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CAS9 and AAV

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https://www.readbyqxmd.com/read/29221488/divergent-susceptibilities-to-aav-sacas9-grna-vector-mediated-genome-editing-in-a-single-cell-derived-cell-population
#1
Salma G Morsy, Jason M Tonne, Yaxi Zhu, Brian Lu, Karol Budzik, James W Krempski, Sherine A Ali, Mohamed A El-Feky, Yasuhiro Ikeda
OBJECTIVE: Recombinant adeno-associated virus (AAV)-based vectors are characterized by their robust and safe transgene delivery. The CRISPR/Cas9 and guide RNA (gRNA) system present a promising genome-editing platform, and a recent development of a shorter Cas9 enzyme from Staphylococcus aureus (SaCas9) allows generation of high titer single AAV vectors which carry both saCas9- and gRNA-expression cassettes. Here, we used two AAV-SaCas9 vectors with distinct GFP-targeted gRNA sequences and determined the impact of AAV-SaCas9-gRNA vector treatment in a single cell clone carrying a GFP-expression cassette...
December 8, 2017: BMC Research Notes
https://www.readbyqxmd.com/read/29204648/aav-crispr-cas9-mediated-depletion-of-vegfr2-blocks-angiogenesis-in-vitro
#2
Wenyi Wu, Yajian Duan, Gaoen Ma, Guohong Zhou, Cindy Windhol, Patricia A D'Amore, Hetian Lei
Purpose: Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)-mediated CRISPR (clustered regularly interspaced short palindromic repeats)-associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events...
December 1, 2017: Investigative Ophthalmology & Visual Science
https://www.readbyqxmd.com/read/29188510/methods-for-in-vivo-crispr-cas-editing-of-the-adult-murine-retina
#3
Sandy S Hung, Fan Li, Jiang-Hui Wang, Anna E King, Bang V Bui, Guei-Sheung Liu, Alex W Hewitt
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) is used by some bacteria and most archaea to protect against viral phage intrusion and has recently been adapted to allow for efficient editing of the mammalian genome. Whilst CRISPR/Cas-based technology has been used to modify genes in mammalian cells in vitro, delivery of CRISPR/Cas system into mammalian tissue and/or organs is more difficult and often requires additional vectors. With the use of adeno-associated virus (AAV) gene delivery system, active CRISPR/Cas enzyme can be maintained for an extended period of time and enable efficient editing of genome in the retina in vivo...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29154869/evaluating-different-dna-binding-domains-to-modulate-l1-orf2p-driven-site-specific-retrotransposition-events-in-human-cells
#4
Catherine M Ade, Rebecca S Derbes, Bradley J Wagstaff, Sara B Linker, Travis B White, Dawn Deharo, Victoria P Belancio, Zoltán Ivics, Astrid M Roy-Engel
DNA binding domains (DBDs) have been used with great success to impart targeting capabilities to a variety of proteins creating highly useful genomic tools. We evaluated the ability of five types of DBDs and strategies (AAV Rep proteins, Cre, TAL effectors, zinc finger proteins, and Cas9/gRNA system) to target the L1 ORF2 protein to drive retrotransposition of Alu inserts to specific sequences in the human genome. First, we find that the L1 ORF2 protein tolerates the addition of protein domains both at the amino- and carboxy-terminus...
November 14, 2017: Gene
https://www.readbyqxmd.com/read/29130152/combining-engineered-nucleases-with-adeno-associated-viral-vectors-for-therapeutic-gene-editing
#5
Benjamin E Epstein, David V Schaffer
With the recent advent of several generations of targeted DNA nucleases, most recently CRISPR/Cas9, genome editing has become broadly accessible across the biomedical community. Importantly, the capacity of these nucleases to modify specific genomic loci associated with human disease could render new classes of genetic disease, including autosomal dominant or even idiopathic disease, accessible to gene therapy. In parallel, the emergence of adeno-associated virus (AAV) as a clinically important vector raises the possibility of integrating these two technologies towards the development of gene editing therapies...
