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https://www.readbyqxmd.com/read/29333573/simultaneous-site-directed-mutagenesis-of-duplicated-loci-in-soybean-using-a-single-guide-rna
#1
Yuhei Kanazashi, Aya Hirose, Ippei Takahashi, Masafumi Mikami, Masaki Endo, Sakiko Hirose, Seiichi Toki, Akito Kaga, Ken Naito, Masao Ishimoto, Jun Abe, Tetsuya Yamada
Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD)...
January 15, 2018: Plant Cell Reports
https://www.readbyqxmd.com/read/29331077/optimized-paired-sgrna-cas9-cloning-and-expression-cassette-triggers-high-efficiency-multiplex-genome-editing-in-kiwifruit
#2
Zupeng Wang, Shuaibin Wang, Dawei Li, Qiong Zhang, Li Li, Caihong Zhong, Yifei Liu, Hongwen Huang
Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing...
January 13, 2018: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/29330168/mrb7260-is-essential-for-productive-protein-rna-interactions-within-the-rna-editing-substrate-binding-complex-during-trypanosome-rna-editing
#3
Natalie M McAdams, Rachel M Simpson, Runpu Chen, Yijun Sun, Laurie Read
The trypanosome RNA Editing Substrate Binding Complex (RESC) acts as the platform for mitochondrial uridine insertion/deletion RNA editing and facilitates the protein-protein and protein-RNA interactions required for the editing process. RESC is broadly comprised of two subcomplexes: GRBC (Guide RNA Binding Complex) and REMC (RNA Editing Mediator Complex). Here, we characterize the function and position in RESC organization of a previously unstudied RESC protein, MRB7260. We show that MRB7260 forms numerous RESC-related complexes, including a novel, small complex with the guide RNA binding protein, GAP1, which is a canonical GRBC component, and REMC components MRB8170 and TbRGG2...
January 12, 2018: RNA
https://www.readbyqxmd.com/read/29321601/crispr-cas9-mediated-gene-modification-and-gene-knock-out-in-the-human-infective-parasite-trichomonas-vaginalis
#4
Brian D Janssen, Yi-Pei Chen, Brenda M Molgora, Shuqi E Wang, Augusto Simoes-Barbosa, Patricia J Johnson
The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T...
January 10, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29321333/microrna-130a-regulates-both-hcv-and-hbv-replication-through-a-central-metabolic-pathway
#5
Xiaoqiong Duan, Shilin Li, Jacinta A Holmes, Zeng Tu, Yujia Li, Dachuan Cai, Xiao Liu, Wenting Li, Chunhui Yang, Baihai Jiao, Esperance A Schaefer, Dahlene N Fusco, Shadi Salloum, Limin Chen, Wenyu Lin, Raymond T Chung
BACKGROUND: HCV infection has been shown to regulate miR-130a in patient biopsies and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and HBV replication.METHODS: We used bioinformatics software including miRanda, TargetScan, PITA and RNAhybrid to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed, or knocked down by siRNA or by CRISPR/Cas9 gRNA, respectively. Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, JFH1 HCV-infected Huh7...
January 10, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29321167/construction-of-designer-selectable-marker-deletions-with-crispr-cas9-toolbox-in-schizosaccharomyces-pombe-and-optimized-design-of-common-entry-vectors
#6
Yu Zhao, Jef D Boeke
Vectors encoding selectable markers have been widely used in yeast to maintain or express exogenous DNA fragments. In the fission yeast Schizosaccharomyces pombe, several engineered markers have been reported and widely used, such as ura4+ and ScLEU2 from Saccharomyces cerevisiae, which complement ura4 and leu1 mutations, respectively. These two auxotrophic markers share no homology with the Sch. pombe genome, however most others can recombine with the genomic due to sequence homology shared between the genomic and plasmid-born copies of the markers...
January 10, 2018: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/29316283/split-cas9-not-hairs-advancing-the-therapeutic-index-of-crispr-technology
#7
REVIEW
Carolin Schmelas, Dirk Grimm
The discovery that the bacterial CRISPR/Cas9 system can be translated into mammalian cells continues to have an unprecedented impact on the biomedical research community, as it largely facilitates efforts to experimentally interrogate or therapeutically modify the cellular genome. In particular, CRISPR promises the ability to correct disease-associated genetic defects, or to target and destroy invading foreign DNA, in a simple, efficient and selective manner directly in affected human cells or tissues. Here, we highlight a set of exciting new strategies that aim at further increasing the therapeutic index of CRISPR technologies, by reducing the size of Cas9 expression cassettes and thus enhancing their compatibility with viral gene delivery vectors...
