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CAS9 and AAV

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https://www.readbyqxmd.com/read/29597895/in-vivo-genome-editing-partially-restores-alpha1-antitrypsin-in-a-murine-model-of-aat-deficiency
#1
Chun-Qing Song, Dan Wang, Tingting Jiang, Kevin O'Connor, Qiushi Tang, Lingling Cai, Xiangrui Li, Zhiping Weng, Hao Yin, Guangping Gao, Christian Mueller, Terence R Flotte, Wen Xue
CRISPR genome editing holds promise in the treatment of genetic diseases that currently lack effective long-term therapies. Patients with Alpha-1 Antitrypsin (AAT) deficiency develop progressive lung disease due to the loss of AAT's antiprotease function and liver disease due to a toxic gain of function of the common mutant allele. However, it remains unknown whether CRISPR-mediated AAT correction in the liver, where AAT is primarily expressed, can correct either or both defects. Here we show that AAV delivery of CRISPR can effectively correct Z-AAT mutation in the liver of a transgenic mouse model...
March 29, 2018: Human Gene Therapy
https://www.readbyqxmd.com/read/29562138/rational-design-of-mini-cas9-for-transcriptional-activation
#2
Dacheng Ma, Shuguang Peng, Weiren Huang, Zhiming Cai, Zhen Xie
Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with variant effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage...
March 21, 2018: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29503867/mapping-a-functional-cancer-genome-atlas-of-tumor-suppressors-in-mouse-liver-using-aav-crispr-mediated-direct-in-vivo-screening
#3
Guangchuan Wang, Ryan D Chow, Lupeng Ye, Christopher D Guzman, Xiaoyun Dai, Matthew B Dong, Feng Zhang, Phillip A Sharp, Randall J Platt, Sidi Chen
Cancer genomics consortia have charted the landscapes of numerous human cancers. Whereas some mutations were found in classical oncogenes and tumor suppressors, others have not yet been functionally studied in vivo. To date, a comprehensive assessment of how these genes influence oncogenesis is lacking. We performed direct high-throughput in vivo mapping of functional variants in an autochthonous mouse model of cancer. Using adeno-associated viruses (AAVs) carrying a single-guide RNA (sgRNA) library targeting putative tumor suppressor genes significantly mutated in human cancers, we directly pool-mutagenized the livers of Cre-inducible CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) mice...
February 2018: Science Advances
https://www.readbyqxmd.com/read/29483550/oligonucleotide-conjugated-multi-functional-adeno-associated-viruses
#4
Dhruva Katrekar, Ana M Moreno, Genghao Chen, Atharv Worlikar, Prashant Mali
Recombinant adeno-associated viruses (AAVs) are among the most commonly used vehicles for in vivo gene delivery. However, their tropism is limited, and additionally their efficacy can be negatively affected by prevalence of neutralizing antibodies in sera. Methodologies to systematically engineer AAV capsid properties would thus be of great relevance. In this regard, we develop here multi-functional AAVs by engineering precision tethering of oligonucleotides onto the AAV surface, and thereby enabling a spectrum of nucleic-acid programmable functionalities...
February 26, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29458131/inhibition-of-hepatitis-b-virus-replication-via-hbv-dna-cleavage-by-cas9-from-staphylococcus-aureus
#5
Yu Liu, Miaoxian Zhao, Mingxing Gong, Ying Xu, Cantao Xie, Haohui Deng, Xueying Li, Hongkai Wu, Zhanhui Wang
Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector...
February 16, 2018: Antiviral Research
https://www.readbyqxmd.com/read/29446741/what%C3%A2-s-new-in-gene-therapy-of-hemophilia
#6
E Carlos Rodriguez-Merchan
BACKGROUND: Several methods have been investigated to effectively and safely transmit genes that stimulate cells to release therapeutic factor VIII (FVIII) and factor IX (FIX) into the circulation of people with hemophilia (PWH). OBJECTIVE: To review the role of gene therapy (GT) in PWH. METHODS: A Cochrane Library and PubMed (MEDLINE) search related to the role of GT in hemophilia was analyzed. RESULTS: The most promising vectors for hemophilia GT are adeno-associated virus (AAV) and lentivirus...