2017: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/29119608/synthetic-aav-crispr-vectors-for-blocking-hiv-1-expression-in-persistently-infected-astrocytes
#6
Christine Kunze, Kathleen Börner, Eike Kienle, Tanja Orschmann, Ejona Rusha, Martha Schneider, Milena Radivojkov-Blagojevic, Micha Drukker, Sabrina Desbordes, Dirk Grimm, Ruth Brack-Werner
Astrocytes, the most abundant cells in the mammalian brain, perform key functions and are involved in several neurodegenerative diseases. The human immunodeficiency virus (HIV) can persist in astrocytes, contributing to the HIV burden and neurological dysfunctions in infected individuals. While a comprehensive approach to HIV cure must include the targeting of HIV-1 in astrocytes, dedicated tools for this purpose are still lacking. Here we report a novel Adeno-associated virus-based vector (AAV9P1) with a synthetic surface peptide for transduction of astrocytes...
November 9, 2017: Glia
https://www.readbyqxmd.com/read/29056297/virus-mediated-genome-editing-via-homology-directed-repair-in-mitotic-and-postmitotic-cells-in-mammalian-brain
#7
Jun Nishiyama, Takayasu Mikuni, Ryohei Yasuda
Precise genome editing via homology-directed repair (HDR) in targeted cells, particularly in vivo, provides an invaluable tool for biomedical research. However, HDR has been considered to be largely restricted to dividing cells, making it challenging to apply the technique in postmitotic neurons. Here we show that precise genome editing via HDR is possible in mature postmitotic neurons as well as mitotic cells in mice brain by combining CRISPR-Cas9-mediated DNA cleavage and the efficient delivery of donor template with adeno-associated virus (AAV)...
November 15, 2017: Neuron
https://www.readbyqxmd.com/read/28967995/casaav-a-crispr-based-platform-for-rapid-dissection-of-gene-function-in-vivo
#8
Nathan J VanDusen, Yuxuan Guo, Weiliang Gu, William T Pu
In vivo loss-of-function studies are currently limited by the need for appropriate conditional knockout alleles. CRISPR/Cas9 is a powerful tool commonly used to induce loss-of-function mutations in vitro. However, CRISPR components have been difficult to deploy in vivo. To address this problem, we developed the CASAAV (CRISPR/Cas9/AAV-based somatic mutagenesis) platform, in which recombinant adeno-associated virus (AAV) is used to deliver tandem guide RNAs and Cre recombinase to Cre-dependent Cas9-P2A-GFP mice...
October 2, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28839205/targeted-genome-replacement-via-homology-directed-repair-in-non-dividing-cardiomyocytes
#9
Takamaru Ishizu, Shuichiro Higo, Yuki Masumura, Yasuaki Kohama, Mikio Shiba, Tomoaki Higo, Masato Shibamoto, Akito Nakagawa, Sachio Morimoto, Seiji Takashima, Shungo Hikoso, Yasushi Sakata
Although high-throughput sequencing can elucidate the genetic basis of hereditary cardiomyopathy, direct interventions targeting pathological mutations have not been established. Furthermore, it remains uncertain whether homology-directed repair (HDR) is effective in non-dividing cardiomyocytes. Here, we demonstrate that HDR-mediated genome editing using CRISPR/Cas9 is effective in non-dividing cardiomyocytes. Transduction of adeno-associated virus (AAV) containing sgRNA and repair template into cardiomyocytes constitutively expressing Cas9 efficiently introduced a fluorescent protein to the C-terminus of Myl2...
August 24, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28805815/aav-mediated-direct-in-vivo-crispr-screen-identifies-functional-suppressors-in-glioblastoma
#10
Ryan D Chow, Christopher D Guzman, Guangchuan Wang, Florian Schmidt, Mark W Youngblood, Lupeng Ye, Youssef Errami, Matthew B Dong, Michael A Martinez, Sensen Zhang, Paul Renauer, Kaya Bilguvar, Murat Gunel, Phillip A Sharp, Feng Zhang, Randall J Platt, Sidi Chen
A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus-mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlated with those in two independent patient cohorts...
