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CRISPR selection

Daniel O'Reilly, Zachary J Kartje, Eman A Ageely, Elise Malek-Adamian, Maryam Habibian, Annabelle Schofield, Christopher L Barkau, Kushal J Rohilla, Lauren B DeRossett, Austin T Weigle, Masad J Damha, Keith T Gagnon
CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity...
December 4, 2018: Nucleic Acids Research
Olga Musharova, Danylo Vyhovskyi, Sofia Medvedeva, Jelena Guzina, Yulia Zhitnyuk, Marko Djordjevic, Konstantin Severinov, Ekaterina Savitskaya
CRISPR DNA arrays of unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. New spacers are acquired into a CRISPR array in a process of CRISPR adaptation. Selection of foreign DNA fragments to be integrated into CRISPR arrays relies on PAM (protospacer adjacent motif) recognition, as only those spacers will be functional against invaders. However, acquisition of different PAM-associated spacers proceeds with markedly different efficiency from the same DNA...
December 4, 2018: MBio
Christian Møller-Olsen, Siu Fung Stanley Ho, Ranti Dev Shukla, Tamas Feher, Antonia P Sagona
Bacterial infections can be treated with bacteriophages that show great specificity towards their bacterial host and can be genetically modified for different applications. However, whether and how bacteriophages can kill intracellular bacteria in human cells remains elusive. Here, using CRISPR/Cas selection, we have engineered a fluorescent bacteriophage specific for E. coli K1, a nosocomial pathogen responsible for urinary tract infections, neonatal meningitis and sepsis. By confocal and live microscopy, we show that engineered bacteriophages K1F-GFP and E...
December 3, 2018: Scientific Reports
Yong-Tao Du, Meng-Jie Zhao, Chang-Tao Wang, Yuan Gao, Yan-Xia Wang, Yong-Wei Liu, Ming Chen, Jun Chen, Yong-Bin Zhou, Zhao-Shi Xu, You-Zhi Ma
BACKGROUND: Abiotic stress severely influences plant growth and development. MYB transcription factors (TFs), which compose one of the largest TF families, play an important role in abiotic stress responses. RESULT: We identified 139 soybean MYB-related genes; these genes were divided into six groups based on their conserved domain and were distributed among 20 chromosomes (Chrs). Quantitative real-time PCR (qRT-PCR) indicated that GmMYB118 highly responsive to drought, salt and high temperature stress; thus, this gene was selected for further analysis...
December 3, 2018: BMC Plant Biology
X Wang, M Nachliely, J S Harrison, M Danilenko, G P Studzinski
Standard therapy for Acute Myeloid Leukemia (AML) is rarely curative, and several suggested improvements have had little success so far. We have reported that in an in vitro model of a potential therapeutic regimen for AML, the activity of cytarabine (AraC) is enhanced by a sequential treatment with a combination of the vitamin D2 analog Doxercalciferol (D2) and the plant-derived antioxidant carnosic acid (CA) Importantly, the enhancement occurred selectively in patient-derived AML blasts, but not in the normal bone marrow cells...
November 30, 2018: Journal of Steroid Biochemistry and Molecular Biology
Fan Yang, Athen N Kimberlin, Christian G Elowsky, Yunfeng Liu, Ariadna Gonzalez-Solis, Edgar B Cahoon, James R Alfano
Plants recycle non-activated immune receptors to maintain a functional immune system. The Arabidopsis immune receptor kinase FLAGELLIN-SENSING 2 (FLS2) recognizes bacterial flagellin. Arabidopsis orosomucoid (ORM) proteins are negative regulators of sphingolipid biosynthesis, but the mechanism by which they do this is not well understood. Here, we provide evidence that ORM proteins act as selective autophagy receptors to degrade FLS2. Arabidopsis plants over-expressing ORM1 or ORM2 lack or have greatly diminished FLS2 levels, lack FLS2 signaling, and are more susceptible to the bacterial pathogen Pseudomonas syringae...
November 30, 2018: Molecular Plant
Julia K Bialek, Thomas Walther, Joachim Hauber, Ulrike C Lange
Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we present a detailed protocol for the generation of novel in vitro models for HIV infection with chosen genomic integration sites using CRISPR-Cas9-based genome engineering technology. With this method, a reporter sequence of choice can be integrated into a targeted, chosen genomic locus, reflecting clinically relevant integration sites. In the protocol, the design of an HIV-derived reporter and choosing of a target site and gRNA sequence are described...