February 14, 2018: Current Gene Therapy
https://www.readbyqxmd.com/read/29400692/hippo-mediated-suppression-of-irs2-akt-signaling-prevents-hepatic-steatosis-and-liver-cancer
#7
Sun-Hye Jeong, Han-Byul Kim, Min-Chul Kim, Ji-Min Lee, Jae Ho Lee, Jeong-Hwan Kim, Jin-Woo Kim, Woong-Yang Park, Seon-Young Kim, Jae Bum Kim, Haeryoung Kim, Jin-Man Kim, Hueng-Sik Choi, Dae-Sik Lim
Nonalcoholic fatty liver disease (NAFLD) is a major risk factor for liver cancer; therefore, its prevention is an important clinical goal. Ablation of phosphatase and tensin homolog (PTEN) or the protein kinase Hippo signaling pathway induces liver cancer via activation of AKT or the transcriptional regulators YAP/TAZ, respectively; however, the potential for crosstalk between the PTEN/AKT and Hippo/YAP/TAZ pathways in liver tumorigenesis has thus far remained unclear. Here, we have shown that deletion of both PTEN and SAV1 in the liver accelerates the development of NAFLD and liver cancer in mice...
March 1, 2018: Journal of Clinical Investigation
https://www.readbyqxmd.com/read/29281027/cas9-sgrna-selective-targeting-of-the-p23h-rhodopsin-mutant-allele-for-treating-retinitis-pigmentosa-by-intravitreal-aav9-php-b-based-delivery
#8
Serena G Giannelli, Mirko Luoni, Valerio Castoldi, Luca Massimino, Tommaso Cabassi, Debora Angeloni, Gian Carlo Demontis, Letizia Leocani, Massimiliano Andreazzoli, Vania Broccoli
P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro...
March 1, 2018: Human Molecular Genetics
https://www.readbyqxmd.com/read/29259521/nuclease-mediated-gene-therapies-for-inherited-metabolic-diseases-of-the-liver
#9
REVIEW
Taylor E Bryson, Caitlin M Anglin, P Hudson Bridges, Renee N Cottle
Inherited metabolic diseases (IMDs) of the liver represent a vast and diverse group of rare genetic diseases characterized by the loss or dysfunction of enzymes or proteins essential for metabolic pathways in the liver. Conventional gene therapy involving adeno-associated virus (AAV) serotype 8 vectors provide therapeutically high levels of hepatic transgene expression facilitating the correction of the disease phenotype in pre-clinical studies and are currently being evaluated in clinical trials for multiple IMDs...
December 2017: Yale Journal of Biology and Medicine
https://www.readbyqxmd.com/read/29233960/multiplexed-in-vivo-homology-directed-repair-and-tumor-barcoding-enables-parallel-quantification-of-kras-variant-oncogenicity
#10
Ian P Winters, Shin-Heng Chiou, Nicole K Paulk, Christopher D McFarland, Pranav V Lalgudi, Rosanna K Ma, Leszek Lisowski, Andrew J Connolly, Dmitri A Petrov, Mark A Kay, Monte M Winslow
Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice...
December 12, 2017: Nature Communications
https://www.readbyqxmd.com/read/29221488/divergent-susceptibilities-to-aav-sacas9-grna-vector-mediated-genome-editing-in-a-single-cell-derived-cell-population
#11
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
#12
Wenyi Wu, Yajian Duan, Gaoen Ma, Guohong Zhou, Cindy Park-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
#13
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
#14
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...
February 5, 2018: Gene
https://www.readbyqxmd.com/read/29130152/combining-engineered-nucleases-with-adeno-associated-viral-vectors-for-therapeutic-gene-editing
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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