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https://www.readbyqxmd.com/read/28448738/site-specific-recruitment-of-epigenetic-factors-with-a-modular-crispr-cas-system
#1
Tobias Anton, Sebastian Bultmann
Dissecting the complex network of epigenetic modifications requires tools that combine precise recognition of DNA sequences with the capability to modify epigenetic marks. The CRISPR/Cas system has been proven to be a valuable addition to existing methodologies that fulfill these tasks. So far, sequence-specific editing of epigenetic modifications such as DNA methylation and histone posttranslational modifications relied on direct fusions of enzymatically inactivated Cas9 (dCas9) with epigenetic effectors. Here, we report a novel, modular system that facilitates the recruitment of any GFP-tagged protein to desired genomic loci...
February 23, 2017: Nucleus
https://www.readbyqxmd.com/read/28448066/structural-basis-of-crispr-spycas9-inhibition-by-an-anti-crispr-protein
#2
De Dong, Minghui Guo, Sihan Wang, Yuwei Zhu, Shuo Wang, Zhi Xiong, Jianzheng Yang, Zengliang Xu, Zhiwei Huang
The CRISPR-Cas9 systems are bacterial encoded adaptive immune systems to defend against phages infection, through RNA-guided endonuclease activity of Cas9 to degrade double-stranded DNA bearing a protospacer adjacent motif (PAM) and complementary sequences to the guide RNA1,2,3,4,5. Recently, two anti-CRISPR proteins AcrIIA2 and AcrIIA4 from Listeria monocytogenes (Lmo) prophages have been identified, both of which potently inhibit Streptococcus pyogenes Cas9 (SpyCas9) and LmoCas9 activity in bacteria and human cells6...
April 27, 2017: Nature
https://www.readbyqxmd.com/read/28448034/using-a-fluorescent-pcr-capillary-gel-electrophoresis-technique-to-genotype-crispr-cas9-mediated-knockout-mutants-in-a-high-throughput-format
#3
Muhammad Khairul Ramlee, Jing Wang, Alice M S Cheung, Shang Li
The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site...
April 8, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28444372/quantifying-the-number-of-independent-organelle-dna-insertions-in-genome-evolution-and-human-health
#4
Einat Hazkani-Covo, William F Martin
Fragments of organelle genomes are often found as insertions in nuclear DNA. These fragments of mitochondrial DNA (numts) and plastid DNA (nupts) are ubiquitous components of eukaryotic genomes. They are, however, often edited out during the genome assembly process, leading to systematic underestimation of their frequency. Numts and nupts, once inserted, can become further fragmented through subsequent insertion of mobile elements or other recombinational events that disrupt the continuity of the inserted sequence relative to the genuine organelle DNA copy...
April 21, 2017: Genome Biology and Evolution
https://www.readbyqxmd.com/read/28439266/a-novel-regulator-of-activation-induced-cytidine-deaminase-apobecs-in-immunity-and-cancer-schr%C3%A3-dinger-s-catalytic-pocket
#5
REVIEW
Justin J King, Mani Larijani
Activation-induced cytidine deaminase (AID) and its relative APOBEC3 cytidine deaminases boost immune response by mutating immune or viral genes. Because of their genome-mutating activities, AID/APOBECs are also drivers of tumorigenesis. Due to highly charged surfaces, extensive non-specific protein-protein/nucleic acid interactions, formation of polydisperse oligomers, and general insolubility, structure elucidation of these proteins by X-ray crystallography and NMR has been challenging. Hence, almost all available AID/APOBEC structures are of mutated and/or truncated versions...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/28435892/dramatic-improvement-of-crispr-cas9-editing-in-candida-albicans-by-increased-single-guide-rna-expression
#6
Henry Ng, Neta Dean
The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans. Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein (RFP) gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing...
March 2017: MSphere
https://www.readbyqxmd.com/read/28434148/advancing-chimeric-antigen-receptor-t-cell-therapy-with-crispr-cas9
#7
REVIEW
Jiangtao Ren, Yangbing Zhao
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (CRISPR/Cas9) system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for therapeutic applications, especially chimeric antigen receptor (CAR) T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared...
April 22, 2017: Protein & Cell
https://www.readbyqxmd.com/read/28433723/elimination-of-the-cryptic-plasmid-in-leuconostoc-citreum-by-crispr-cas9-system
#8
Ye-Ji Jang, Seung-Oh Seo, Seul-Ah Kim, Ling Li, Tae-Jip Kim, Sun Chang Kim, Yong-Su Jin, Nam Soo Han
Leuconostoc spp. are important lactic acid bacteria for the fermentation of foods. In particular, L. citreum strains isolated from various foods have been used as host strains for genetic and metabolic engineering studies. In order to develop a food-grade genetic engineering system, L. citreum CB2567 was isolated from Kimchi. However, the isolated bacterium contained a cryptic plasmid which was difficult to eliminate. As the existence of the plasmid might hinder strain engineering, we eliminated the plasmid using an RNA-guided DNA endonuclease CRISPR/Cas9 system...
