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Bibliography on: CRISPR-Cas

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Robert J. Robbins is a biologist, an educator, a science administrator, a publisher, an information technologist, and an IT leader and manager who specializes in advancing biomedical knowledge and supporting education through the application of information technology. More About:  RJR | OUR TEAM | OUR SERVICES | THIS WEBSITE

RJR: Recommended Bibliography 17 Jan 2019 at 01:35 Created: 

CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: "CRISPR.CAS" OR "crispr/cas" NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

RevDate: 2019-01-15

Nakamura M, Srinivasan P, Chavez M, et al (2019)

Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells.

Nature communications, 10(1):194 pii:10.1038/s41467-018-08158-x.

Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.

RevDate: 2019-01-15
CmpDate: 2019-01-15

Gay S, Bugeon J, Bouchareb A, et al (2018)

MiR-202 controls female fecundity by regulating medaka oogenesis.

PLoS genetics, 14(9):e1007593.

Female gamete production relies on coordinated molecular and cellular processes that occur in the ovary throughout oogenesis. In fish, as in other vertebrates, these processes have been extensively studied both in terms of endocrine/paracrine regulation and protein expression and activity. The role of small non-coding RNAs in the regulation of animal reproduction remains however largely unknown and poorly investigated, despite a growing interest for the importance of miRNAs in a wide variety of biological processes. Here, we analyzed the role of miR-202, a miRNA predominantly expressed in male and female gonads in several vertebrate species. We studied its expression in the medaka ovary and generated a mutant line (using CRISPR/Cas9 genome editing) to determine its importance for reproductive success with special interest for egg production. Our results show that miR-202-5p is the most abundant mature form of the miRNA and that it is expressed in granulosa cells and in the unfertilized egg. The knock out (KO) of mir-202 gene resulted in a strong phenotype both in terms of number and quality of eggs produced. Mutant females exhibited either no egg production or produced a dramatically reduced number of eggs that could not be fertilized, ultimately leading to no reproductive success. We quantified the size distribution of the oocytes in the ovary of KO females and performed a large-scale transcriptomic analysis approach to identified dysregulated molecular pathways. Together, cellular and molecular analyses indicate that the lack of miR-202 impairs the early steps of oogenesis/folliculogenesis and decreases the number of large (i.e. vitellogenic) follicles, ultimately leading to dramatically reduced female fecundity. This study sheds new light on the regulatory mechanisms that control the early steps of follicular development, including possible targets of miR-202-5p, and provides the first in vivo functional evidence that a gonad-predominant microRNA may have a major role in female reproduction.

RevDate: 2019-01-15
CmpDate: 2019-01-15

Ravi P, Trivedi D, G Hasan (2018)

FMRFa receptor stimulated Ca2+ signals alter the activity of flight modulating central dopaminergic neurons in Drosophila melanogaster.

PLoS genetics, 14(8):e1007459.

Neuropeptide signaling influences animal behavior by modulating neuronal activity and thus altering circuit dynamics. Insect flight is a key innate behavior that very likely requires robust neuromodulation. Cellular and molecular components that help modulate flight behavior are therefore of interest and require investigation. In a genetic RNAi screen for G-protein coupled receptors that regulate flight bout durations, we earlier identified several receptors, including the receptor for the neuropeptide FMRFa (FMRFaR). To further investigate modulation of insect flight by FMRFa we generated CRISPR-Cas9 mutants in the gene encoding the Drosophila FMRFaR. The mutants exhibit significant flight deficits with a focus in dopaminergic cells. Expression of a receptor specific RNAi in adult central dopaminergic neurons resulted in progressive loss of sustained flight. Further, genetic and cellular assays demonstrated that FMRFaR stimulates intracellular calcium signaling through the IP3R and helps maintain neuronal excitability in a subset of dopaminergic neurons for positive modulation of flight bout durations.

RevDate: 2019-01-15
CmpDate: 2019-01-15

Yang D, Zheng X, Chen S, et al (2018)

Sensing of cytosolic LPS through caspy2 pyrin domain mediates noncanonical inflammasome activation in zebrafish.

Nature communications, 9(1):3052.

The noncanonical inflammasome is critical for cytosolic sensing of Gram-negative pathogens. Here, we show that bacterial infection induces caspy2 activation in zebrafish fibroblasts, which mediates pyroptosis via a caspase-5-like activity. Zebrafish caspy2 binds directly to lipopolysaccharide via the N-terminal pyrin death domain, resulting in caspy2 oligomerization, which is critical for pyroptosis. Furthermore, we show that caspy2 is highly expressed in the zebrafish gut and is activated during infection. Knockdown of caspy2 expression impairs the ability of zebrafish to restrict bacterial invasion in vivo, and protects larvae from lethal sepsis. Collectively, our results identify a crucial event in the evolution of pattern recognition into the death domain superfamily-mediated intracellular lipopolysaccharide-sensing pathway in innate immunity.

RevDate: 2019-01-15
CmpDate: 2019-01-15

Shi X, Kitano A, Jiang Y, et al (2018)

Clonal expansion and myeloid leukemia progression modeled by multiplex gene editing of murine hematopoietic progenitor cells.

Experimental hematology, 64:33-44.e5.

Recent advances in next-generation sequencing have identified novel mutations and revealed complex genetic architectures in human hematological malignancies. Moving forward, new methods to quickly generate animal models that recapitulate the complex genetics of human hematological disorders are needed to transform the genetic information to new therapies. Here, we used a ribonucleoprotein-based CRISPR/Cas9 system to model human clonal hematopoiesis of indeterminate potential and acute myeloid leukemia (AML). We edited multiple genes recurrently mutated in hematological disorders, including those encoding epigenetic regulators, transcriptional regulators, and signaling components in murine hematopoietic stem/progenitor cells. Tracking the clonal dynamics by sequencing the indels induced by CRISPR/Cas9 revealed clonal expansion in some recipient mice that progressed to AML initiated by leukemia-initiating cells. Our results establish that the CRISPR/Cas9-mediated multiplex mutagenesis can be used to engineer a variety of murine models of hematological malignancies with complex genetic architectures seen in human disease.

RevDate: 2019-01-15
CmpDate: 2019-01-15

Naert T, K Vleminckx (2018)

CRISPR/Cas9-Mediated Knockout of Rb1 in Xenopus tropicalis.

Methods in molecular biology (Clifton, N.J.), 1726:177-193.

At this time, no molecular targeted therapies exist for treatment of retinoblastoma. This can be, in part, attributed to the lack of animal models that allow for both rapid identification of novel therapeutic targets and hypothesis driven drug testing. Within this scope, we have recently reported the first genuine genetic nonmammalian retinoblastoma cancer model within the aquatic model organism Xenopus tropicalis (Naert et al., Sci Rep 6: 35263, 2016). Here we describe the methods to generate rb1 mosaic mutant Xenopus tropicalis by employing the CRISPR/Cas9 technology. In depth, we discuss short guide RNA (sgRNA) design parameters, generation, quality control, quantification, and delivery followed by several methods for assessing genome editing efficiencies. As such the reader should be capable, by minor changes to the methods described here, to (co-) target rb1 or any one or multiple gene(s) within the Xenopus tropicalis genome by multiplex CRISPR/Cas9 methodology.

RevDate: 2019-01-15
CmpDate: 2019-01-15

Rahim K, Huo L, Li C, et al (2017)

Identification of a basidiomycete-specific Vilse-like GTPase activating proteins (GAPs) and its roles in the production of virulence factors in Cryptococcus neoformans.

FEMS yeast research, 17(8):.

Cryptococcus neoformans is a basidiomycetous pathogenic yeast that causes fatal infections in both immunocompetent and immunocompromised patients. Regulation on the production of its virulence factors is not fully understood. Here we reported the characterization of a gene, named CVH1(CNA06260), encoding a Drosophila Vilse-like RhoGAP homolog, which is hallmarked by three conserved functional domains: WW, MyTH4 and RhoGAP. Phylogenetic analysis suggests that CVH1 is highly conserved from protists to mammals and interestingly in basidiomycetes, but absent in plants or Ascomycota and other lower fungi. This phylogenetic distribution indicates an evolutionary link among these groups of organisms. Functional analyses demonstrated that CVH1 was involved in stress tolerance and virulence factor production. By disrupting CVH1, we created a second mutant cvh1Δ with the CRISPR-Cas9 editing tool. The mutant strain exhibited hypersensitivity to osmotic stress by 2 M sorbitol and NaCl, suggesting defects in the HOG signaling pathway and an interaction of Cvh1 with the HOG pathway. Hypersensitivity of cvh1Δ to 1% Congo red and 0.01% SDS suggests that the cell wall integrity was impaired in the mutant. And cvh1Δ hardly produced the pigment melanin and capsule. Our study for the first time demonstrates that the fungal Vilse-like RhoGAP CVH1 is an important regulator of multiple biological processes in C. neoformans, and provides novel insights into the regulatory circuit of stress resistance/cell wall integrity, and laccase and capsule synthesis in C. neoformans.

RevDate: 2019-01-15
CmpDate: 2019-01-15

Mans R, Hassing EJ, Wijsman M, et al (2017)

A CRISPR/Cas9-based exploration into the elusive mechanism for lactate export in Saccharomyces cerevisiae.

FEMS yeast research, 17(8):.

