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https://www.readbyqxmd.com/read/29150930/photoautotrophic-production-of-macular-pigment-in-a-chlamydomonas-reinhardtii-strain-generated-by-using-dna-free-crispr-cas9-rnp-mediated-mutagenesis
#1
Kwangryul Baek, Jihyeon Yu, Jooyeon Jeong, Sang Jun Sim, Sangsu Bae, EonSeon Jin
Lutein and zeaxanthin are dietary carotenoids reported to be protective against age-related macular degeneration. Recently, the green alga Chlamydomonas reinhardtii has received attention as a photosynthetic cell factory, but the potential of this alga for carotenoid production has not yet been evaluated. In this study, we selected the C. reinhardtii CC-4349 strain as the best candidate among seven laboratory strains tested for carotenoid production. A knock-out mutant of the zeaxanthin epoxidase gene induced by preassembled DNA-free CRISPR-Cas9 ribonucleoproteins in the CC-4349 strain had a significantly higher zeaxanthin content (56-fold) and productivity (47-fold) than the wild type without the reduction in lutein level...
November 18, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/29150593/rapid-and-efficient-crispr-cas9-based-mating-type-switching-of-saccharomyces-cerevisiae
#2
Ze-Xiong Xie, Leslie A Mitchell, Hui-Min Liu, Bing-Zhi Li, Duo Liu, Neta Agmon, Yi Wu, Xia Li, Xiao Zhou, Bo Li, Wen-Hai Xiao, Ming-Zhu Ding, Ying Wang, Ying-Jin Yuan, Jef D Boeke
Rapid and highly efficient mating-type switching of Saccharomyces cerevisiae enables a wide variety of genetic manipulations such as the construction of strains, for instance isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break (DSB) at the MAT locus, and in a single co-transformation, both haploid and diploid cells were switched to the specified mating-type at ~80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking HMLα and HMRa cryptic mating loci...
November 17, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/29150011/optimization-of-crispr-cas9-genome-editing-for-loss-of-function-in-the-early-chick-embryo
#3
Shashank Gandhi, Michael L Piacentino, Felipe M Vieceli, Marianne E Bronner
The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions...
December 1, 2017: Developmental Biology
https://www.readbyqxmd.com/read/29150007/crispr-in-animals-and-animal-models
#4
Ellen Shrock, Marc Güell
CRISPR-Cas9 has revolutionized the generation of transgenic animals. This system has demonstrated an unprecedented efficiency, multiplexability, and ease of use, thereby reducing the time and cost required for genome editing and enabling the production of animals with more extensive genetic modifications. It has also been shown to be applicable to a wide variety of animals, from early-branching metazoans to primates. Genome-wide screens in model organisms have been performed, accurate models of human diseases have been constructed, and potential therapies have been tested and validated in animal models...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/29150005/crispr-libraries-and-screening
#5
John T Poirier
CRISPR-Cas9 technology has revolutionized large-scale functional genomic screening in mammalian cell-culture systems. Due in part to optimized lentiviral delivery vectors; it is now possible to perform CRISPR-Cas9 screens in animals in order to study biological processes in the context of a whole organism and within more physiologically relevant environment. This chapter focuses primarily on mouse models of human cancers; viral vectors used for simultaneous tumor initiation and genome editing and sgRNA library design considerations...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/29150004/dynamics-of-indel-profiles-induced-by-various-crispr-cas9-delivery-methods
#6
Michael Kosicki, Sandeep S Rajan, Flaminia C Lorenzetti, Hans H Wandall, Yoshiki Narimatsu, Emmanouil Metzakopian, Eric P Bennett
The introduction of CRISPR/Cas9 gene editing in mammalian cells is a scientific breakthrough, which has greatly affected basic research and gene therapy. The simplicity and general access to CRISPR/Cas9 reagents has in an unprecedented manner "democratized" gene targeting in biomedical research, enabling genetic engineering of any gene in any cell, tissue, organ, and organism. The ability for fast, precise, and efficient profiling of the double-stranded break induced insertions and deletions (indels), mediated by any of the available programmable nucleases, is paramount to any given gene targeting approach...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/29150003/crispr-cas9-technology-applications-and-human-disease-modeling
#7
Marta Martinez-Lage, Raúl Torres-Ruiz, Sandra Rodriguez-Perales
The CRISPR/Cas9 system development has revolutionized the field of genome engineering through the efficient creation of targeted breaks in the DNA of almost any organism and cell type, opening an avenue for a wide range of applications in biomedical research and medicine. Apart from gene edition through knock-in or knock-out approaches, CRISPR/Cas9 technology has been used for many other purposes, including regulation of endogenous gene expression, epigenome editing, live-cell imaging of chromosomal loci, edition of RNA and high-throughput screening...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/29147778/genome-wide-investigation-of-transcription-factors-provides-insights-into-transcriptional-regulation-in-plutella-xylostella
#8
Qian Zhao, Dongna Ma, Yuping Huang, Weiyi He, Yiying Li, Liette Vasseur, Minsheng You
Transcription factors (TFs), which play a vital role in regulating gene expression, are prevalent in all organisms and characterization of them may provide important clues for understanding regulation in vivo. The present study reports a genome-wide investigation of TFs in the diamondback moth, Plutella xylostella (L.), a worldwide pest of crucifers. A total of 940 TFs distributed among 133 families were identified. Phylogenetic analysis of insect species showed that some of these families were found to have expanded during the evolution of P...