November 14, 2018: Journal of Visualized Experiments: JoVE
Jung-Wook Kim, Hong Zhang, Figen Seymen, Mine Koruyucu, Yuanyuan Hu, Jenny Kang, Youn Jung Kim, Atsushi Ikeda, Yelda Kasimoglu, Merve Bayram, Chuhua Zhang, Kazuhiko Kawasaki, John D Bartlett, Thomas L Saunders, James P Simmer, Jan C-C Hu
Amelogenesis imperfecta (AI) is a collection of isolated (non-syndromic) inherited diseases affecting dental enamel formation or a clinical phenotype in syndromic conditions. We characterized three consanguineous AI families with generalized irregular hypoplastic enamel with rapid attrition that perfectly segregated with homozygous defects in a novel gene: RELT that is a member of the tumor necrosis factor receptor superfamily (TNFRSF). RNAscope in situ hybridization of wild-type mouse molars and incisors demonstrated specific Relt mRNA expression by secretory stage ameloblasts and by odontoblasts...
December 3, 2018: Clinical Genetics
Kazuya Ikeda, Nobuko Uchida, Toshinobu Nishimura, Joseph White, Renata M Martin, Hiromitsu Nakauchi, Vittorio Sebastiano, Kenneth I Weinberg, Matthew H Porteus
Scarless genome editing in human pluripotent stem cells (hPSCs) represents a goal for both precise research applications and clinical translation of hPSC-derived therapies. Here we established a versatile and efficient method that combines CRISPR-Cas9-mediated homologous recombination with positive-negative selection of edited clones to generate scarless genetic changes in hPSCs.
December 2018: Nature Methods
Behnom Farboud, Aaron F Severson, Barbara J Meyer
The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and non-model organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes for both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-stranded breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans We also devised strategies to insert long (10 kb) exogenous sequences and incorporate multiple nucleotide substitutions at considerable distance from DSBs...
November 30, 2018: Genetics
Cai Liang, Zhenlei Zhang, Qinfu Chen, Haiyan Yan, Miao Zhang, Xingfeng Xiang, Qi Yi, Xuan Pan, Hankun Cheng, Fangwei Wang
The inner centromere region of a mitotic chromosome critically regulates sister chromatid cohesion and kinetochore-microtubule attachments. However, the molecular mechanism underlying inner centromere assembly remains elusive. Here, using CRISPR/Cas9-based gene editing in HeLa cells, we disrupted the interaction of Shugoshin 1 (Sgo1) with histone H2A phosphorylated on Thr-120 (H2ApT120) to selectively release Sgo1 from mitotic centromeres. Interestingly, cells expressing the H2ApT120-binding defective mutant of Sgo1 have an elevated rate of chromosome missegregation accompanied by weakened centromeric cohesion and decreased centromere accumulation of the chromosomal passenger complex (CPC), an integral part of the inner centromere and a key player in the correction of erroneous kinetochore-microtubule attachments...
November 29, 2018: Journal of Biological Chemistry
Alexander Nash, Giulia Mignini Urdaneta, Andrea K Beaghton, Astrid Hoermann, Philippos Aris Papathanos, George K Christophides, Nikolai Windbichler
A first generation of CRISPR-based gene drives have now been tested in the laboratory in a number of organisms including malaria vector mosquitoes. Challenges for their use in the area-wide genetic control of vector-borne disease have been identified, including the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. We have evaluated theoretically the concept of Integral Gene Drive (IGD) as an alternative paradigm for population replacement...
November 29, 2018: Biology Open
Kai Miao, Xin Zhang, Sek Man Su, Jianming Zeng, Zebin Huang, Un In Chan, Xiaoling Xu, Chu-Xia Deng
CRISPR/Cas9 is a powerful technology widely used for genome editing, with the potential to be used for correcting a wide variety of deleterious disease-causing mutations. However, the technique tends to generate more indels (insertions and deletions) than precise modifications at the target sites, which might not resolve the mutation and could instead exacerbate the initial genetic disruption. We sought to develop an improved protocol for CRISPR/Cas9 that would correct mutations without unintended consequences...
November 28, 2018: Journal of Biological Chemistry
Takaya Ichikawa, Yusuke Okuno, Yoshitaka Sato, Fumi Goshima, Hironori Yoshiyama, Teru Kanda, Hiroshi Kimura, Takayuki Murata
Epigenetic modifications play a pivotal role in the expression of the genes of Epstein-Barr virus (EBV). We found that de novo EBV infection of primary B cells caused moderate induction of enhancer of zeste homolog 2 (EZH2), the major histone H3 lysine 27 (K27) methyltransferase. To investigate the role of EZH2, we knocked out the EZH2 gene in EBV-negative Akata cells by the CRISPR/Cas9 system and infected the cells with EBV, followed by selection of EBV-positive cells. During the latent state, growth of EZH2-knockout (KO) cells was significantly slower after infection compared to wild-type controls, despite similar levels of viral gene expression between cell lines...