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). In anaerobic, glucose-grown batch cultures this strain exhibited a lower specific growth rate (0.15 vs. 0.25 h-1) and biomass-specific lactate production rate (0.7 vs. 2.4 mmol g biomass-1 h-1) than an LcLDH-expressing reference strain. However, a comparison of the two strains in anaerobic glucose-limited chemostat cultures (dilution rate 0.10 h-1) showed identical lactate production rates. These results indicate that, although deletion of the 25 transporter genes affected the maximum specific growth rate, it did not impact lactate export rates when analysed at a fixed specific growth rate. The 25-deletion strain provides a first step towards a 'minimal transportome' yeast platform, which can be applied for functional analysis of specific (heterologous) transport proteins as well as for evaluation of metabolic engineering strategies.

RevDate: 2019-01-15
CmpDate: 2019-01-15

Borodina I, ZK Zhao (2017)

Editorial: Yeast cell factories for production of fuels and chemicals.

FEMS yeast research, 17(8):.

RevDate: 2019-01-14

Gürtler S, Wolke C, Otto O, et al (2019)

Tafazzin-dependent cardiolipin composition in C6 glioma cells correlates with changes in mitochondrial and cellular functions, and cellular proliferation.

Biochimica et biophysica acta. Molecular and cell biology of lipids pii:S1388-1981(19)30003-4 [Epub ahead of print].

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function and, more recently, with cellular proliferation. Tafazzin, an acyltransferase with key functions in CL remodeling determining actual CL composition, affects mitochondrial oxidative phosphorylation. Here, we show that the CRISPR-Cas9 mediated knock-out of tafazzin (Taz) is associated with substantial alterations of various mitochondrial and cellular characteristics in C6 glioma cells. The knock-out of tafazzin substantially changed the profile of fatty acids incorporated in CL and the distribution of molecular CL species. Taz knock-out was further associated with decreased capacity of oxidative phosphorylation that mainly originates from impaired complex I associated energy metabolism in C6 glioma cells. The lack of tafazzin switched energy metabolism from oxidative phosphorylation to glycolysis indicated by lower respiration rates, membrane potential and higher levels of mitochondria-derived reactive oxygen species but keeping the cellular ATP content unchanged. The impact of tafazzin on mitochondria was also indicated by altered morphology and arrangement in tafazzin deficient C6 glioma cells. In the cells we observed tafazzin-dependent changes in the distribution of cellular fatty acids as an indication of altered lipid metabolism as well as in stability/morphology. Most impressive is the dramatic reduction in cell proliferation in tafazzin deficient C6 glioma cells that is not mediated by reactive oxygen species. Our data clearly indicate that defects in CL phospholipid remodeling trigger a cascade of events including modifications in CL linked to subsequent alterations in mitochondrial and cellular functions.

RevDate: 2019-01-14

Takeshita D, Sato M, Inanaga H, et al (2019)

Crystal Structures of Csm2 and Csm3 in the Type III-A CRISPR-Cas Effector Complex.

Journal of molecular biology pii:S0022-2836(18)30903-3 [Epub ahead of print].

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and CRISPR-associated (Cas) genes encode CRISPR RNAs (crRNA) and Cas proteins, respectively, which play important roles in the adaptive immunity system (CRISPR-Cas system) in prokaryotes. The crRNA and Cas proteins form ribonucleoprotein effector complexes to capture and degrade invading genetic materials with base complementarity to the crRNA guide sequences. The Csm complex, a type III-A effector complex, comprises five Cas proteins (Csm1-Csm5) and a crRNA, which co-transcriptionally degrades invading DNA and RNA. Here we report the crystal structures of the Staphylococcus epidermidis Csm2 (SeCsm2) and Thermoplasma volcanium Csm3 (TvCsm3) at 2.4 and 2.7 Å resolutions, respectively. SeCsm2 adopts a monomeric globular fold by itself, in striking contrast to the previously reported Thermotoga maritima Csm2, which adopted an extended conformation and formed a dimeric structure. We propose that the globular monomeric form is the bona fide structure of Csm2. TvCsm3 forms a filamentous structure in the crystals. The molecular arrangement of TvCsm3 is similar to that of the stacked Cmr4 proteins in the Cmr complex, suggesting the functionally relevant architecture of the present Csm3 structure. We constructed model structures of the Csm complex, which revealed that Csm3 binds the crRNA and periodically deforms the crRNA-target duplex by a similar mechanism to that of Cmr4 in the Cmr complex. The model and mutational analysis suggest that the conserved lysine residue of Csm2 is important for target RNA binding, and Csm2 stabilizes the active structure of the Csm complex to facilitate the reaction.

RevDate: 2019-01-14

Swarts DC, M Jinek (2018)

Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a.

Molecular cell pii:S1097-2765(18)30991-2 [Epub ahead of print].

CRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA in cis and non-target single-stranded DNAs (ssDNAs) in trans. To elucidate the molecular basis for both DNase cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that guide RNA-target strand DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded DNase activity. In turn, cis cleavage of double-stranded DNA targets is a result of protospacer adjacent motif (PAM)-dependent DNA duplex unwinding, electrostatic stabilization of the displaced non-target DNA strand, and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent, trans-active state. Together, these results provide a revised model for the molecular mechanisms of both the cis- and the trans-acting DNase activities of Cas12a enzymes, enabling their further exploitation as genome editing tools.

RevDate: 2019-01-14

Bayer K, Jahn MT, Slaby BM, et al (2018)

Marine Sponges as Chloroflexi Hot Spots: Genomic Insights and High-Resolution Visualization of an Abundant and Diverse Symbiotic Clade.

mSystems, 3(6): pii:mSystems00150-18.

Members of the widespread bacterial phylum Chloroflexi can dominate high-microbial-abundance (HMA) sponge microbiomes. In the Sponge Microbiome Project, Chloroflexi sequences amounted to 20 to 30% of the total microbiome of certain HMA sponge genera with the classes/clades SAR202, Caldilineae, and Anaerolineae being the most prominent. We performed metagenomic and single-cell genomic analyses to elucidate the functional gene repertoire of Chloroflexi symbionts of Aplysina aerophoba. Eighteen draft genomes were reconstructed and placed into phylogenetic context of which six were investigated in detail. Common genomic features of Chloroflexi sponge symbionts were related to central energy and carbon converting pathways, amino acid and fatty acid metabolism, and respiration. Clade-specific metabolic features included a massively expanded genomic repertoire for carbohydrate degradation in Anaerolineae and Caldilineae genomes, but only amino acid utilization by SAR202. While Anaerolineae and Caldilineae import cofactors and vitamins, SAR202 genomes harbor genes encoding components involved in cofactor biosynthesis. A number of features relevant to symbiosis were further identified, including CRISPR-Cas systems, eukaryote-like repeat proteins, and secondary metabolite gene clusters. Chloroflexi symbionts were visualized in the sponge extracellular matrix at ultrastructural resolution by the fluorescence in situ hybridization-correlative light and electron microscopy (FISH-CLEM) method. Carbohydrate degradation potential was reported previously for "Candidatus Poribacteria" and SAUL, typical symbionts of HMA sponges, and we propose here that HMA sponge symbionts collectively engage in degradation of dissolved organic matter, both labile and recalcitrant. Thus, sponge microbes may not only provide nutrients to the sponge host, but they may also contribute to dissolved organic matter (DOM) recycling and primary productivity in reef ecosystems via a pathway termed the sponge loop. IMPORTANCEChloroflexi represent a widespread, yet enigmatic bacterial phylum with few cultivated members. We used metagenomic and single-cell genomic approaches to characterize the functional gene repertoire of Chloroflexi symbionts in marine sponges. The results of this study suggest clade-specific metabolic specialization and that Chloroflexi symbionts have the genomic potential for dissolved organic matter (DOM) degradation from seawater. Considering the abundance and dominance of sponges in many benthic environments, we predict that the role of sponge symbionts in biogeochemical cycles is larger than previously thought.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Dzau VJ, McNutt M, C Bai (2018)

Wake-up call from Hong Kong.

Science (New York, N.Y.), 362(6420):1215.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Farooq R, Hussain K, Nazir S, et al (2018)

CRISPR/Cas9; A robust technology for producing genetically engineered plants.

Cellular and molecular biology (Noisy-le-Grand, France), 64(14):31-38.

CRISPR/Cas9 is a technology evolved from modified type II immune system of bacteria and archaea. Exploitation of this bacterial immune system in all eukaryotes including plants may lead to site-specific targeted genome engineering. Genome engineering is objectively utilized to express/silence a trait harbouring gene in the plant genome. In this review, different genetic engineering techniques including classical breeding, RNAi and genetic transformation and synthetic sequence-specific nucleases (zinc finger nucleases; ZFNs and transcription activator-like effector nuclease; TALENs) techniques have been described and compared with advanced genome editing technique CRISPR/Cas9, on the basis of their merits and drawbacks. This revolutionary genome engineering technology has edge over all other approaches because of its simplicity, stability, specificity of the target and multiple genes can be engineered at a time. CRISPR/Cas9 requires only Cas9 endonuclease and single guide RNA, which are directly delivered into plant cells via either vector-mediated stable transformation or transient delivery of ribonucleoproteins (RNPs) and generate double-strand breaks (DSBs) at target site. These DSBs are further repaired by cell endogenous repairing pathways via HDR or NHEJ. The major advantage of CRISPR/Cas9 system is that engineered plants are considered Non-GM; can be achieved using in vitro expressed RNPs transient delivery. Different variants of Cas9 genes cloned in different plasmid vectors can be used to achieve different objectives of genome editing including double-stranded DNA break, single-stranded break, activate/repress the gene expression. Fusion of Cas9 with fluorescent protein can lead to visualize the expression of the CRISPR/Cas9 system. The applications of this technology in plant genome editing to improve different plant traits are comprehensively described.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Wahiduzzaman M, Karnan S, Ota A, et al (2019)

Establishment and characterization of CRISPR/Cas9-mediated NF2-/- human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma.