April 19, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28433382/applications-of-the-crispr-cas9-system-in-kidney-research
#9
REVIEW
Yoshiki Higashijima, Seiichi Hirano, Masaomi Nangaku, Osamu Nureki
The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) is an RNA-guided DNA nuclease, and has been harnessed for the development of simple, efficient, and relatively inexpensive technologies to precisely manipulate the genomic information in virtually all cell types and organisms. The CRIPSR-Cas9 systems have already been effectively used to disrupt multiple genes simultaneously, create conditional alleles, and generate reporter proteins, even in vivo...
April 19, 2017: Kidney International
https://www.readbyqxmd.com/read/28431230/structural-basis-for-guide-rna-processing-and-seed-dependent-dna-targeting-by-crispr-cas12a
#10
Daan C Swarts, John van der Oost, Martin Jinek
The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding...
April 20, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28429755/opossum-apobec1-is-a-dna-mutator-with-retrovirus-and-retroelement-restriction-activity
#11
Terumasa Ikeda, Mayuko Shimoda, Diako Ebrahimi, John L VandeBerg, Reuben S Harris, Atsushi Koito, Kazuhiko Maeda
APOBEC3s (A3s) are single-stranded DNA cytosine deaminases that provide innate immune defences against retroviruses and mobile elements. A3s are specific to eutherian mammals because no direct homologs exist at the syntenic genomic locus in metatherian (marsupial) or prototherian (monotreme) mammals. However, the A3s in these species have the likely evolutionary precursors, the antibody gene deaminase AID and the RNA/DNA editing enzyme APOBEC1 (A1). Here, we used cell culture-based assays to determine whether opossum A1 restricts the infectivity of retroviruses including human immunodeficiency virus type 1 (HIV-1) and the mobility of LTR/non-LTR retrotransposons...
April 21, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28429156/crystallization-and-preliminary-x-ray-diffraction-analysis-of-a-mammal-inositol-1-3-4-5-6-pentakisphosphate-2-kinase
#12
Elsa Franco-Echevarría, Julia Sanz-Aparicio, Nathalie Troffer-Charlier, Arnaud Poterszman, Beatriz González
Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5 2-K) is an enzyme that catalyses the formation of phytic acid (IP6) from IP5 and ATP. In mammals, IP6 is involved in multiple events such as DNA repair and mRNA edit and it is the precursor of inositol pyrophosphates, emerging compounds shown to have an essential role in apoptosis. In addition, IP5 2-K have functions in cells independently of its catalytic activity, for example in rRNA biogenesis. We pursue the structure determination of a mammal IP5 2-K by Protein Crystallography...
April 21, 2017: Protein Journal
https://www.readbyqxmd.com/read/28426191/genome-wide-abolishment-of-mobile-genetic-elements-using-genome-shuffling-and-crispr-cas-assisted-mage-allows-the-efficient-stabilization-of-a-bacterial-chassis
#13
Kinga Umenhoffer, Gábor Draskovits, Ákos Nyerges, Ildikó Karcagi, Balázs Bogos, Edit Tímár, Bálint Csörgő, Róbert Herczeg, István Nagy, Tamás Fehér, Csaba Pál, György Pósfai
The ideal bacterial chassis provides a simplified, stable and predictable host environment for synthetic biological circuits. Mutability and evolution can, however, compromise stability, leading to deterioration of artificial genetic constructs. By eliminating certain sources of instability, these undesired genetic changes can be mitigated. Specifically, deletion of prophages and insertion sequences, nonessential constituents of bacterial genomes, has been shown to be beneficial in cellular and genetic stabilization...
April 26, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28420610/the-diversity-of-dna-fragment-editing-by-crispr-cas9-in-highly-homologous-or-repetitive-sequences
#14
Wang Leyang, Huang Haiyan, Wu Qiang
In complex genomes, there are a large number of duplicated genes in the coding regions and many more repetitive sequences in the non-coding regions. Repetitive sequences can exert great impacts on the heredity and evolution of the organisms, as well as their genome 3D architecture and transcriptional regulation. The high homology nature of repetitive sequences renders their editing by CRISPR/Cas9 very complex. At diploid or polyploid situations, such repetitive sequences could be edited differently on each chromosome or chromatid...
