJ)-mediated DNA repair...
August 16, 2017: Proceedings. Biological Sciences
https://www.readbyqxmd.com/read/28790199/in-vivo-genome-editing-restores-dystrophin-expression-and-cardiac-function-in-dystrophic-mice
#5
Mona El Refaey, Li Xu, Yandi Gao, Benjamin D Canan, Tm A Adesanya, Sarah C Warner, Keiko Akagi, David E Symer, Peter J Mohler, Jianjie Ma, Paul M Janssen, Renzhi Han
Rationale: Duchenne muscular dystrophy (DMD) is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic cardiomyopathy is a leading cause of death in DMD patients and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on DMD cardiac function has yet to be evaluated...
August 8, 2017: Circulation Research
https://www.readbyqxmd.com/read/28757163/evaluating-synthetic-activation-and-repression-of-neuropsychiatric-related-genes-in-hipsc-derived-npcs-neurons-and-astrocytes
#6
Seok-Man Ho, Brigham J Hartley, Erin Flaherty, Prashanth Rajarajan, Rawan Abdelaal, Ifeanyi Obiorah, Natalie Barretto, Hamza Muhammad, Hemali P Phatnani, Schahram Akbarian, Kristen J Brennand
Modulation of transcription, either synthetic activation or repression, via dCas9-fusion proteins is a relatively new methodology with the potential to facilitate high-throughput up- or downregulation studies of gene function. Genetic studies of neurodevelopmental disorders have identified a growing list of risk variants, including both common single-nucleotide variants and rare copy-number variations, many of which are associated with genes having limited functional annotations. By applying a CRISPR-mediated gene-activation/repression platform to populations of human-induced pluripotent stem cell-derived neural progenitor cells, neurons, and astrocytes, we demonstrate that it is possible to manipulate endogenous expression levels of candidate neuropsychiatric risk genes across these three cell types...
August 8, 2017: Stem Cell Reports
https://www.readbyqxmd.com/read/28750323/differential-use-of-3-cites-by-the-subgenomic-rna-of-pea-enation-mosaic-virus-2
#7
Feng Gao, Anne E Simon
The genomic RNA (gRNA) of Pea enation mosaic virus 2 (PEMV2) is the template for p33 and -1 frameshift product p94. The PEMV2 subgenomic RNA (sgRNA) encodes two overlapping ORFs, p26 and p27, which are required for movement and stability of the gRNA. Efficient translation of p33 requires two of three 3' proximal cap-independent translation enhancers (3'CITEs): the kl-TSS, which binds ribosomes and engages in a long-distance interaction with the 5'end; and the adjacent eIF4E-binding PTE. Unlike the gRNA, all three 3'CITEs were required for efficient translation of the sgRNA, which included the ribosome-binding 3'TSS...
July 24, 2017: Virology
https://www.readbyqxmd.com/read/28745625/a-protocol-for-multiple-gene-knockout-in-mouse-small-intestinal-organoids-using-a-crispr-concatemer
#8
Alessandra Merenda, Amanda Andersson-Rolf, Roxana C Mustata, Taibo Li, Hyunki Kim, Bon-Kyoung Koo
CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids...
July 12, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28742008/development-of-a-crispr-cas9-mediated-gene-editing-tool-in-streptomyces-rimosus
#9
Haiyan Jia, Longmei Zhang, Tongtong Wang, Jin Han, Hui Tang, Liping Zhang
Clustered regularly interspaced short palindromic repeats, associated proteins (CRISPR/Cas), has been developed into a powerful, targeted genome-editing tool in a wide variety of species. Here, we report an extensive investigation of the type II CRISPR/Cas9 system for targeted gene editing in Streptomyces rimosus. S. rimosus is used in the production of the antibiotic oxytetracycline, and its genome differs greatly from other species of the genus Streptomyces in the conserved chromosome terminal and core regions, which is of major production and scientific research value...
July 26, 2017: Microbiology
https://www.readbyqxmd.com/read/28730433/computational-prediction-of-the-immunomodulatory-potential-of-rna-sequences
#10
Gandharva Nagpal, Kumardeep Chaudhary, Sandeep Kumar Dhanda, Gajendra Pal Singh Raghava
Advances in the knowledge of various roles played by non-coding RNAs have stimulated the application of RNA molecules as therapeutics. Among these molecules, miRNA, siRNA, and CRISPR-Cas9 associated gRNA have been identified as the most potent RNA molecule classes with diverse therapeutic applications. One of the major limitations of RNA-based therapeutics is immunotoxicity of RNA molecules as it may induce the innate immune system. In contrast, RNA molecules that are potent immunostimulators are strong candidates for use in vaccine adjuvants...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28729655/a-crispr-cas9-guidance-rna-screen-platform-for-hiv-provirus-disruption-and-hiv-aids-gene-therapy-in-astrocytes
#11
Zaohua Huang, Madahavan Nair
HIV/AIDS remains a major health threat despite significant advances in the prevention and treatment of HIV infection. The major reason is the inability of existing treatments to eradicate the multiple HIV reservoirs in the human body, including astrocytes in the human brain. CRISPR/Cas9 system is an emerging gene-editing technique with the potential to eliminate or disrupt HIV provirus in HIV reservoir cells, which may lead to a complete cure of HIV/AIDS. The key components of CRISPR/Cas9 are guide RNAs (gRNAs) which determine specific sequence targeting of DNAs...