Cancer science, 110(1):180-193.

Malignant pleural mesothelioma (MPM), a highly refractory tumor, is currently incurable due to the lack of an early diagnosis method and medication, both of which are urgently needed to improve the survival and/or quality of life of patients. NF2 is a tumor suppressor gene and is frequently mutated in MPM. Using a CRISPR/Cas9 system, we generated an NF2-knockout human mesothelial cell line, MeT-5A (NF2-KO). In NF2-KO cell clones, cell growth, clonogenic activity, migration activity, and invasion activity significantly increased compared with those in NF2-WT cell clones. Complementary DNA microarray analysis clearly revealed the differences in global gene expression profile between NF2-WT and NF2-KO cell clones. Quantitative PCR analysis and western blot analysis showed that the upregulation of fibroblast growth factor receptor 2 (FGFR2) was concomitant with the increases in phosphorylation levels of JNK, c-Jun, and retinoblastoma (Rb) in NF2-KO cell clones. These increases were all abrogated by the exogenous expression of NF2 in the NF2-KO clone. In addition, the disruption of FGFR2 in the NF2-KO cell clone suppressed cell proliferation as well as the phosphorylation levels of JNK, c-Jun, and Rb. Notably, FGFR2 was found to be highly expressed in NF2-negative human mesothelioma tissues (11/12 cases, 91.7%) but less expressed in NF2-positive tissues. Collectively, these findings suggest that NF2 deficiency might play a role in the tumorigenesis of human mesothelium through mediating FGFR2 expression; FGFR2 would be a candidate molecule to develop therapeutic and diagnostic strategies for targeting MPM with NF2 loss.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Chen G, Xiong L, Wang Y, et al (2018)

ITGB1b-Deficient Rare Minnows Delay Grass Carp Reovirus (GCRV) Entry and Attenuate GCRV-Triggered Apoptosis.

International journal of molecular sciences, 19(10):.

Integrin β-1 (ITGB1) is a transmembrane protein belonging to the integrin family and it plays an important role in viral entry. In this study, the itgb1b gene of the rare minnow, Gobiocypris rarus, was cloned and analyzed. To investigate the possible role of itgb1b on grass carp reovirus (GCRV) infection, we generated an ITGB1b-deficient rare minnow (ITGB1b-/-) using the CRISPR/Cas9 system. Following stimulation with GCRV, the survival time of the -ITGB1b-/- rare minnows was extended in comparison to the wild-type minnows. Moreover, the relative copy number of GCRV and the level of clathrin-mediated endocytosis-associated and apoptosis-related gene expression in the ITGB1b-/- rare minnows was significantly lower than that of the wild-type minnows. These results suggested that the absence of itgb1b reduced viral entry efficiency and the expression of apoptosis-related genes. Moreover, the data suggested that itgb1b played an important role in mediating the entry of viruses into the cells via clathrin. Therefore, these findings provide novel insight into the function of itgb1b in the process of GCRV infection.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Serif M, Dubois G, Finoux AL, et al (2018)

One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing.

Nature communications, 9(1):3924.

Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome. Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene. Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells. This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Xiao D, Wang H, Hao L, et al (2018)

The roles of SMYD4 in epigenetic regulation of cardiac development in zebrafish.

PLoS genetics, 14(8):e1007578.

SMYD4 belongs to a family of lysine methyltransferases. We analyzed the role of smyd4 in zebrafish development by generating a smyd4 mutant zebrafish line (smyd4L544Efs*1) using the CRISPR/Cas9 technology. The maternal and zygotic smyd4L544Efs*1 mutants demonstrated severe cardiac malformations, including defects in left-right patterning and looping and hypoplastic ventricles, suggesting that smyd4 was critical for heart development. Importantly, we identified two rare SMYD4 genetic variants in a 208-patient cohort with congenital heart defects. Both biochemical and functional analyses indicated that SMYD4(G345D) was pathogenic. Our data suggested that smyd4 functions as a histone methyltransferase and, by interacting with HDAC1, also serves as a potential modulator for histone acetylation. Transcriptome and bioinformatics analyses of smyd4L544Efs*1 and wild-type developing hearts suggested that smyd4 is a key epigenetic regulator involved in regulating endoplasmic reticulum-mediated protein processing and several important metabolic pathways in developing zebrafish hearts.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Torbey P, Thierion E, Collombet S, et al (2018)

Cooperation, cis-interactions, versatility and evolutionary plasticity of multiple cis-acting elements underlie krox20 hindbrain regulation.

PLoS genetics, 14(8):e1007581.

Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Callaway E (2018)

Controversial CRISPR 'gene drives' tested in mammals for the first time.

Nature, 559(7713):164.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Iyer V, Boroviak K, Thomas M, et al (2018)

No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice.

PLoS genetics, 14(7):e1007503.

CRISPR-Cas9 technologies have transformed genome-editing of experimental organisms and have immense therapeutic potential. Despite significant advances in our understanding of the CRISPR-Cas9 system, concerns remain over the potential for off-target effects. Recent studies have addressed these concerns using whole-genome sequencing (WGS) of gene-edited embryos or animals to search for de novo mutations (DNMs), which may represent candidate changes introduced by poor editing fidelity. Critically, these studies used strain-matched, but not pedigree-matched controls and thus were unable to reliably distinguish generational or colony-related differences from true DNMs. Here we used a trio design and whole genome sequenced 8 parents and 19 embryos, where 10 of the embryos were mutagenised with well-characterised gRNAs targeting the coat colour Tyrosinase (Tyr) locus. Detailed analyses of these whole genome data allowed us to conclude that if CRISPR mutagenesis were causing SNV or indel off-target mutations in treated embryos, then the number of these mutations is not statistically distinguishable from the background rate of DNMs occurring due to other processes.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Zimmermann M, Murina O, Reijns MAM, et al (2018)

CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.

Nature, 559(7713):285-289.

The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1-4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Xie S, Cooley A, Armendariz D, et al (2018)

Frequent sgRNA-barcode recombination in single-cell perturbation assays.

PloS one, 13(6):e0198635.

Simultaneously detecting CRISPR-based perturbations and induced transcriptional changes in the same cell is a powerful approach to unraveling genome function. Several lentiviral approaches have been developed, some of which rely on the detection of distally located genetic barcodes as an indirect proxy of sgRNA identity. Since barcodes are often several kilobases from their corresponding sgRNAs, viral recombination-mediated swapping of barcodes and sgRNAs is feasible. Using a self-circularization-based sgRNA-barcode library preparation protocol, we estimate the recombination rate to be ~50% and we trace this phenomenon to the pooled viral packaging step. Recombination is random, and decreases the signal-to-noise ratio of the assay. Our results suggest that alternative approaches can increase the throughput and sensitivity of single-cell perturbation assays.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Qian P, Wang X, Yang Z, et al (2018)

A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing.

Molecular and biochemical parasitology, 222:21-28.

The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Nitika , AW Truman (2018)

Endogenous epitope tagging of heat shock protein 70 isoform Hsc70 using CRISPR/Cas9.

Cell stress & chaperones, 23(3):347-355.

Heat shock protein 70 (Hsp70) is an evolutionarily well-conserved molecular chaperone involved in several cellular processes such as folding of proteins, modulating protein-protein interactions, and transport of proteins across the membrane. Binding partners of Hsp70 (known as "clients") are identified on an individual basis as researchers discover their particular protein of interest binds to Hsp70. A full complement of Hsp70 interactors under multiple stress conditions remains to be determined. A promising approach to characterizing the Hsp70 "interactome" is the use of protein epitope tagging and then affinity purification followed by mass spectrometry (AP-MS/MS). AP-MS analysis is a widely used method to decipher protein-protein interaction networks and identifying protein functions. Conventionally, the proteins are overexpressed ectopically which interferes with protein complex stoichiometry, skewing AP-MS/MS data. In an attempt to solve this issue, we used CRISPR/Cas9-mediated gene editing to integrate a tandem-affinity (TAP) epitope tag into the genomic locus of HSC70. This system offers several benefits over existing expression systems including native expression, no requirement for selection, and homogeneity between cells. This cell line, freely available to chaperone researchers, will aid in small and large-scale protein interaction studies as well as the study of biochemical activities and structure-function relationships of the Hsc70 protein.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Jiang C, Mei M, Li B, et al (2017)

A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo.

Cell research, 27(3):440-443.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Zhou Y, Wang P, Tian F, et al (2017)

Painting a specific chromosome with CRISPR/Cas9 for live-cell imaging.

Cell research, 27(2):298-301.

RevDate: 2019-01-14
CmpDate: 2019-01-14

Liu X, Zhang Y, Cheng C, et al (2017)

CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells.

Cell research, 27(1):154-157.

RevDate: 2019-01-12

Schuster JA, Vogel RF, MA Ehrmann (2019)

Characterization and distribution of CRISPR-Cas systems in Lactobacillus sakei.

Archives of microbiology pii:10.1007/s00203-019-01619-x [Epub ahead of print].

