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CAS9 and AAV

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https://www.readbyqxmd.com/read/27898094/crispr-cas9-aav-mediated-knock-in-at-nrl-locus-in-human-embryonic-stem-cells
#1
Xianglian Ge, Haitao Xi, Fayu Yang, Xiao Zhi, Yanghua Fu, Ding Chen, Ren-He Xu, Ge Lin, Jia Qu, Junzhao Zhao, Feng Gu
Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low...
November 29, 2016: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/27837149/chronic-hyperphosphatemia-and-vascular-calcification-are-reduced-by-stable-delivery-of-soluble-klotho
#2
Julia M Hum, Linda M O'Bryan, Arun K Tatiparthi, Taryn A Cass, Erica L Clinkenbeard, Martin S Cramer, Manoj Bhaskaran, Robert L Johnson, Jonathan M Wilson, Rosamund C Smith, Kenneth E White
αKlotho (αKL) regulates mineral metabolism, and diseases associated with αKL deficiency are characterized by hyperphosphatemia and vascular calcification (VC). αKL is expressed as a membrane-bound protein (mKL) and recognized as the coreceptor for fibroblast growth factor-23 (FGF23) and a circulating soluble form (cKL) created by endoproteolytic cleavage of mKL. The functions of cKL with regard to phosphate metabolism are unclear. We tested the ability of cKL to regulate pathways and phenotypes associated with hyperphosphatemia in a mouse model of CKD-mineral bone disorder and αKL-null mice...
November 11, 2016: Journal of the American Society of Nephrology: JASN
https://www.readbyqxmd.com/read/27803249/adeno-associated-virus-mediated-delivery-of-crispr-cas-systems-for-genome-engineering-in-mammalian-cells
#3
Thomas Gaj, David V Schaffer
The CRISPR-Cas9 system has emerged as a highly versatile platform for introducing targeted genome modifications into mammalian cells and model organisms. However, fully capitalizing on the therapeutic potential for this system requires its safe and efficient delivery into relevant cell types. Adeno-associated virus (AAV) vectors are a clinically promising class of engineered gene-delivery vehicles capable of safely infecting a broad range of dividing and nondividing cell types, while also serving as a highly effective donor template for homology-directed repair...
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27784063/a-novel-method-combining-vitreous-aspiration-and-intravitreal-aav2-8-injection-results-in-retina-wide-transduction-in-adult-mice
#4
Romain Da Costa, Carsten Röger, Jasmin Segelken, Maya Barben, Christian Grimm, John Neidhardt
Purpose: Gene therapies to treat eye disorders have been extensively studied in the past 20 years. Frequently, adeno-associated viruses were applied to the subretinal or intravitreal space of the eye to transduce retinal cells with nucleotide sequences of therapeutic potential. In this study we describe a novel intravitreal injection procedure that leads to a reproducible adeno-associated virus (AAV)2/8-mediated transduction of more than 70% of the retina. Methods: Prior to a single intravitreal injection of a enhanced green fluorescent protien (GFP)-expressing viral suspension, we performed an aspiration of vitreous tissue from wild-type C57Bl/6J mice...
October 1, 2016: Investigative Ophthalmology & Visual Science
https://www.readbyqxmd.com/read/27775045/supernova-a-versatile-vector-system-for-single-cell-labeling-and-gene-function-studies-in-vivo
#5
Wenshu Luo, Hidenobu Mizuno, Ryohei Iwata, Shingo Nakazawa, Kosuke Yasuda, Shigeyoshi Itohara, Takuji Iwasato
Here we describe "Supernova" series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood...
October 24, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27742910/crispr-cas9-with-single-guide-rna-expression-driven-by-small-trna-promoters-showed-reduced-editing-efficiency-compared-to-u6-promoter
#6
Yuda Wei, Yan Qiu, Yanhao Chen, Gaigai Liu, Yongxian Zhang, Luwei Xu, Qiurong Ding
Multiplex genome engineering in vivo with CRISPR/Cas9 shows great promise as a potential therapeutic approach. The ability to incorporate multiple single guide RNA (sgRNA) cassettes together with Cas9 gene expression in one AAV vector could greatly enhance the efficiency. In a recent Method article, Mefferd et al indicated that small tRNA promoters could be used to drive sgRNA expression to facilitate the construction of a more effective AAV vector. In contrast, we found that when targeting endogenous genomic loci, CRISPR/Cas9 with tRNA promoter-driven sgRNA expression showed much reduced genome editing activity, compared with significant cleavage with U6 promoter-driven sgRNA expression...