April 20, 2017: Yi Chuan, Hereditas
https://www.readbyqxmd.com/read/28416802/rna-editing-dependent-epitranscriptome-diversity-in-cancer-stem-cells
#15
REVIEW
Qingfei Jiang, Leslie A Crews, Frida Holm, Catriona H M Jamieson
Cancer stem cells (CSCs) can regenerate all facets of a tumour as a result of their stem cell-like capacity to self-renew, survive and become dormant in protective microenvironments. CSCs evolve during tumour progression in a manner that conforms to Charles Darwin's principle of natural selection. Although somatic DNA mutations and epigenetic alterations promote evolution, post-transcriptional RNA modifications together with RNA binding protein activity (the 'epitranscriptome') might also contribute to clonal evolution through dynamic determination of RNA function and gene expression diversity in response to environmental stimuli...
April 18, 2017: Nature Reviews. Cancer
https://www.readbyqxmd.com/read/28412170/lipid-nanoparticle-systems-for-enabling-gene-therapies
#16
REVIEW
Pieter R Cullis, Michael J Hope
Genetic drugs such as small interfering RNA (siRNA), mRNA, or plasmid DNA provide potential gene therapies to treat most diseases by silencing pathological genes, expressing therapeutic proteins, or through gene-editing applications. In order for genetic drugs to be used clinically, however, sophisticated delivery systems are required. Lipid nanoparticle (LNP) systems are currently the lead non-viral delivery systems for enabling the clinical potential of genetic drugs. Application will be made to the Food and Drug Administration (FDA) in 2017 for approval of an LNP siRNA drug to treat transthyretin-induced amyloidosis, presently an untreatable disease...
April 13, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/28411843/the-molecular-revolution-in-cutaneous-biology-emerging-landscape-in-genomic-dermatology-new-mechanistic-ideas-gene-editing-and-therapeutic-breakthroughs
#17
REVIEW
Matthias Titeux, Araksya Izmiryan, Alain Hovnanian
Stunning technological advances in genomics have led to spectacular breakthroughs in the understanding of the underlying defects, biological pathways and therapeutic targets of skin diseases leading to new therapeutic interventions. Next-generation sequencing has revolutionized the identification of disease-causing genes and has a profound impact in deciphering gene and protein signatures in rare and frequent skin diseases. Gene addition strategies have shown efficacy in junctional EB and in recessive dystrophic EB (RDEB)...
May 2017: Journal of Investigative Dermatology
https://www.readbyqxmd.com/read/28410976/genome-editing-via-delivery-of-cas9-ribonucleoprotein
#18
Mark DeWitt, Jacob E Corn, Dana Carroll
The CRISPR-Cas genome editing system is very powerful. The format of the CRISPR reagents and the means of delivery are often important factors in targeting efficiency. Delivery of recombinant Cas9 protein and guide RNA (gRNA) as a preformed ribonucleoprotein (RNP) complex has recently emerged as a powerful and general approach to genome editing. Here we outline methods to produce and deliver Cas9 RNPs. A donor DNA carrying desired sequence changes can also be included to program precise sequence introduction or replacement...
April 11, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28408207/transactivator-protein-an-alternative-for-delivery-of-recombinant-proteins-for-safer-reprogramming-of-induced-pluripotent-stem-cell
#19
REVIEW
Fazlina Nordin, Raja Norazireen Raja Ahmad, Farzin Farzaneh
Induced pluripotent stem cells (iPSC) are somatic cells reprogrammed to pluripotency by forced expression of pluripotency factors. These cells are shown to have the same pluripotent potential as embryonic stem cells (ESC) and considered as an alternative to the much controversial usage of ESC which involved human embryos. However, the traditional method in reprogramming cells into iPSC using genome-integrating retro- or lenti- viruses remains an obstacle for its application in clinical setting. Although numerous studies have been conducted for a safer DNA-based reprogramming, reprogramming of iPSC by genetic modifications may raise the possibility of malignant transformation and has been a major limitation for its usage in clinical applications...
April 10, 2017: Virus Research
https://www.readbyqxmd.com/read/28405721/genome-editing-a-robust-technology-for-human-stem-cells
#20
REVIEW
Arun Pandian Chandrasekaran, Minjung Song, Suresh Ramakrishna
Human pluripotent stem cells comprise induced pluripotent and embryonic stem cells, which have tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. Genome editing of stem cells is possible with the help of synthetic nucleases that facilitate site-specific modification of a gene of interest. Recent advances in genome editing techniques have improved the efficiency and speed of the development of stem cells for human disease models...
April 12, 2017: Cellular and Molecular Life Sciences: CMLS
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