Clustered regularly interspaced palindromic repeats (CRISPR)-Cas (CRISPR-associated) structures, known as prokaryotes 'immune system', have been successfully applied for genetic engineering and genotyping purposes for a variety of microorganisms. Here we investigated 50 Lactobacillus (L.) sakei genomes and found 13 of them as CRISPR-Cas positive. The majority of positive genomes contain type II-A system, which appears to be widespread across food born lactic acid bacteria. However, a type II-C system with low similarity in Cas protein sequence to related II-C structures is rarely present in the genomes. We depicted a correlation between prophages integrated in the genomes and the presence/absence of CRISPR-Cas systems and identified the novel protospacer adjunction motifs (PAMs) (a/g)AAA for the II-A and (g/a)(c/t)AC for the II-C system including the corresponding tracrRNAs, creating the basis for the development of new Cas-mediated genome editing tools. Moreover, we performed a PCR screening for 81 selected L. sakei isolates and identified 25 (31%) isolates as CRISPR-Cas positive with hypervariable spacer content. Comparative sequence analysis of 33 repeat-spacer arrays resulted in 18 CRISPR genotypes, revealing insights into evolutionary relationships between different strains and illustrating possible applications for the research and development of starter cultures, e.g., the usage for strain differentiation in assertiveness experiments or the development of bacteriophage-resistant strains.

RevDate: 2019-01-11

Oakes BL, Fellmann C, Rishi H, et al (2019)

CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification.

Cell, 176(1-2):254-267.e16.

The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Lee SH, Kim S, JK Hur (2018)

CRISPR and Target-Specific DNA Endonucleases for Efficient DNA Knock-in in Eukaryotic Genomes.

Molecules and cells, 41(11):943-952.

The discovery and mechanistic understanding of target-specific genome engineering technologies has led to extremely effective and specific genome editing in higher organisms. Target-specific genetic modification technology is expected to have a leading position in future gene therapy development, and has a ripple effect on various basic and applied studies. However, several problems remain and hinder efficient and specific editing of target genomic loci. The issues are particularly critical in precise targeted insertion of external DNA sequences into genomes. Here, we discuss some recent efforts to overcome such problems and present a perspective of future genome editing technologies.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Nachmanson D, Lian S, Schmidt EK, et al (2018)

Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS).

Genome research, 28(10):1589-1599.

Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Sun Q, Lin L, Liu D, et al (2018)

CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L.

International journal of molecular sciences, 19(9):.

Targeted genome editing is a desirable means of basic science and crop improvement. The clustered, regularly interspaced, palindromic repeat (CRISPR)/Cas9 (CRISPR-associated 9) system is currently the simplest and most commonly used system in targeted genomic editing in plants. Single and multiplex genome editing in plants can be achieved under this system. In Arabidopsis, AtWRKY11 and AtWRKY70 genes were involved in JA- and SA-induced resistance to pathogens, in rapeseed (Brassica napus L.), BnWRKY11 and BnWRKY70 genes were found to be differently expressed after inoculated with the pathogenic fungus, Sclerotinia sclerotiorum (Lib.) de Bary. In this study, two Cas9/sgRNA constructs targeting two copies of BnWRKY11 and four copies of BnWRKY70 were designed to generate BnWRKY11 and BnWRKY70 mutants respectively. As a result, twenty-two BnWRKY11 and eight BnWRKY70 independent transformants (T₀) were obtained, with the mutation ratios of 54.5% (12/22) and 50% (4/8) in BnWRKY11 and BnWRKY70 transformants respectively. Eight and two plants with two copies of mutated BnWRKY11 and BnWRKY70 were obtained respectively. In T₁ generation of each plant examined, new mutations on target genes were detected with high efficiency. The vast majority of BnWRKY70 mutants showed editing in three copies of BnWRKY70 in examined T₁ plants. BnWRKY70 mutants exhibited enhanced resistance to Sclerotinia, while BnWRKY11 mutants showed no significant difference in Sclerotinia resistance when compared to non-transgenic plants. In addition, plants that overexpressed BnWRKY70 showed increased sensitivity when compared to non-transgenic plants. Altogether, our results demonstrated that BnWRKY70 may function as a regulating factor to negatively control the Sclerotinia resistance and CRISPR/Cas9 system could be used to generate germplasm in B. napus with high resistance against Sclerotinia.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Alper HS, CL Beisel (2018)

Advances in CRISPR Technologies for Microbial Strain Engineering.

Biotechnology journal, 13(9):e1800460.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Liu K, Yu W, Tang M, et al (2018)

A dual genetic tracing system identifies diverse and dynamic origins of cardiac valve mesenchyme.

Development (Cambridge, England), 145(18): pii:dev.167775.

In vivo genomic engineering is instrumental for studying developmental biology and regenerative medicine. Development of novel systems with more site-specific recombinases (SSRs) that complement with the commonly used Cre-loxP would be valuable for more precise lineage tracing and genome editing. Here, we introduce a new SSR system via Nigri-nox. By generating tissue-specific Nigri knock-in and its responding nox reporter mice, we show that the Nigri-nox system works efficiently in vivo by targeting specific tissues. As a new orthogonal system to Cre-loxP, Nigri-nox provides an additional control of genetic manipulation. We also demonstrate how the two orthogonal systems Nigri-nox and Cre-loxP could be used simultaneously to map the cell fate of two distinct developmental origins of cardiac valve mesenchyme in the mouse heart, providing dynamics of cellular contribution from different origins for cardiac valve mesenchyme during development. This work provides a proof-of-principle application of the Nigri-nox system for in vivo mouse genomic engineering. Coupled with other SSR systems, Nigri-nox would be valuable for more precise delineation of origins and cell fates during development, diseases and regeneration.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Ryu N, Kim MA, Park D, et al (2018)

Effective PEI-mediated delivery of CRISPR-Cas9 complex for targeted gene therapy.

Nanomedicine : nanotechnology, biology, and medicine, 14(7):2095-2102.

The-state-of-art CRISPR/Cas9 is one of the most powerful among the approaches being developed to rescue fundamental causes of gene-based inheritable diseases. Several strategies for delivering such genome editing materials have been developed, but the safety, efficacy over time, cost of production, and gene size limitations are still under debate and must be addressed to further improve applications. In this study, we evaluated branched forms of the polyethylenimine (PEI) - branched PEI 25 kDa (BPEI-25K) - and found that it could efficiently deliver CRISPR/Cas9 plasmids. Plasmid DNA expressing both guide RNA and Cas9 to target the Slc26a4 locus was successfully delivered into Neuro2a cells and meditated genome editing within the targeted locus. Our results demonstrated that BPEI-25K is a promising non-viral vector to deliver the CRISPR/Cas9 system in vitro to mediate targeted gene therapy, and these findings contribute to an understanding of CRISPR/Cas9 delivery that may enable development of successful in vivo techniques.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Lu IL, Chen C, Tung CY, et al (2018)

Identification of genes associated with cortical malformation using a transposon-mediated somatic mutagenesis screen in mice.

Nature communications, 9(1):2498.

Mutations in genes involved in the production, migration, or differentiation of cortical neurons often lead to malformations of cortical development (MCDs). However, many genetic mutations involved in MCD pathogenesis remain unidentified. Here we developed a genetic screening paradigm based on transposon-mediated somatic mutagenesis by in utero electroporation and the inability of mutant neuronal precursors to migrate to the cortex and identified 33 candidate MCD genes. Consistent with the screen, several genes have already been implicated in neural development and disorders. Functional disruption of the candidate genes by RNAi or CRISPR/Cas9 causes altered neuronal distributions that resemble human cortical dysplasia. To verify potential clinical relevance of these candidate genes, we analyzed somatic mutations in brain tissue from patients with focal cortical dysplasia and found that mutations are enriched in these candidate genes. These results demonstrate that this approach is able to identify potential mouse genes involved in cortical development and MCD pathogenesis.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Cao M, Gao M, Ploessl D, et al (2018)

CRISPR-Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis.

Biotechnology journal, 13(9):e1700598.

Scheffersomyces stipitis, renowned for its native xylose-utilizing capacity, has recently demonstrated its potential in producing health-promoting shikimate pathway derivatives. However, its broader application is hampered by the low transformation efficiency and the lack of genetic engineering tools to enable sophisticated genomic manipulations. S. stipitis employs the predominant non-homologous end joining (NHEJ) mechanism for repairing DNA double-strand breaks (DSB), which is less desired due to its incompetence in achieving precise genome editing. Using CRISPR technology, here a ku70Δku80Δ deficient strain in which homologous recombination (HR)-based genome editing appeared dominant for the first time in S. stipitis is constructed. To build all essential tools for efficiently manipulating this highly promising nonconventional microbial host, the gene knockdown tool is also established, and repression efficiency is improved by incorporating a transcriptional repressor Mxi1 into the CRISPR-dCas9 platform. All these results are obtained with the improved transformation efficiency, which is 191-fold higher than that obtained with the traditional parameters used in yeast transformation. This work paves the way for advancing a new microbial chassis and provides a guideline for developing efficient CRISPR tools in other nonconventional yeasts.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Stratigopoulos G, De Rosa MC, LeDuc CA, et al (2018)

DMSO increases efficiency of genome editing at two non-coding loci.

PloS one, 13(6):e0198637.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) has become the tool of choice for genome editing. Despite the fact that it has evolved as a highly efficient means to edit/replace coding sequence, CRISPR/Cas9 efficiency for "clean" editing of non-coding DNA remains low. We set out to introduce a single base-pair substitution in two intronic SNPs at the FTO locus without altering nearby non-coding sequence. Substitution efficiency increased up to 10-fold by treatment of human embryonic stem cells (ESC) with non-toxic levels of DMSO (1%) before CRISPR/Cas9 delivery. Treatment with DMSO did not result in CRISPR/Cas9 off-target effects or compromise the chromosomal stability of the ESC. Twenty-four hour treatment of human ESC with DMSO before CRISPR/Cas9 delivery may prove a simple means to increase editing efficiency of non-coding DNA without incorporation of undesirable mutations.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Zhao Y, Li L, Zheng G, et al (2018)

CRISPR/dCas9-Mediated Multiplex Gene Repression in Streptomyces.