January 5, 2018: Biotechnology Journal
https://www.readbyqxmd.com/read/29315387/a-validated-grna-library-for-crispr-cas9-targeting-of-the-human-glycosyltransferase-genome
#8
Yoshiki Narimatsu, Hiren J Joshi, Yang Zhang, Catarina Gomes, Yen-Hsi Chen, Flaminia Lorenzetti, Sanae Furukawa, Katrine Schjoldager, Lars Hansen, Henrik Clausen, Eric P Bennett, Hans H Wandall
Over 200 glycosyltransferases are involved in the orchestration of the biosynthesis of the human glycome , which is comprised of all glycan structures found on different glycoconjugates in cells. The glycome is vast, and despite advancements in analytic strategies it continues to be difficult to decipher biological roles of glycans with respect to specific glycan structures, type of glycoconjugate, particular glycoproteins, and distinct glycosites on proteins. In contrast to this, the number of glycosyltransferase genes involved in the biosynthesis of the human glycome is manageable, and the biosynthetic roles of most of these enzymes are defined or can be predicted with reasonable confidence...
January 5, 2018: Glycobiology
https://www.readbyqxmd.com/read/29286403/a-universal-protocol-for-large-scale-grna-library-production-from-any-dna-source
#9
Anna Köferle, Stefan H Stricker
The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment...
December 6, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29250878/precise-editing-of-clavata-genes-in-brassica-napus-l-regulates-multilocular-silique-development
#10
Yang Yang, Kaiyu Zhu, Huailin Li, Shaoqing Han, Qingwei Meng, Shahid Ullah Khan, Chuchuan Fan, Kabin Xie, Yongming Zhou
Multilocular silique is a desirable agricultural trait with great potential for the development of high-yield varieties of Brassica. To date, no spontaneous or induced multilocular mutants have been reported in Brassica napus, which likely reflects its allotetraploid nature and the extremely low probability of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we present evidence for the efficient knockout of rapeseed homologues of CLAVATA3 (CLV3) for a secreted peptide and its related receptors CLV1 and CLV2 in the CLV signalling pathway using the CRISPR/Cas9 system and achieved stable transmission of the mutations across three generations...
December 18, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/29229813/human-genetic-variation-alters-crispr-cas9-on-and-off-targeting-specificity-at-therapeutically-implicated-loci
#11
Samuel Lessard, Laurent Francioli, Jessica Alfoldi, Jean-Claude Tardif, Patrick T Ellinor, Daniel G MacArthur, Guillaume Lettre, Stuart H Orkin, Matthew C Canver
The CRISPR-Cas9 nuclease system holds enormous potential for therapeutic genome editing of a wide spectrum of diseases. Large efforts have been made to further understanding of on- and off-target activity to assist the design of CRISPR-based therapies with optimized efficacy and safety. However, current efforts have largely focused on the reference genome or the genome of cell lines to evaluate guide RNA (gRNA) efficiency, safety, and toxicity. Here, we examine the effect of human genetic variation on both on- and off-target specificity...
December 11, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29221488/divergent-susceptibilities-to-aav-sacas9-grna-vector-mediated-genome-editing-in-a-single-cell-derived-cell-population
#12
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/29215041/crispr-cas9-delivery-with-one-single-adenoviral-vector-devoid-of-all-viral-genes
#13
Eric Ehrke-Schulz, Maren Schiwon, Theo Leitner, Stephan Dávid, Thorsten Bergmann, Jing Liu, Anja Ehrhardt
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing...
December 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29212218/optimized-guide-rna-structure-for-genome-editing-via-cas9
#14
Jianyong Xu, Wei Lian, Yuning Jia, Lingyun Li, Zhong Huang
The genome editing tool Cas9-gRNA (guide RNA) has been successfully applied in different cell types and organisms with high efficiency. However, more efforts need to be made to enhance both efficiency and specificity. In the current study, we optimized the guide RNA structure of Streptococcus pyogenes CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system to improve its genome editing efficiency. Comparing with the original functional structure of guide RNA, which is composed of crRNA and tracrRNA, the widely used chimeric gRNA has shorter crRNA and tracrRNA sequence...