November 28, 2018: MSphere
Li Xue, Bin Tang, Wei Chen, Jiesi Luo
The CRISPR-Cas9 system derived from adaptive immunity in bacteria and archaea has been developed into a powerful tool for genome engineering with wide-ranging applications. Optimizing single guide RNA (sgRNA) design to improve efficiency of target cleavage is a key step for successful gene editing using the CRISPR-Cas9 system. Because not all sgRNAs that cognate to a given target gene are equally effective, computational tools have been developed based on experimental data to increase the likelihood of selecting effective sgRNAs...
November 28, 2018: Journal of Chemical Information and Modeling
Tonći Šuštić, Sake van Wageningen, Evert Bosdriesz, Robert J D Reid, John Dittmar, Cor Lieftink, Roderick L Beijersbergen, Lodewyk F A Wessels, Rodney Rothstein, René Bernards
BACKGROUND: Mutations in KRAS are frequent in human cancer, yet effective targeted therapeutics for these cancers are still lacking. Attempts to drug the MEK kinases downstream of KRAS have had limited success in clinical trials. Understanding the specific genomic vulnerabilities of KRAS-driven cancers may uncover novel patient-tailored treatment options. METHODS: We first searched for synthetic lethal (SL) genetic interactions with mutant RAS in yeast with the ultimate aim to identify novel cancer-specific targets for therapy...
November 27, 2018: Genome Medicine
Takuya Katayama, Hidetoshi Nakamura, Yue Zhang, Arnaud Pascal, Wataru Fujii, Jun-Ichi Maruyama
Filamentous fungi are used for food fermentation and industrial production of recombinant proteins. They also serve as a source of secondary metabolites, and are recently expected as hosts for heterologous production of useful secondary metabolites. Multiple-step genetic engineering is required to enhance industrial production involving these fungi, but traditional sequential modification of multiple genes using a limited number of selection markers is laborious. Moreover, efficient genetic engineering techniques for industrial strains have not yet been established...
November 26, 2018: Applied and Environmental Microbiology
Shujuan Zhang, Rongzhi Zhang, Guoqi Song, Jie Gao, Wei Li, Xiaodong Han, Mingli Chen, Yulian Li, Genying Li
BACKGROUND: Recently, the CRISPR/Cas9 system has been widely used to precisely edit plant genomes. Due to the difficulty in Agrobacterium-mediated genetic transformation of wheat, the reported applications in CRISPR/Cas9 system were all based on the biolistic transformation. RESULTS: In the present study, we efficiently applied targeted mutagenesis in common wheat (Triticum aestivum L.) protoplasts and transgenic T0 plants using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens...
November 26, 2018: BMC Plant Biology
Anja Hofmann, Johannes Falk, Tim Prangemeier, Dominic Happel, Adrian Köber, Andreas Christmann, Heinz Koeppl, Harald Kolmar
The robust and precise on and off switching of one or more genes of interest, followed by expression or repression is essential for many biological circuits as well as for industrial applications. However, many regulated systems published to date influence the viability of the host cell, show high basal expression or enable only the overexpression of the target gene without the possibility of fine regulation. Herein, we describe an AND gate designed to overcome these limitations by combining the advantages of three well established systems, namely the scaffold RNA CRISPR/dCas9 platform that is controlled by Gal10 as a natural and by LexA-ER-AD as heterologous transcription factor...
November 22, 2018: Nucleic Acids Research
Yanjun Li, Fangqing Yan, Heyun Wu, Guoliang Li, Yakun Han, Qian Ma, Xiaoguang Fan, Chenglin Zhang, Qingyang Xu, Xixian Xie, Ning Chen
Although CRISPR/Cas9-mediated gene editing technology has developed vastly in Escherichia coli, the chromosomal integration of large DNA fragment is still challenging compared with gene deletion and small fragment integration. Moreover, to guarantee sufficient Cas9-induced double-strand breaks, it is usually necessary to design several gRNAs to select the appropriate one. Accordingly, we established a practical daily routine in the laboratory work, involving multiple-step chromosomal integration of the divided segments from a large DNA fragment...
November 23, 2018: Journal of Industrial Microbiology & Biotechnology
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