Biotechnology journal, 13(9):e1800121.

Streptomycetes are Gram-positive bacteria with the capacity to produce copious bioactive secondary metabolites, which are the main source of medically and industrially relevant drugs. However, genetic manipulation of Streptomyces strains is much more difficult than other model microorganisms like Escherichia coli and Saccharomyces cerevisiae. Recently, CRISPR/Cas9 or dCas9-mediated genetic manipulation tools have been developed and facilitated Streptomyces genome editing. However, till now, CRISPR/dCas9-based interference system (CRISPRi) is only designed to repress single gene expression. Herein, the authors developed a novel CRISPRi system for multiplex gene repression in the model strain Streptomyces coelicolor. In this system, the integrative plasmid pSET152 is used as the backbone for the expression of the dCas9/sgRNA complex and both dCas9 and sgRNAs are designed to be under the control of constitutive promoters. Using the integrative CRISPRi system, the authors achieved efficient repression of multiple genes simultaneously; the mRNA levels of four targets are reduced to 2-32% of the control. Furthermore, it is successfully employed for functional gene screening, and an orphan response regulator (RR) (encoded by SCO2013) containing an RNA-binding ANTAR domain is identified being involved in bacterial growth. Collectively, this integrative CRISPRi system is very effective for multiplex gene repression in S. coelicolor, which could be extended to other Streptomyces strains for functional gene screening as well as for metabolic engineering.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Gao D, Smith S, Spagnuolo M, et al (2018)

Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

Biotechnology journal, 13(9):e1700590.

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Grenier A, Sujobert P, Olivier S, et al (2018)

Knockdown of Human AMPK Using the CRISPR/Cas9 Genome-Editing System.

Methods in molecular biology (Clifton, N.J.), 1732:171-194.

AMP-activated protein kinase (AMPK) is a critical energy sensor, regulating signaling networks involved in pathology including metabolic diseases and cancer. This increasingly recognized role of AMPK has prompted tremendous research efforts to develop new pharmacological AMPK activators. To precisely study the role of AMPK, and the specificity and activity of AMPK activators in cellular models, genetic AMPK inactivating tools are required. We report here methods for genetic inactivation of AMPK α1/α2 catalytic subunits in human cell lines by the CRISPR/Cas9 technology, a recent breakthrough technique for genome editing.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Xin Y, C Duan (2018)

Microinjection of Antisense Morpholinos, CRISPR/Cas9 RNP, and RNA/DNA into Zebrafish Embryos.

Methods in molecular biology (Clifton, N.J.), 1742:205-211.

In this chapter, we describe a stepwise protocol of microinjection. Using this method, antisense morpholinos, CRISPR-Cas9 ribonucleoprotein complexes, capped mRNA, and DNA can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. This protocol can also be adapted for microinjection in other fish and amphibian species.

RevDate: 2019-01-11
CmpDate: 2019-01-11

Sun S, Xiao J, Huo J, et al (2018)

Targeting ectodysplasin promotor by CRISPR/dCas9-effector effectively induces the reprogramming of human bone marrow-derived mesenchymal stem cells into sweat gland-like cells.

Stem cell research & therapy, 9(1):8.

BACKGROUND: Patients with a deep burn injury are characterized by losing the function of perspiration and being unable to regenerate the sweat glands. Because of their easy accession, multipotency, and lower immunogenicity, bone marrow-derived mesenchymal stem cells (BM-MSCs) represent as an ideal biological source for cell therapy. The aim of this study was to identify whether targeting the promotor of ectodysplasin (EDA) by CRISPR/dCas9-effector (dCas9-E) could induce the BM-MSCs to differentiate into sweat gland-like cells (SGCs).

METHODS: Activation of EDA transcription in BM-MSCs was attained by transfection of naive BM-MSCs with the lenti-CRISPR/dCas9-effector and single-guide RNAs (sgRNAs). The impact of dCas9-E BM-MSCs on the formation of SGCs and repair of burn injury was identified and evaluated both in vitro and in a mouse model.

RESULTS: After transfection with sgRNA-guided dCas9-E, the BM-MSCs acquired significantly higher transcription and expression of EDA by doxycycline (Dox) induction. Intriguingly, the specific markers (CEA, CK7, CK14, and CK19) of sweat glands were also positive in the transfected BM-MSCs, suggesting that EDA plays a critical role in promoting BM-MSC differentiation into sweat glands. Furthermore, when the dCas9-E BM-MSCs with Dox induction were implanted into a wound in a laboratory animal model, iodine-starch perspiration tests revealed that the treated paws were positive for perspiration, while the paws treated with saline showed a negative manifestation. For the regulatory mechanism, the expression of downstream genes of NF-κB (Shh and cyclin D1) was also enhanced accordingly.

CONCLUSIONS: These results suggest that EDA is a pivotal factor for sweat gland regeneration from BM-MSCs and may also offer a new approach for destroyed sweat glands and extensive deep burns.

RevDate: 2019-01-10

Hai L, Szwarc MM, Lanza DG, et al (2019)

Using CRISPR/Cas9 engineering to generate a mouse with a conditional knockout allele for the promyelocytic leukemia zinc finger transcription factor.

Genesis (New York, N.Y. : 2000) [Epub ahead of print].

The promyelocytic leukemia zinc finger (PLZF) transcription factor mediates a wide-range of biological processes. Accordingly, perturbation of PLZF function results in a myriad of physiologic defects, the most conspicuous of which is abnormal skeletal patterning. Although whole body knockout of Plzf in the mouse (PlzfKO) has significantly expanded our understanding of Plzf function in vivo, a conditional knockout mouse model that enables tissue or cell-type specific ablation of Plzf has not been developed. Therefore, we used CRISPR/Cas 9 gene editing to generate a mouse model in which exon 2 of the murine Plzf gene is specifically flanked (or floxed) by LoxP sites (Plzff/f). Crossing our Plzff/f mouse with a global cre-driver mouse to generate the Plzfd/d bigenic mouse, we demonstrate that exon 2 of the Plzf gene is ablated in the Plzfd/d bigenic. Similar to the previously reported PlzfKO mouse, the Plzfd/d mouse exhibits a severe defect in skeletal patterning of the hindlimb, indicating that the Plzff/f mouse functions as designed. Therefore, studies in this short technical report demonstrate that the Plzff/f mouse will be useful to investigators who wish to explore the role of the Plzf transcription factor in a specific tissue or cell-type. This article is protected by copyright. All rights reserved.

RevDate: 2019-01-10
CmpDate: 2019-01-10

Libby AR, Joy DA, So PL, et al (2018)

Spatiotemporal mosaic self-patterning of pluripotent stem cells using CRISPR interference.

eLife, 7:.

Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our in vitro system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference. Mosaic knockdown of ROCK1 or CDH1 resulted in differential patterning within hiPSC colonies due to cellular self-organization, while retaining an epithelial pluripotent phenotype. Knockdown induction stimulates a transient wave of differential gene expression within the mixed populations that stabilized in coordination with observed self-organization. Mosaic patterning enables genetic interrogation of emergent multicellular properties, which can facilitate better understanding of the molecular pathways that regulate symmetry-breaking during morphogenesis.

RevDate: 2019-01-10
CmpDate: 2019-01-10

Montoliu L, CBA Whitelaw (2018)

Unexpected mutations were expected and unrelated to CRISPR-Cas9 activity.

Transgenic research, 27(4):315-319.

The scientific journal Nature Methods have just retracted a publication that reported numerous unexpected mutations after a CRISPR-Cas9 experiment based on collecting whole genome sequencing information from one control and two experimental genome edited mice. In the intervening 10 months since publication the data presented have been strongly contested and criticized by the scientific and biotech communities, through publications, open science channels and social networks. The criticism focused on the animal used as control, which was derived from the same mouse strain as the experimental individuals but from an unrelated sub-colony, hence control and experimental mice were genetically divergent. The most plausible explanation for the vast majority of the reported unexpected mutations were the expected underlying genetic polymorphisms that normally accumulate in two different colonies of the same mouse strain which occur as a result of spontaneous mutations and genetic drift. Therefore, the reported mutations were most likely not related to CRISPR-Cas9 activity.

RevDate: 2019-01-10
CmpDate: 2019-01-10

D'Ambrosio C, Stigliani AL, G Giorio (2018)

CRISPR/Cas9 editing of carotenoid genes in tomato.

Transgenic research, 27(4):367-378.

CRISPR/Cas9 technology is rapidly spreading as genome editing system in crop breeding. The efficacy of CRISPR/Cas9 in tomato was tested on Psy1 and CrtR-b2, two key genes of carotenoid biosynthesis. Carotenoids are plant secondary metabolites that must be present in the diet of higher animals because they exert irreplaceable functions in important physiological processes. Psy1 and CrtR-b2 were chosen because their impairment is easily detectable as a change of fruit or flower color. Two CRISPR/Cas9 constructs were designed to target neighboring sequences on the first exon of each gene. Thirty-four out of forty-nine (69%) transformed plants showed the expected loss-of-function phenotypes due to the editing of both alleles of a locus. However, by including the seven plants edited only at one of the two homologs and showing a normal phenotype, the editing rate reaches the 84%. Although none chimeric phenotype was observed, the cloning of target region amplified fragments revealed that in the 40% of analyzed DNA samples were present more than two alleles. As concerning the type of mutation, it was possible to identify 34 new different alleles across the four transformation experiments. The sequence characterization of the CRISPR/Cas9-induced mutations showed that the most frequent repair errors were the insertion and the deletion of one base. The results of this study prove that the CRISPRCas9 system can be an efficient and quick method for the generation of useful mutations in tomato to be implemented in breeding programs.