October 2017: Nature Neuroscience
https://www.readbyqxmd.com/read/28790199/in-vivo-genome-editing-restores-dystrophin-expression-and-cardiac-function-in-dystrophic-mice
#11
Mona El Refaey, Li Xu, Yandi Gao, Benjamin D Canan, T M Ayodele Adesanya, Sarah C Warner, Keiko Akagi, David E Symer, Peter J Mohler, Jianjie Ma, Paul M L Janssen, Renzhi Han
RATIONALE: Duchenne muscular dystrophy 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 Duchenne muscular dystrophy 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 Duchenne muscular dystrophy cardiac function has yet to be evaluated...
September 29, 2017: Circulation Research
https://www.readbyqxmd.com/read/28740073/genome-editing-abrogates-angiogenesis-in-vivo
#12
Xionggao Huang, Guohong Zhou, Wenyi Wu, Yajian Duan, Gaoen Ma, Jingyuan Song, Ru Xiao, Luk Vandenberghe, Feng Zhang, Patricia A D'Amore, Hetian Lei
Angiogenesis, in which vascular endothelial growth factor receptor (VEGFR) 2 plays an essential role, is associated with a variety of human diseases including proliferative diabetic retinopathy and wet age-related macular degeneration. Here we report that a system of adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) is used to deplete VEGFR2 in vascular endothelial cells (ECs), whereby the expression of SpCas9 is driven by an endothelial-specific promoter of intercellular adhesion molecule 2...
July 24, 2017: Nature Communications
https://www.readbyqxmd.com/read/28723575/crispr-mediated-integration-of-large-gene-cassettes-using-aav-donor-vectors
#13
Rasmus O Bak, Matthew H Porteus
The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno-associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of ∼4.5 kb limits the donor size. Here, we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for the integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing...
July 18, 2017: Cell Reports
https://www.readbyqxmd.com/read/28714989/correction-of-a-splicing-defect-in-a-mouse-model-of-congenital-muscular-dystrophy-type-1a-using-a-homology-directed-repair-independent-mechanism
#14
Dwi U Kemaladewi, Eleonora Maino, Elzbieta Hyatt, Huayun Hou, Maylynn Ding, Kara M Place, Xinyi Zhu, Prabhpreet Bassi, Zahra Baghestani, Amit G Deshwar, Daniele Merico, Hui Y Xiong, Brendan J Frey, Michael D Wilson, Evgueni A Ivakine, Ronald D Cohn
Splice-site defects account for about 10% of pathogenic mutations that cause Mendelian diseases. Prevalence is higher in neuromuscular disorders (NMDs), owing to the unusually large size and multi-exonic nature of genes encoding muscle structural proteins. Therapeutic genome editing to correct disease-causing splice-site mutations has been accomplished only through the homology-directed repair pathway, which is extremely inefficient in postmitotic tissues such as skeletal muscle. Here we describe a strategy using nonhomologous end-joining (NHEJ) to correct a pathogenic splice-site mutation...
August 2017: Nature Medicine
https://www.readbyqxmd.com/read/28646206/crispr-cas9-mediated-genome-editing-via-postnatal-administration-of-aav-vector-cures-haemophilia-b-mice
#15
Tsukasa Ohmori, Yasumitsu Nagao, Hiroaki Mizukami, Asuka Sakata, Shin-Ichi Muramatsu, Keiya Ozawa, Shin-Ichi Tominaga, Yutaka Hanazono, Satoshi Nishimura, Osamu Nureki, Yoichi Sakata
Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B...
