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CAS9 and AAV

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https://www.readbyqxmd.com/read/28967995/casaav-a-crispr-based-platform-for-rapid-dissection-of-gene-function-in-vivo
#1
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
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
#10
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
#11
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
#12
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
#13
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
https://www.readbyqxmd.com/read/28334779/targeted-gene-knock-in-by-homology-directed-genome-editing-using-cas9-ribonucleoprotein-and-aav-donor-delivery
#14
Thomas Gaj, Brett T Staahl, Gonçalo M C Rodrigues, Prajit Limsirichai, Freja K Ekman, Jennifer A Doudna, David V Schaffer
Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide...
June 20, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28300165/somatic-genome-editing-with-crispr-cas9-generates-and-corrects-a-metabolic-disease
#15
Kelsey E Jarrett, Ciaran M Lee, Yi-Hsien Yeh, Rachel H Hsu, Rajat Gupta, Min Zhang, Perla J Rodriguez, Chang Seok Lee, Baiba K Gillard, Karl-Dimiter Bissig, Henry J Pownall, James F Martin, Gang Bao, William R Lagor
Germline manipulation using CRISPR/Cas9 genome editing has dramatically accelerated the generation of new mouse models. Nonetheless, many metabolic disease models still depend upon laborious germline targeting, and are further complicated by the need to avoid developmental phenotypes. We sought to address these experimental limitations by generating somatic mutations in the adult liver using CRISPR/Cas9, as a new strategy to model metabolic disorders. As proof-of-principle, we targeted the low-density lipoprotein receptor (Ldlr), which when deleted, leads to severe hypercholesterolemia and atherosclerosis...
March 16, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28291770/nrl-knockdown-by-aav-delivered-crispr-cas9-prevents-retinal-degeneration-in-mice
#16
Wenhan Yu, Suddhasil Mookherjee, Vijender Chaitankar, Suja Hiriyanna, Jung-Woong Kim, Matthew Brooks, Yasaman Ataeijannati, Xun Sun, Lijin Dong, Tiansen Li, Anand Swaroop, Zhijian Wu
In retinitis pigmentosa, loss of cone photoreceptors leads to blindness, and preservation of cone function is a major therapeutic goal. However, cone loss is thought to occur as a secondary event resulting from degeneration of rod photoreceptors. Here we report a genome editing approach in which adeno-associated virus (AAV)-mediated CRISPR/Cas9 delivery to postmitotic photoreceptors is used to target the Nrl gene, encoding for Neural retina-specific leucine zipper protein, a rod fate determinant during photoreceptor development...
March 14, 2017: Nature Communications
https://www.readbyqxmd.com/read/28220790/in-vivo-genome-editing-with-a-small-cas9-orthologue-derived-from-campylobacter-jejuni
#17
Eunji Kim, Taeyoung Koo, Sung Wook Park, Daesik Kim, Kyoungmi Kim, Hee-Yeon Cho, Dong Woo Song, Kyu Jun Lee, Min Hee Jung, Seokjoong Kim, Jin Hyoung Kim, Jeong Hun Kim, Jin-Soo Kim
Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome...
February 21, 2017: Nature Communications
https://www.readbyqxmd.com/read/28195574/muscle-specific-crispr-cas9-dystrophin-gene-editing-ameliorates-pathophysiology-in-a-mouse-model-for-duchenne-muscular-dystrophy
#18
Niclas E Bengtsson, John K Hall, Guy L Odom, Michael P Phelps, Colin R Andrus, R David Hawkins, Stephen D Hauschka, Joel R Chamberlain, Jeffrey S Chamberlain
Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx(4cv) mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation...
February 14, 2017: Nature Communications
https://www.readbyqxmd.com/read/28109959/crispr-cas9-mediated-genome-editing-as-a-therapeutic-approach-for-leber-congenital-amaurosis-10
#19
Guo-Xiang Ruan, Elizabeth Barry, Dan Yu, Michael Lukason, Seng H Cheng, Abraham Scaria
As the most common subtype of Leber congenital amaurosis (LCA), LCA10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. The most frequent mutation found in patients with LCA10 is a deep intronic mutation in CEP290 that generates a cryptic splice donor site. The large size of the CEP290 gene prevents its use in adeno-associated virus (AAV)-mediated gene augmentation therapy. Here, we show that targeted genomic deletion using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system represents a promising therapeutic approach for the treatment of patients with LCA10 bearing the CEP290 splice mutation...
February 1, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/27995572/intramuscular-delivery-of-scaav9-higf1-prolongs-survival-in-the-hsod1-g93a-als-mouse-model-via-upregulation-of-d-amino-acid-oxidase
#20
HuiQian Lin, HaoJie Hu, WeiSong Duan, YaLing Liu, GuoJun Tan, ZhongYao Li, YaKun Liu, BinBin Deng, XueQin Song, Wan Wang, Di Wen, Ying Wang, ChunYan Li
Self-complementary adeno-associated viral vector 9 (scAAV9) has been confirmed to be an efficient AAV serotype for gene transfer to the central nervous system (CNS). Neurotrophic factors have been considered to be therapeutic targets for amyotrophic lateral sclerosis (ALS). In the present study, we intramuscularly injected scAAV9 encoding human insulin-like growth factor 1 (hIGF1) into an hSOD1(G93A) ALS mouse model. We observed that scAAV9-hIGF1 significantly reduced the loss of motor neurons of the anterior horn in the lumbar spinal cord and delayed muscle atrophy in ALS mice...
December 19, 2016: Molecular Neurobiology
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