RevDate: 2019-01-10
CmpDate: 2019-01-10

Zhang Z, Ursin R, Mahapatra S, et al (2018)

CRISPR/CAS9 ablation of individual miRNAs from a miRNA family reveals their individual efficacies for regulating cardiac differentiation.

Mechanisms of development, 150:10-20.

Although it is well understood that genetic mutations, chromosomal abnormalities, and epigenetic miscues can cause congenital birth defects, many defects are still labeled idiopathic, meaning their origin is not yet understood. microRNAs are quickly entering the causal fray of developmental defects. miRNAs use a 7-8 base-pair seed sequence to target a corresponding sequence on one or multiple mRNAs resulting in rapid down-regulation of translation. miRNAs can also control protein 'amounts' in cells. As a result if miRNAs are over or under expressed during development protein homeostasis can be compromised resulting in defects in the development of organ systems. Here, we show that during differentiation of embryonic stem cells, individual miRNAs that reside in the miRNA17 family (composed of 14 miRNAs) do not share the same function even though they have the same seed sequence. The advent of CRISPR/CAS9 technology has not only yielded a true observation of individual miRNA function, it has also reconnected advanced molecular biology approaches to classical cell biology approaches such as gene rescue. We show that miRNA106a and to a lesser extent miR17 and 93 target the cardiac suppressor gene Fog2, which specifically suppress Gata-4 and Coup-TF2. However, when each miRNA is knocked out, we find that their targeting efficacies for Fog2 differ resulting in varying degrees of cardiac differentiation.

RevDate: 2019-01-10
CmpDate: 2019-01-10

Heinze SD, Kohlbrenner T, Ippolito D, et al (2017)

CRISPR-Cas9 targeted disruption of the yellow ortholog in the housefly identifies the brown body locus.

Scientific reports, 7(1):4582.

The classic brown body (bwb) mutation in the housefly Musca domestica impairs normal melanization of the adult cuticle. In Drosophila melanogaster, a reminiscent pigmentation defect results from mutations in the yellow gene encoding dopachrome conversion enzyme (DCE). Here, we demonstrate that the bwb locus structurally and functionally represents the yellow ortholog of Musca domestica, MdY. In bwb Musca strains, we identified two mutant MdY alleles that contain lesions predicted to result in premature truncation of the MdY open reading frame. We targeted wildtype MdY by CRISPR-Cas9 RNPs and generated new mutant alleles that fail to complement existing MdY alleles, genetically confirming that MdY is the bwb locus. We further found evidence for Cas9-mediated interchromosomal recombination between wildtype and mutant bwb alleles. Our work resolves the molecular identity of the classic bwb mutation in Musca domestica and establishes the feasibility of Cas9-mediated genome editing in the Musca model.

RevDate: 2019-01-10
CmpDate: 2019-01-10

Van der Hoek KH, Eyre NS, Shue B, et al (2017)

Viperin is an important host restriction factor in control of Zika virus infection.

Scientific reports, 7(1):4475.

Zika virus (ZIKV) infection has emerged as a global health threat and infection of pregnant women causes intrauterine growth restriction, spontaneous abortion and microcephaly in newborns. Here we show using biologically relevant cells of neural and placental origin that following ZIKV infection, there is attenuation of the cellular innate response characterised by reduced expression of IFN-β and associated interferon stimulated genes (ISGs). One such ISG is viperin that has well documented antiviral activity against a wide range of viruses. Expression of viperin in cultured cells resulted in significant impairment of ZIKV replication, while MEFs derived from CRISPR/Cas9 derived viperin-/- mice replicated ZIKV to higher titers compared to their WT counterparts. These results suggest that ZIKV can attenuate ISG expression to avoid the cellular antiviral innate response, thus allowing the virus to replicate unchecked. Moreover, we have identified that the ISG viperin has significant anti-ZIKV activity. Further understanding of how ZIKV perturbs the ISG response and the molecular mechanisms utilised by viperin to suppress ZIKV replication will aid in our understanding of ZIKV biology, pathogenesis and possible design of novel antiviral strategies.

RevDate: 2019-01-09

Domenici G, Aurrekoetxea-Rodríguez I, Simões BM, et al (2019)

A Sox2-Sox9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells.

Oncogene pii:10.1038/s41388-018-0656-7 [Epub ahead of print].

Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer.

RevDate: 2019-01-09

Homanics GE (2019)

Gene-edited CRISPy Critters for alcohol research.

Alcohol (Fayetteville, N.Y.), 74:11-19.

Genetically engineered animals are powerful tools that have provided invaluable insights into mechanisms of alcohol action and alcohol-use disorder. Traditionally, production of gene-targeted animals was a tremendously expensive, time consuming, and technically demanding undertaking. However, the recent advent of facile methods for editing the genome at very high efficiency is revolutionizing how these animals are made. While pioneering approaches to create gene-edited animals first used zinc finger nucleases and subsequently used transcription activator-like effector nucleases, these approaches have been largely supplanted in an extremely short period of time with the recent discovery and precocious maturation of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. CRISPR uses a short RNA sequence to guide a non-specific CRISPR-associated nuclease (Cas) to a precise, single location in the genome. Because the CRISPR/Cas system can be cheaply, rapidly, and easily reprogrammed to target nearly any genomic locus of interest simply by recoding the sequence of the guide RNA, this gene-editing system has been rapidly adopted by numerous labs around the world. With CRISPR/Cas, it is now possible to perform gene editing directly in early embryos from every species of animals that is of interest to the alcohol field. Techniques have been developed that enable the rapid production of animals in which a gene has been inactivated (knockout) or modified to harbor specific nucleotide changes (knockins). This system has also been used to insert specific DNA sequences such as reporter or recombinase genes into specific loci of interest. Genetically engineered animals created with the CRISPR/Cas system (CRISPy Critters) are being produced at an astounding pace. Animal production is no longer a significant bottleneck to new discoveries. CRISPy animal studies are just beginning to appear in the alcohol literature, but their use is expected to explode in the near future. CRISPy mice, rats, and other model organisms are sure to facilitate advances in our understanding of alcohol-use disorder.

RevDate: 2019-01-09
CmpDate: 2019-01-09

Chen F, Yang P, J Zhu (2018)

[Construction and application of PGRN and Rev-erbβ double genes knockout HEK293 cell lines].

Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 34(10):1679-1692.

In order to study the molecular mechanism and physiological significance of the interaction between PGRN and Rev-erbβ, the PGRN gene in HEK293 (Rev-erbβ-/-) marked as C3-6 cell lines was knocked out by CRISPR/Cas9 system to generate the Rev-erbβ and PGRN double genes knockout HEK293 cell lines. First, four sgRNAs were designed for PGRN gene, and PGRN sgRNA2 and sgRNA3 with the higher activity were used to construct the Lentiviral vector, pLenti/CMV-Loxp-Cas9-sgRNA2-U6-sgRNA3-U6-Loxp-EF1α-Puro. Then, the lentivirus vector carrying Cas9 and double PGRN sgRNA were used to infect HEK293 C3-6 cells. Through drug screening, cloning and sequencing, we obtained the monoclonal HEK293 (Rev-erbβ-/-; PGRN-/-) marked as C3-6/23 cell lines. Using qRT-PCR and Western blotting, we detected PGRN mRNA and protein expression in C3-6/23 cell lines. Finally, genetic complementation was used to study the effect of PGRN-mediated Rev-erbβ on the regulation of the target gene promoter transcriptional activity in the C3-6/23 cell lines. In HEK293 C3-6/23 cell lines, the two DNA chains of PGRN gene were both deletion mutagenesis, and the expression mRNA and protein of PGRN did not reach the detection level. At the same time, the interaction between PGRN and Rev-erbβ enhanced the regulation of Rev-erbβ on the transcription of target gene promoter in the cell lines. Using CRISPR/Cas9 system, we successfully constructed the double knockout HEK293 (Rev-erbβ-/-; PGRN-/-) monoclonal cell lines. The study found that PGRN could affect Rev-erbβ on the regulation of target gene promoter transcription in the C3-6/23 cell lines; however, the mechanism of PGRN involvement in mediating Rev-erbβ in transcriptional regulation remains to be further studied.

RevDate: 2019-01-09
CmpDate: 2019-01-09

Amoasii L, Hildyard JCW, Li H, et al (2018)

Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

Science (New York, N.Y.), 362(6410):86-91.

Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational "hotspot" in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD.

RevDate: 2019-01-09
CmpDate: 2019-01-09

Ivanov Kavkova E, Blöchl C, R Tenhaken (2019)

The Myo-inositol pathway does not contribute to ascorbic acid synthesis.

Plant biology (Stuttgart, Germany), 21 Suppl 1:95-102.

Ascorbic acid (AsA) biosynthesis in plants predominantly occurs via a pathway with d-mannose and l-galactose as intermediates. One alternative pathway for AsA synthesis, which is similar to the biosynthesis route in mammals, is controversially discussed for plants. Here, myo-inositol is cleaved to glucuronic acid and then converted via l-gulonate to AsA. In contrast to animals, plants have an effective recycling pathway for glucuronic acid, being a competitor for the metabolic rate. Recycling involves a phosphorylation at C1 by the enzyme glucuronokinase. Two previously described T-DNA insertion lines in the gene coding for glucuronokinase1 show wild type-like expression levels of the mRNA in our experiments and do not accumulate glucuronic acid in labelling experiments disproving that these lines are true knockouts. As suitable T-DNA insertion lines were not available, we generated frameshift mutations in the major expressed isoform glucuronokinase1 (At3g01640) to potentially redirect metabolites to AsA. However, radiotracer experiments with 3 H-myo-inositol revealed that the mutants in glucuronokinase1 accumulate only glucuronic acid and incorporate less metabolite into cell wall polymers. AsA was not labelled, suggesting that Arabidopsis cannot efficiently use glucuronic acid for AsA biosynthesis. All four mutants in glucuronokinase as well as the wild type have the same level of AsA in leaves.