November 16, 2017: Molecular Genetics and Genomics: MGG
https://www.readbyqxmd.com/read/29145843/development-of-a-crispr-cas9-genome-editing-toolbox-for-corynebacterium-glutamicum
#9
Jiao Liu, Yu Wang, Yujiao Lu, Ping Zheng, Jibin Sun, Yanhe Ma
BACKGROUND: Corynebacterium glutamicum is an important industrial workhorse and advanced genetic engineering tools are urgently demanded. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) have revolutionized the field of genome engineering. The CRISPR/Cas9 system that utilizes NGG as protospacer adjacent motif (PAM) and has good targeting specificity can be developed into a powerful tool for efficient and precise genome editing of C...
November 16, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/29145633/the-initiation-propagation-and-dynamics-of-crispr-spycas9-r-loop-complex
#10
Yan Zeng, Yang Cui, Yong Zhang, Yanruo Zhang, Meng Liang, Hui Chen, Jie Lan, Guangtao Song, Jizhong Lou
CRISPR-Cas9 system has been widely used for efficient genome editing. Although the structures of Cas9 protein in complex with single-guided RNA (sgRNA) and target DNA have been resolved, the molecular details about the formation of Cas9 endonuclease R-loop structure remain elusive. Here we examine the DNA cleavage activities of Streptococcus pyogenes Cas9 (SpyCas9) and its mutants using various target sequences and study the conformational dynamics of R-loop structure during target binding using single-molecule fluorescence energy transfer (smFRET) technique...
November 14, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29145596/a-crispr-cas9-based-exploration-into-the-elusive-mechanism-for-lactate-export-in-saccharomyces-cerevisiae
#11
Robert Mans, Else-Jasmijn Hassing, Melanie Wijsman, Annabel Giezekamp, Jack T Pronk, Jean-Marc Daran, Antonius J A van Maris
CRISPR/Cas9-based genome editing allows rapid, simultaneous modification of multiple genetic loci in Saccharomyces cerevisiae. Here, this technique was used in a functional analysis study aimed at identifying the hitherto unknown mechanism of lactate export in this yeast. First, an S. cerevisiae strain was constructed with deletions in 25 genes encoding transport proteins, including the complete aqua(glycero)porin family and all known carboxylic-acid transporters. The 25-deletion strain was then transformed with an expression cassette for Lactobacillus casei lactate dehydrogenase (LcLDH)...
November 14, 2017: FEMS Yeast Research
https://www.readbyqxmd.com/read/29143984/crispr-cas9-mediated-knockout-of-lim-domain-only-4-retards-organ-of-corti-cell-growth
#12
Rajamani Rathinam, Rita Rosati, Samson Jamesdaniel
Lim-domain only 4 (LMO4) plays a critical role in mediating the ototoxic side-effects of cisplatin, a highly effective anti-cancer drug. However, the signaling mechanism by which cochlear LMO4 mediates otopathology is yet to be fully understood. Knockout cell culture models are useful tools for investigating the functional roles of novel genes and delineating associated signaling pathways. Therefore, LMO4 knockout organ of Corti cells were generated by using the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system...
November 16, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/29141997/crispr-cas9-mediated-genome-editing-reveals-the-synergistic-effects-of-%C3%AE-defensin-family-members-on-sperm-maturation-in-rat-epididymis
#13
Chaobao Zhang, Yuchuan Zhou, Shengsong Xie, Qianqian Yin, Chunhua Tang, Zimei Ni, Jian Fei, Yonglian Zhang
The epididymis is a male reproductive organ involved in posttesticular sperm maturation and storage, but the mechanism underlying sperm maturation remains unclear. β-Defensins (Defbs) belong to a family of small, cysteine-rich, cationic peptides that are antimicrobial and modulate the immune response. A large number of Defb genes are expressed abundantly in the male reproductive tract, especially in the epididymis. We and other groups have shown the involvement of several Defb genes in regulation of sperm function...