October 14, 2016: RNA
https://www.readbyqxmd.com/read/27595405/a-multifunctional-aav-crispr-cas9-and-its-host-response
#7
Wei Leong Chew, Mohammadsharif Tabebordbar, Jason K W Cheng, Prashant Mali, Elizabeth Y Wu, Alex H M Ng, Kexian Zhu, Amy J Wagers, George M Church
CRISPR-Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV-CRISPR-Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes...
October 2016: Nature Methods
https://www.readbyqxmd.com/read/27587996/the-development-of-a-viral-mediated-crispr-cas9-system-with-doxycycline-dependent-grna-expression-for-inducible-in-vitro-and-in-vivo-genome-editing
#8
Christopher A de Solis, Anthony Ho, Roopashri Holehonnur, Jonathan E Ploski
The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter...
2016: Frontiers in Molecular Neuroscience
https://www.readbyqxmd.com/read/27434683/cns-restricted-transduction-and-crispr-cas9-mediated-gene-deletion-with-an-engineered-aav-vector
#9
Giridhar Murlidharan, Kensuke Sakamoto, Lavanya Rao, Travis Corriher, Dan Wang, Guangping Gao, Patrick Sullivan, Aravind Asokan
Gene therapy using recombinant adeno-associated viral (AAV) vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9), which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9...
2016: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/27367513/aav-mediated-crispr-cas-gene-editing-of-retinal-cells-in-vivo
#10
Sandy S C Hung, Vicki Chrysostomou, Fan Li, Jeremiah K H Lim, Jiang-Hui Wang, Joseph E Powell, Leilei Tu, Maciej Daniszewski, Camden Lo, Raymond C Wong, Jonathan G Crowston, Alice Pébay, Anna E King, Bang V Bui, Guei-Sheung Liu, Alex W Hewitt
PURPOSE: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo...
June 1, 2016: Investigative Ophthalmology & Visual Science
https://www.readbyqxmd.com/read/27280971/aav-vectorization-of-dsb-mediated-gene-editing-technologies
#11
Rachel J Moser, Matthew L Hirsch
Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome...
2016: Current Gene Therapy
https://www.readbyqxmd.com/read/27246850/the-clustered-regularly-interspaced-short-palindromic-repeats-associated-endonuclease-9-crispr-cas9-created-mdm2-t309g-mutation-enhances-vitreous-induced-expression-of-mdm2-and-proliferation-and-survival-of-cells
#12
Yajian Duan, Gaoen Ma, Xionggao Huang, Patricia A D'Amore, Feng Zhang, Hetian Lei
The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T...
July 29, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27203437/high-efficiency-crispr-cas9-mediated-gene-editing-in-primary-human-t-cells-using-mutant-adenoviral-e4orf6-e1b55k-helper-proteins
#13
Kamila S Gwiazda, Alexandra E Grier, Jaya Sahni, Stephen M Burleigh, Unja Martin, Julia G Yang, Nicholas A Popp, Michelle C Krutein, Iram F Khan, Kyle Jacoby, Michael C Jensen, David J Rawlings, Andrew M Scharenberg
Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates...
September 29, 2016: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/26829317/a-dual-aav-system-enables-the-cas9-mediated-correction-of-a-metabolic-liver-disease-in-newborn-mice
#14
Yang Yang, Lili Wang, Peter Bell, Deirdre McMenamin, Zhenning He, John White, Hongwei Yu, Chenyu Xu, Hiroki Morizono, Kiran Musunuru, Mark L Batshaw, James M Wilson
Many genetic liver diseases in newborns cause repeated, often lethal, metabolic crises. Gene therapy using nonintegrating viruses such as adeno-associated virus (AAV) is not optimal in this setting because the nonintegrating genome is lost as developing hepatocytes proliferate. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR-Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC)...