RevDate: 2019-01-09
CmpDate: 2019-01-09

Reber S, Mechtersheimer J, Nasif S, et al (2018)

CRISPR-Trap: a clean approach for the generation of gene knockouts and gene replacements in human cells.

Molecular biology of the cell, 29(2):75-83.

CRISPR/Cas9-based genome editing offers the possibility to knock out almost any gene of interest in an affordable and simple manner. The most common strategy is the introduction of a frameshift into the open reading frame (ORF) of the target gene which truncates the coding sequence (CDS) and targets the corresponding transcript for degradation by nonsense-mediated mRNA decay (NMD). However, we show that transcripts containing premature termination codons (PTCs) are not always degraded efficiently and can generate C-terminally truncated proteins which might have residual or dominant negative functions. Therefore, we recommend an alternative approach for knocking out genes, which combines CRISPR/Cas9 with gene traps (CRISPR-Trap) and is applicable to ∼50% of all spliced human protein-coding genes and a large subset of lncRNAs. CRISPR-Trap completely prevents the expression of the ORF and avoids expression of C-terminal truncated proteins. We demonstrate the feasibility of CRISPR-Trap by utilizing it to knock out several genes in different human cell lines. Finally, we also show that this approach can be used to efficiently generate gene replacements allowing for modulation of protein levels for otherwise lethal knockouts (KOs). Thus, CRISPR-Trap offers several advantages over conventional KO approaches and allows for generation of clean CRISPR/Cas9-based KOs.

RevDate: 2019-01-08

Berges M, Michel AM, Lassek C, et al (2018)

Iron Regulation in Clostridioides difficile.

Frontiers in microbiology, 9:3183.

The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen Clostridioides difficile were investigated by a combined RNA sequencing, proteomic, metabolomic and electron microscopy approach. At high iron conditions (15 μM) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron and siderophore transporter genes were induced by Fur during low iron (0.2 μM) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some intermediates (phenylpyruvate, 2-oxo-isocaproate, 3-hydroxy-butyryl-CoA, crotonyl-CoA) accumulated, while end products like isocaproate and butyrate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were enhanced, most likely to replace the missing ferredoxins. Proline reductase (prd), the corresponding ion pumping RNF complex (rnf) and the reaction product 5-aminovalerate were significantly enhanced. An ATP forming ATPase (atpCDGAHFEB) of the F0F1-type was induced while the formation of a ATP-consuming, proton-pumping V-type ATPase (atpDBAFCEKI) was decreased. The [Fe-S] enzyme-dependent pyruvate formate lyase (pfl), formate dehydrogenase (fdh) and hydrogenase (hyd) branch of glucose utilization and glycogen biosynthesis (glg) were significantly reduced, leading to an accumulation of glucose and pyruvate. The formation of [Fe-S] enzyme carbon monoxide dehydrogenase (coo) was inhibited. The fur mutant showed an increased sensitivity to vancomycin and polymyxin B. An intensive remodeling of the cell wall was observed, Polyamine biosynthesis (spe) was induced leading to an accumulation of spermine, spermidine, and putrescine. The fur mutant lost most of its flagella and motility. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of around 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, a significant replacement of iron requiring metabolic pathways and the restructuring of the cell surface for protection during the complex adaptation phase and was only partly directly regulated by Fur.

RevDate: 2019-01-08
CmpDate: 2019-01-08

Koslová A, Kučerová D, Reinišová M, et al (2018)

Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells.

Viruses, 10(11):.

Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.

RevDate: 2019-01-08
CmpDate: 2019-01-08

Yokoyama CC, Baldridge MT, Leung DW, et al (2018)

LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens.

The Journal of biological chemistry, 293(16):6022-6038.

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

RevDate: 2019-01-08
CmpDate: 2019-01-08

Peng R, Wang Y, Feng WW, et al (2018)

CRISPR/dCas9-mediated transcriptional improvement of the biosynthetic gene cluster for the epothilone production in Myxococcus xanthus.

Microbial cell factories, 17(1):15.

BACKGROUND: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites.

RESULTS: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects.

CONCLUSIONS: Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.

RevDate: 2019-01-08
CmpDate: 2019-01-08

Park J, Shin H, Lee SM, et al (2018)

RNA-guided single/double gene repressions in Corynebacterium glutamicum using an efficient CRISPR interference and its application to industrial strain.

Microbial cell factories, 17(1):4.

BACKGROUND: The construction of microbial cell factories requires cost-effective and rapid strain development through metabolic engineering. Recently, RNA-guided CRISPR technologies have been developed for metabolic engineering of industrially-relevant host.

RESULTS: To demonstrate the application of the CRISPR interference (CRISPRi), we developed two-plasmid CRISPRi vectors and applied the CRISPRi in Corynebacterium glutamicum to repress single target genes and double target genes simultaneously. Four-different single genes (the pyc, gltA, idsA, and glgC genes) repressions were successfully performed using the CRISPRi vectors, resulting significant mRNA reductions of the targets compared to a control. Subsequently, the phenotypes for the target gene-repressed strains were analyzed, showing the expected cell growth behaviors with different carbon sources. In addition, double gene repression (the idsA and glgC genes in a different order) by the CRISPRi resulted in an independent gene repression to each target gene simultaneously. To demonstrate an industrial application of the CRISPRi, citrate synthase (CS)-targeting DM1919 (L-lysine producer) strains with a sgRNA-gltA-r showed reduced CS activity, resulting in the improvement of L-lysine yield by 1.39-fold than the parental DM1919 (a lysine producer).

CONCLUSIONS: Single or double gene repression were successfully performed using the CRISPRi vectors and sequence specific sgRNAs. The CRISPRi can be applied for multiplex metabolic engineering to enhanced lysine production and it will promote the further rapid development of microbial cell factories of C. glutamicum.

RevDate: 2019-01-07

Price MA, Cruz R, Baxter S, et al (2019)

CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis.

PloS one, 14(1):e0210121 pii:PONE-D-18-27547.

CRISPR-Cas systems have become widely used across all fields of biology as a genome engineering tool. With its recent demonstration in the Gram positive industrial workhorse Bacillus subtilis, this tool has become an attractive option for rapid, markerless strain engineering of industrial production hosts. Previously described strategies for CRISPR-Cas9 genome editing in B. subtilis have involved chromosomal integrations of Cas9 and single guide RNA expression cassettes, or construction of large plasmids for simultaneous transformation of both single guide RNA and donor DNA. Here we use a flexible, co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%. This allowed multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homolog, M-protease. A novel subtilisin E variant was obtained with increased thermotolerance and activity.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Emmer BT, Hesketh GG, Kotnik E, et al (2018)

The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9.

eLife, 7:.

PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, we apply a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify SURF4, a homologue of the yeast cargo receptor Erv29p, as a primary mediator of PCSK9 secretion in HEK293T cells. The functional contribution of SURF4 to PCSK9 secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. SURF4 was found to localize to the early secretory pathway where it physically interacts with PCSK9. Deletion of SURF4 resulted in ER accumulation and decreased extracellular secretion of PCSK9. These findings support a model in which SURF4 functions as an ER cargo receptor mediating the efficient cellular secretion of PCSK9.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Palin K, Pitkänen E, Turunen M, et al (2018)

Contribution of allelic imbalance to colorectal cancer.

Nature communications, 9(1):3664.

Point mutations in cancer have been extensively studied but chromosomal gains and losses have been more challenging to interpret due to their unspecific nature. Here we examine high-resolution allelic imbalance (AI) landscape in 1699 colorectal cancers, 256 of which have been whole-genome sequenced (WGSed). The imbalances pinpoint 38 genes as plausible AI targets based on previous knowledge. Unbiased CRISPR-Cas9 knockout and activation screens identified in total 79 genes within AI peaks regulating cell growth. Genetic and functional data implicate loss of TP53 as a sufficient driver of AI. The WGS highlights an influence of copy number aberrations on the rate of detected somatic point mutations. Importantly, the data reveal several associations between AI target genes, suggesting a role for a network of lineage-determining transcription factors in colorectal tumorigenesis. Overall, the results unravel the contribution of AI in colorectal cancer and provide a plausible explanation why so few genes are commonly affected by point mutations in cancers.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Li R, Bernau K, Sandbo N, et al (2018)

Pdgfra marks a cellular lineage with distinct contributions to myofibroblasts in lung maturation and injury response.

eLife, 7:.

Pdgfra-expressing (Pdgfra+) cells have been implicated as progenitors in many mesenchymal tissues. To determine lineage potential, we generated PdgfrartTA knockin mice using CRISPR/Cas9. During lung maturation, counter to a prior study reporting that Pdgfra+ cells give rise equally to myofibroblasts and lipofibroblasts, lineage tracing using PdgfrartTA;tetO-cre mice indicated that ~95% of the lineaged cells are myofibroblasts. Genetic ablation of Pdgfra+ cells using PdgfrartTA-driven diphtheria toxin (DTA) led to alveolar simplification, demonstrating that these cells are essential for building the gas exchange surface area. In the adult bleomycin model of lung fibrosis, lineaged cells increased to contribute to pathological myofibroblasts. In contrast, in a neonatal hyperoxia model of bronchopulmonary dysplasia (BPD), lineaged cells decreased and do not substantially contribute to pathological myofibroblasts. Our findings revealed complexity in the behavior of the Pdgfra-lineaged cells as exemplified by their distinct contributions to myofibroblasts in normal maturation, BPD and adult fibrosis.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Pankowicz FP, Barzi M, Kim KH, et al (2018)

Rapid Disruption of Genes Specifically in Livers of Mice Using Multiplex CRISPR/Cas9 Editing.