November 15, 2017: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/29141958/dna-methylation-defines-regional-identity-of-human-intestinal-epithelial-organoids-and-undergoes-dynamic-changes-during-development
#14
Judith Kraiczy, Komal M Nayak, Kate J Howell, Alexander Ross, Jessica Forbester, Camilla Salvestrini, Roxana Mustata, Sally Perkins, Amanda Andersson-Rolf, Esther Leenen, Anke Liebert, Ludovic Vallier, Philip C Rosenstiel, Oliver Stegle, Gordon Dougan, Robert Heuschkel, Bon-Kyoung Koo, Matthias Zilbauer
OBJECTIVE: Human intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular characteristics and behaviour in culture remains limited. DESIGN: We performed genome-wide DNA methylation and transcriptomic profiling of human IEOs derived from paediatric/adult and fetal small and large bowel as well as matching purified human gut epithelium. Furthermore, organoids were subjected to in vitro differentiation and genome editing using CRISPR/Cas9 technology...
November 15, 2017: Gut
https://www.readbyqxmd.com/read/29141633/genome-modification-of-cxcr4-by-staphylococcus-aureus-cas9-renders-cells-resistance-to-hiv-1-infection
#15
Qiankun Wang, Shuliang Chen, Qiaoqiao Xiao, Zhepeng Liu, Shuai Liu, Panpan Hou, Li Zhou, Wei Hou, Wenzhe Ho, Chunmei Li, Li Wu, Deyin Guo
BACKGROUND: The CRISPR/Cas9 system has been widely used for genome editing in mammalian cells. CXCR4 is a co-receptor for human immunodeficiency virus type 1 (HIV-1) entry, and loss of CXCR4 function can protect cells from CXCR4 (X4)-tropic HIV-1 infection, making CXCR4 an important target for HIV-1 gene therapy. However, the large size of the CRISPR/SpCas9 system presents an obstacle to its efficient delivery into primary CD4(+) T cells. Recently, a small Staphylococcus aureus Cas9 (SaCas9) has been developed as a genome editing tool can address this question...
November 15, 2017: Retrovirology
https://www.readbyqxmd.com/read/29141013/indcaps-a-tool-for-designing-screening-primers-for-crispr-cas9-mutagenesis-events
#16
Charles Hodgens, Zachary L Nimchuk, Joseph J Kieber
Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles...
2017: PloS One
https://www.readbyqxmd.com/read/29140662/re-framing-biotechnology-regulation
#17
Alison Peck
Biotechnology is about to spill the banks of federal regulation. New genetic engineering techniques like CRISPR-Cas9 promise revolutionary breakthroughs in medicine, agriculture, and public health—but those techniques would not be regulated under the terms of the Coordinated Framework for Regulation of Biotechnology. This revolutionary moment in biotechnology offers an opportunity to correct the flaws in the framework, which was hastily patched together at the advent of the technology. The framework has never captured all relevant technologies, has never satisfied the public that risk is being effectively managed, and has never been accessible to small companies and publicly-funded labs that increasingly are positioned to make radical, life-saving innovations...
2017: Food and Drug Law Journal
https://www.readbyqxmd.com/read/29140568/crispr-cas9-based-safe-harbor-gene-editing-in-rhesus-ipscs
#18
Ravi Chandra Yada, John W Ostrominski, Ilker Tunc, So Gun Hong, Jizhong Zou, Cynthia E Dunbar
NHP iPSCs provide a unique opportunity to test safety and efficacy of iPSC-derived therapies in clinically relevant NHP models. To monitor these cells in vivo, there is a need for safe and efficient labeling methods. Gene insertion into genomic safe harbors (GSHs) supports reliable transgene expression while minimizing the risk the modification poses to the host genome or target cell. Specifically, this protocol demonstrates targeting of the adeno-associated virus site 1 (AAVS1), one of the most widely used GSH loci in the human genome, with CRISPR/Cas9, allowing targeted marker or therapeutic gene insertion in rhesus macaque induced pluripotent stem cells (RhiPSCs)...
November 15, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29138316/germline-cas9-expression-yields-highly-efficient-genome-engineering-in-a-major-worldwide-disease-vector-aedes-aegypti
#19
Ming Li, Michelle Bui, Ting Yang, Christian S Bowman, Bradley J White, Omar S Akbari
The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR...
November 14, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29137643/efficient-gene-editing-in-corynebacterium-glutamicum-using-the-crispr-cas9-system
#20
Feng Peng, Xinyue Wang, Yang Sun, Guibin Dong, Yankun Yang, Xiuxia Liu, Zhonghu Bai
BACKGROUND: Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes...
November 14, 2017: Microbial Cell Factories
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