July 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28726630/dissection-of-specific-binding-of-hiv-1-gag-to-the-packaging-signal-in-viral-rna
#12
Mauricio Comas-Garcia, Siddhartha Ak Datta, Laura Baker, Rajat Varma, Prabhakar R Gudla, Alan Rein
Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a cis-acting RNA element called the 'packaging signal' (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength...
July 20, 2017: ELife
https://www.readbyqxmd.com/read/28712493/crispr-cas9-enabled-multiplex-genome-editing-and-its-application
#13
Bastian Minkenberg, Matthew Wheatley, Yinong Yang
The CRISPR/Cas9 system is a prevalent and versatile genome-editing tool of choice for basic and applied biological research. An exchange of a 20-bp spacer sequence in the gRNA can easily reprogram Cas9 to target a different DNA site. By expressing or providing multiple gRNAs, the system also enables multiplex genome editing at high efficiencies. Current approaches for providing multiple gRNAs in vivo include the use of multigene cassettes to express several gRNAs, Csy4-based excision, arrays of crRNAs, ribozyme-flanked gRNAs, tRNA-dependent cleavage of gRNAs, and direct introduction of Cas9 proteins preloaded with different gRNAs...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/28712454/crispr-cas9-mediated-scanning-for-regulatory-elements-required-for-hprt1-expression-via-thousands-of-large-programmed-genomic-deletions
#14
Molly Gasperini, Gregory M Findlay, Aaron McKenna, Jennifer H Milbank, Choli Lee, Melissa D Zhang, Darren A Cusanovich, Jay Shendure
The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel"). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder...
August 3, 2017: American Journal of Human Genetics
https://www.readbyqxmd.com/read/28671645/qtl-mapping-and-crispr-cas9-editing-to-identify-a-drug-resistance-gene-in-toxoplasma-gondii
#15
Bang Shen, Robin H Powell, Michael S Behnke
Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype...
June 22, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28655737/evaluation-and-design-of-genome-wide-crispr-spcas9-knockout-screens
#16
Traver Hart, Amy Hin Yan Tong, Katie Chan, Jolanda Van Leeuwen, Ashwin Seetharaman, Michael Aregger, Megha Chandrashekhar, Nicole Hustedt, Sahil Seth, Avery Noonan, Andrea Habsid, Olga Sizova, Lyudmilla Nedyalkova, Ryan Climie, Leanne Tworzyanski, Keith Lawson, Maria Augusta Sartori, Sabriyeh Alibai, David Tieu, Sanna Masud, Patricia Mero, Alexander Weiss, Kevin R Brown, Matej Ušaj, Maximilian Billmann, Mahfuzur Rahman, Michael Costanzo, Chad L Myers, Brenda J Andrews, Charles Boone, Daniel Durocher, Jason Moffat
The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols...
June 27, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28646085/generation-of-a-collection-of-mutant-tomato-lines-using-pooled-crispr-libraries
#17
Thomas B Jacobs, Ning Zhang, Dhruv Patel, Gregory B Martin
The high efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mutagenesis in plants enables the development of high-throughput mutagenesis strategies. By transforming pooled CRISPR libraries into tomato (Solanum lycopersicum), collections of mutant lines were generated with minimal transformation attempts and in a relatively short period of time. Identification of the targeted gene(s) was easily determined by sequencing the incorporated guide RNA(s) in the primary transgenic events...
August 2017: Plant Physiology
https://www.readbyqxmd.com/read/28643249/genome-editing-of-mouse-by-cytoplasmic-injection
#18
Takuro Horii, Izuho Hatada
CRISPR/Cas enables rapid production of genome-edited animals. The Cas9/gRNA component can be introduced to fertilized eggs in several ways. Here, we provide an instructional guide for the generation of knockout mice by cytoplasmic injection using in vitro transcribed Cas9 and gRNA.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28643248/computational-prediction-of-crispr-cas9-target-sites-reveals-potential-off-target-risks-in-human-and-mouse
#19
Qingbo Wang, Kumiko Ui-Tei
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a prominent genome engineering technology. In the CRISPR/Cas system, the RNA-guided endonuclease Cas protein introduces a DNA double-stranded break at the genome position recognized by a guide RNA (gRNA) based on complementary base-pairing of about 20-nucleotides in length. The 8- or 12-mer gRNA sequence in the proximal region is especially important for target recognition, and the genes with sequence complementarity to such regions are often disrupted...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28640612/crispr-cas9-based-genome-editing-for-disease-modeling-and-therapy-challenges-and-opportunities-for-nonviral-delivery
#20
REVIEW
Hong-Xia Wang, Mingqiang Li, Ciaran M Lee, Syandan Chakraborty, Hae-Won Kim, Gang Bao, Kam W Leong
Genome editing offers promising solutions to genetic disorders by editing DNA sequences or modulating gene expression. The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology can be used to edit single or multiple genes in a wide variety of cell types and organisms in vitro and in vivo. Herein, we review the rapidly developing CRISPR/Cas9-based technologies for disease modeling and gene correction and recent progress toward Cas9/guide RNA (gRNA) delivery based on viral and nonviral vectors...
August 9, 2017: Chemical Reviews
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