June 23, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28596937/harnessing-the-potential-of-human-pluripotent-stem-cells-and-gene-editing-for-the-treatment-of-retinal-degeneration
#16
REVIEW
Patrick Ovando-Roche, Anastasios Georgiadis, Alexander J Smith, Rachael A Pearson, Robin R Ali
PURPOSE OF REVIEW: A major cause of visual disorders is dysfunction and/or loss of the light-sensitive cells of the retina, the photoreceptors. To develop better treatments for patients, we need to understand how inherited retinal disease mutations result in the dysfunction of photoreceptors. New advances in the field of stem cell and gene editing research offer novel ways to model retinal dystrophies in vitro and present opportunities to translate basic biological insights into therapies...
2017: Current Stem Cell Reports
https://www.readbyqxmd.com/read/28503441/applications-of-crispr-cas9-in-retinal-degenerative-diseases
#17
REVIEW
Ying-Qian Peng, Luo-Sheng Tang, Shigeo Yoshida, Ye-Di Zhou
Gene therapy is a potentially effective treatment for retinal degenerative diseases. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been developed as a new genome-editing tool in ophthalmic studies. Recent advances in researches showed that CRISPR/Cas9 has been applied in generating animal models as well as gene therapy in vivo of retinitis pigmentosa (RP) and leber congenital amaurosis (LCA). It has also been shown as a potential attempt for clinic by combining with other technologies such as adeno-associated virus (AAV) and induced pluripotent stem cells (iPSCs)...
2017: International Journal of Ophthalmology
https://www.readbyqxmd.com/read/28390761/the-aav-mediated-and-rna-guided-crispr-cas9-system-for-gene-therapy-of-dmd-and-bmd
#18
REVIEW
Jing-Zhang Wang, Peng Wu, Zhi-Min Shi, Yan-Li Xu, Zhi-Jun Liu
Mutations in the dystrophin gene (Dmd) result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), which afflict many newborn boys. In 2016, Brain and Development published several interesting articles on DMD treatment with antisense oligonucleotide, kinase inhibitor, and prednisolone. Even more strikingly, three articles in the issue 6271 of Science in 2016 provide new insights into gene therapy of DMD and BMD via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)...
April 5, 2017: Brain & Development
https://www.readbyqxmd.com/read/28366764/in%C3%A2-vivo-excision-of-hiv-1-provirus-by-sacas9-and-multiplex-single-guide-rnas-in-animal-models
#19
Chaoran Yin, Ting Zhang, Xiying Qu, Yonggang Zhang, Raj Putatunda, Xiao Xiao, Fang Li, Weidong Xiao, Huaqing Zhao, Shen Dai, Xuebin Qin, Xianming Mo, Won-Bin Young, Kamel Khalili, Wenhui Hu
CRISPR-associated protein 9 (Cas9)-mediated genome editing provides a promising cure for HIV-1/AIDS; however, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an all-in-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9). The quadruplex sgRNAs/saCas9 vector outperformed the duplex vector in excising the integrated HIV-1 genome in cultured neural stem/progenitor cells from HIV-1 Tg26 transgenic mice...
May 3, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/28356340/analysis-of-cardiac-myocyte-maturation-using-casaav-a-platform-for-rapid-dissection-of-cardiac-myocyte-gene-function-in-vivo
#20
REVIEW
Yuxuan Guo, Nathan J VanDusen, Lina Zhang, Weiliang Gu, Isha Sethi, Silvia Guatimosim, Qing Ma, Blake D Jardin, Yulan Ai, Donghui Zhang, Biyi Chen, Ang Guo, Guo-Cheng Yuan, Long-Sheng Song, William T Pu
RATIONALE: Loss-of-function studies in cardiac myocytes (CMs) are currently limited by the need for appropriate conditional knockout alleles. The factors that regulate CM maturation are poorly understood. Previous studies on CM maturation have been confounded by heart dysfunction caused by whole organ gene inactivation. OBJECTIVE: To develop a new technical platform to rapidly characterize cell-autonomous gene function in postnatal murine CMs and apply it to identify genes that regulate transverse tubules (T-tubules), a hallmark of mature CMs...
June 9, 2017: Circulation Research
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