November 7, 2017: Oncotarget
https://www.readbyqxmd.com/read/29211736/versatile-single-step-assembly-crispr-cas9-vectors-for-dual-grna-expression
#15
Fatwa Adikusuma, Chandran Pfitzner, Paul Quinton Thomas
CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable multiplex expression of gRNAs have been developed, these require multiple rounds of cloning and/or PCR for generation of the desired construct. Here, we describe a series of vectors that enable generation of customized dual-gRNA expression constructs via an easy one-step golden gate cloning reaction using two annealed oligonucleotide inserts with different overhangs...
2017: PloS One
https://www.readbyqxmd.com/read/29207996/crispr-cas9-mediated-gene-deletions-in-lager-yeast-saccharomyces-pastorianus
#16
Arthur R Gorter de Vries, Philip A de Groot, Marcel van den Broek, Jean-Marc G Daran
BACKGROUND: The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S...
December 5, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/29202040/a-simple-and-universal-system-for-gene-manipulation-in-aspergillus-fumigatus-in-vitro-assembled-cas9-guide-rna-ribonucleoproteins-coupled-with-microhomology-repair-templates
#17
Qusai Al Abdallah, Wenbo Ge, Jarrod R Fortwendel
CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a novel genome-editing system that has been successfully established in Aspergillus fumigatus. However, the current state of the technology relies heavily on DNA-based expression cassettes for delivering Cas9 and the guide RNA (gRNA) to the cell. Therefore, the power of the technology is limited to strains that are engineered to express Cas9 and gRNA. To overcome such limitations, we developed a simple and universal CRISPR-Cas9 system for gene deletion that works across different genetic backgrounds of A...
November 2017: MSphere
https://www.readbyqxmd.com/read/29196743/repair-of-the-tgfbi-gene-in-human-corneal-keratocytes-derived-from-a-granular-corneal-dystrophy-patient-via-crispr-cas9-induced-homology-directed-repair
#18
Yukako Taketani, Kohdai Kitamoto, Toshihiro Sakisaka, Mikiko Kimakura, Tetsuya Toyono, Satoru Yamagami, Shiro Amano, Masahiko Kuroda, Tara Moore, Tomohiko Usui, Yasuo Ouchi
Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused by a point mutation in the transforming growth factor-β-induced (TGFBI) gene, located on chromosome 5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing for the correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homology-directed repair (HDR)...
December 1, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29192199/crispr-cas9-assisted-grna-free-one-step-genome-editing-with-no-sequence-limitations-and-improved-targeting-efficiency
#19
Dongdong Zhao, Xu Feng, Xinna Zhu, Tao Wu, Xueli Zhang, Changhao Bi
The CRISPR/Cas9 system is a powerful, revolutionary tool for genome editing. However, it is not without limitations. There are PAM-free and CRISPR-tolerant regions that cannot be modified by the standard CRISPR/Cas9 system, and off-target activity impedes its broader applications. To avoid these drawbacks, we developed a very simple CRISPR/Cas9-assisted gRNA-free one-step (CAGO) genome editing technique which does not require the construction of a plasmid to express a specific gRNA. Instead, a universal N20 sequence with a very high targeting efficiency is inserted into the E...
November 30, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29188477/crispr-cas9-mediated-efficient-editing-in-phytoene-desaturase-pds-demonstrates-precise-manipulation-in-banana-cv-rasthali-genome
#20
Navneet Kaur, Anshu Alok, Shivani, Navjot Kaur, Pankaj Pandey, Praveen Awasthi, Siddharth Tiwari
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been reported for precise genome modification in many plants. In the current study, we demonstrate a successful mutation in phytoene desaturase (RAS-PDS) of banana cv. Rasthali using the CRISPR/Cas9 system. Two PDS genes were isolated from Rasthali (RAS-PDS1 and RAS-PDS2), and their protein sequence analysis confirmed that both PDS comprises conserved motifs for enzyme activity. Phylogenetic analysis of RAS-PDS1 and RAS-PDS2 revealed a close evolutionary relationship with other monocot species...
November 29, 2017: Functional & Integrative Genomics
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