March 2016: Nature Biotechnology
https://www.readbyqxmd.com/read/26721686/in-vivo-gene-editing-in-dystrophic-mouse-muscle-and-muscle-stem-cells
#15
Mohammadsharif Tabebordbar, Kexian Zhu, Jason K W Cheng, Wei Leong Chew, Jeffrey J Widrick, Winston X Yan, Claire Maesner, Elizabeth Y Wu, Ru Xiao, F Ann Ran, Le Cong, Feng Zhang, Luk H Vandenberghe, George M Church, Amy J Wagers
Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery...
January 22, 2016: Science
https://www.readbyqxmd.com/read/26657635/improved-methods-of-aav-mediated-gene-targeting-for-human-cell-lines-using-ribosome-skipping-2a-peptide
#16
Sivasundaram Karnan, Akinobu Ota, Yuko Konishi, Md Wahiduzzaman, Yoshitaka Hosokawa, Hiroyuki Konishi
The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1-4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs...
April 7, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/26596280/characterization-of-staphylococcus-aureus-cas9-a-smaller-cas9-for-all-in-one-adeno-associated-virus-delivery-and-paired-nickase-applications
#17
Ari E Friedland, Reshica Baral, Pankhuri Singhal, Katherine Loveluck, Shen Shen, Minerva Sanchez, Eugenio Marco, Gregory M Gotta, Morgan L Maeder, Edward M Kennedy, Anand V R Kornepati, Alexander Sousa, McKensie A Collins, Hari Jayaram, Bryan R Cullen, David Bumcrot
BACKGROUND: CRISPR-Cas systems have been broadly embraced as effective tools for genome engineering applications, with most studies to date utilizing the Streptococcus pyogenes Cas9. Here we characterize and manipulate the smaller, 1053 amino acid nuclease Staphylococcus aureus Cas9. RESULTS: We find that the S. aureus Cas9 recognizes an NNGRRT protospacer adjacent motif (PAM) and cleaves target DNA at high efficiency with a variety of guide RNA (gRNA) spacer lengths...
2015: Genome Biology
https://www.readbyqxmd.com/read/26291065/optimization-of-a-multiplex-crispr-cas-system-for-use-as-an-antiviral-therapeutic
#18
Edward M Kennedy, Anand V R Kornepati, Adam L Mefferd, Joy B Marshall, Kevin Tsai, Hal P Bogerd, Bryan R Cullen
RNA-guided endonucleases or CRISPR/Cas systems have been widely employed for gene engineering/DNA editing applications, and have recently been used against a variety of dsDNA viruses as a potential therapeutic. However, in vivo delivery to specific tissue reservoirs using adeno-associated virus (AAV) vectors is problematic due to the large coding requirement for the principal effector commonly used in these applications, Streptococcus pyogenes (Spy) Cas9. Here we describe design of a minimal CRISPR/Cas system that is capable of multiplexing and can be packaged into a single AAV vector...
December 2015: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/26239654/gene-therapy-for-cardiovascular-disease-advances-in-vector-development-targeting-and-delivery-for-clinical-translation
#19
REVIEW
Melvin Y Rincon, Thierry VandenDriessche, Marinee K Chuah
Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements...
October 1, 2015: Cardiovascular Research
https://www.readbyqxmd.com/read/26187160/expression-of-crispr-cas-single-guide-rnas-using-small-trna-promoters
#20
Adam L Mefferd, Anand V R Kornepati, Hal P Bogerd, Edward M Kennedy, Bryan R Cullen
The in vivo application of CRISPR/Cas-based DNA editing technology will require the development of efficient delivery methods that likely will be dependent on adeno-associated virus (AAV)-based viral vectors. However, AAV vectors have only a modest, ∼4.7-kb packaging capacity, which will necessitate the identification and characterization of highly active Cas9 proteins that are substantially smaller than the prototypic Streptococcus pyogenes Cas9 protein, which covers ∼4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current ∼360 bp size...
September 2015: RNA
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