Gastroenterology, 155(6):1967-1970.e6.

BACKGROUND & AIMS: Despite advances in gene editing technologies, generation of tissue-specific knockout mice is time-consuming. We used CRISPR/Cas9-mediated genome editing to disrupt genes in livers of adult mice in just a few months, which we refer to as somatic liver knockouts.

METHODS: In this system, Fah-/- mice are given hydrodynamic tail vein injections of plasmids carrying CRISPR/Cas9 designed to excise exons in Hpd; the Hpd-edited hepatocytes have a survival advantage in these mice. Plasmids that target Hpd and a separate gene of interest can therefore be used to rapidly generate mice with liver-specific deletion of nearly any gene product.

RESULTS: We used this system to create mice with liver-specific knockout of argininosuccinate lyase, which develop hyperammonemia, observed in humans with mutations in this gene. We also created mice with liver-specific knockout of ATP binding cassette subfamily B member 11, which encodes the bile salt export pump. We found that these mice have a biochemical phenotype similar to that of Abcb11-/- mice. We then used this system to knock out expression of 5 different enzymes involved in drug metabolism within the same mouse.

CONCLUSIONS: This approach might be used to develop new models of liver diseases and study liver functions of genes that are required during development.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Lee JK, Jeong E, Lee J, et al (2018)

Directed evolution of CRISPR-Cas9 to increase its specificity.

Nature communications, 9(1):3048.

The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we develop a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without killing on-target activities in human cells. Unlike other engineered Cas9 variants, Sniper-Cas9 shows WT-level on-target activities with extended or truncated sgRNAs with further reduced off-target activities and works well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Elster D, Tollot M, Schlegelmilch K, et al (2018)

TRPS1 shapes YAP/TEAD-dependent transcription in breast cancer cells.

Nature communications, 9(1):3115.

Yes-associated protein (YAP), the downstream transducer of the Hippo pathway, is a key regulator of organ size, differentiation and tumorigenesis. To uncover Hippo-independent YAP regulators, we performed a genome-wide CRISPR screen that identifies the transcriptional repressor protein Trichorhinophalangeal Syndrome 1 (TRPS1) as a potent repressor of YAP-dependent transactivation. We show that TRPS1 globally regulates YAP-dependent transcription by binding to a large set of joint genomic sites, mainly enhancers. TRPS1 represses YAP-dependent function by recruiting a spectrum of corepressor complexes to joint sites. Loss of TRPS1 leads to activation of enhancers due to increased H3K27 acetylation and an altered promoter-enhancer interaction landscape. TRPS1 is commonly amplified in breast cancer, which suggests that restrained YAP activity favours tumour growth. High TRPS1 activity is associated with decreased YAP activity and leads to decreased frequency of tumour-infiltrating immune cells. Our study uncovers TRPS1 as an epigenetic regulator of YAP activity in breast cancer.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Guo X, Chavez A, Tung A, et al (2018)

High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast.

Nature biotechnology, 36(6):540-546.

Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80-100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, we created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Our strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Bao Z, HamediRad M, Xue P, et al (2018)

Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision.

Nature biotechnology, 36(6):505-508.

We developed a CRISPR-Cas9- and homology-directed-repair-assisted genome-scale engineering method named CHAnGE that can rapidly output tens of thousands of specific genetic variants in yeast. More than 98% of target sequences were efficiently edited with an average frequency of 82%. We validate the single-nucleotide resolution genome-editing capability of this technology by creating a genome-wide gene disruption collection and apply our method to improve tolerance to growth inhibitors.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Roy KR, Smith JD, Vonesch SC, et al (2018)

Multiplexed precision genome editing with trackable genomic barcodes in yeast.

Nature biotechnology, 36(6):512-520.

Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here we describe a CRISPR-Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide-donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA-Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Maass PG, Barutcu AR, Weiner CL, et al (2018)

Inter-chromosomal Contact Properties in Live-Cell Imaging and in Hi-C.

Molecular cell, 69(6):1039-1045.e3.

Imaging (fluorescence in situ hybridization [FISH]) and genome-wide chromosome conformation capture (Hi-C) are two major approaches to the study of higher-order genome organization in the nucleus. Intra-chromosomal and inter-chromosomal interactions (referred to as non-homologous chromosomal contacts [NHCCs]) have been observed by several FISH-based studies, but locus-specific NHCCs have not been detected by Hi-C. Due to crosslinking, neither of these approaches assesses spatiotemporal properties. Toward resolving the discrepancies between imaging and Hi-C, we sought to understand the spatiotemporal properties of NHCCs in living cells by CRISPR/Cas9 live-cell imaging (CLING). In mammalian cells, we find that NHCCs are stable and occur as frequently as intra-chromosomal interactions, but NHCCs occur at farther spatial distance that could explain their lack of detection in Hi-C. By revealing the spatiotemporal properties in living cells, our study provides fundamental insights into the biology of NHCCs.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Tamura I, Jozaki K, Sato S, et al (2018)

The distal upstream region of insulin-like growth factor-binding protein-1 enhances its expression in endometrial stromal cells during decidualization.

The Journal of biological chemistry, 293(14):5270-5280.

We have previously shown that decidualization of human endometrial stromal cells (ESCs) causes a genome-wide increase in the levels of acetylation of histone-H3 Lys-27 (H3K27ac). We also reported that the distal gene regions, more than 3 kb up- or downstream of gene transcription start sites have increased H3K27ac levels. Insulin-like growth factor-binding protein-1 (IGFBP-1) is a specific decidualization marker and has increased H3K27ac levels in its distal upstream region (-4701 to -7501 bp). Here, using a luciferase reporter gene construct containing this IGFBP-1 upstream region, we tested the hypothesis that it is an IGFBP-1 enhancer. To induce decidualization, we incubated ESCs with cAMP and found that cAMP increased luciferase expression, indicating that decidualization increased the transcriptional activity from the IGFBP-1 upstream region. Furthermore, CRISPR/Cas9-mediated deletion of this region in HepG2 cells significantly reduced IGFBP-1 expression, confirming its role as an IGFBP-1 enhancer. A ChIP assay revealed that cAMP increased the recruitment of the transcriptional regulators CCAAT enhancer-binding protein β (C/EBPβ), forkhead box O1 (FOXO1), and p300 to the IGFBP-1 enhancer in ESCs. Of note, C/EBPβ knockdown inhibited the stimulatory effects of cAMP on the levels of H3K27ac, chromatin opening, and p300 recruitment at the IGFBP-1 enhancer. These results indicate that the region -4701 to -7501 bp upstream of IGFBP-1 functions as an enhancer for IGFBP-1 expression in ESCs undergoing decidualization, that C/EBPβ and FOXO1 bind to the enhancer region to up-regulate IGFBP-1 expression, and that C/EBPβ induces H3K27ac by recruiting p300 to the IGFBP-1 enhancer.

RevDate: 2019-01-07
CmpDate: 2019-01-07

Nihongaki Y, Otabe T, M Sato (2018)

Emerging Approaches for Spatiotemporal Control of Targeted Genome with Inducible CRISPR-Cas9.

Analytical chemistry, 90(1):429-439.

RevDate: 2019-01-05

Park J, Choi S, Park S, et al (2019)

DNA-Free Genome Editing via Ribonucleoprotein (RNP) Delivery of CRISPR/Cas in Lettuce.

Methods in molecular biology (Clifton, N.J.), 1917:337-354.

CRISPR/Cas9 nuclease system is getting popular in precise genome editing of both eukaryotic and prokaryotic systems due to its accuracy, programmability, and relative ease of use. CRISPR/Cas systems can be delivered into live cells via plasmid DNA, RNA, and ribonucleoprotein (RNP). Of these, the RNP method is of special interest due to enzymatic action in shorter time and controllability over their activity. In addition, because RNP does not involve DNA, none of unwanted DNA footprints are left in the host genome. Previously, we demonstrated that plant protoplasts can be transfected with functional RNPs and the whole plants can be regenerated from an engineered protoplast. Relative to the published methods, the revised protocols described here should help increase the success rate of whole plant regeneration by reducing damages to the naked protoplast cells.

RevDate: 2019-01-05

Lee K, Zhu H, Yang B, et al (2019)

An Agrobacterium-Mediated CRISPR/Cas9 Platform for Genome Editing in Maize.

Methods in molecular biology (Clifton, N.J.), 1917:121-143.

Precise genome engineering can be efficiently made using the revolutionary tool named CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) systems. Adapted from the bacterial immune system, CRISPR/Cas systems can generate highly specific double-strand breaks (DSBs) at the target site, and desired sequence modifications can be introduced during the DSB repair process, such as nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. CRISPR/Cas9 is the most widely used genome editing tool for targeted mutagenesis, precise sequence modification, transcriptional reprogramming, epigenome editing, disease treatment, and many more. The ease of use and high specificity make CRISPR/Cas9 a great tool not only for basic researches but also for crop trait improvements, such as higher grain yield, better tolerance to abiotic stresses, enhanced disease resistance, and better nutritional contents. In this protocol, we present a step-by-step guide to the CRISPR/Cas9-mediated targeted mutagenesis in maize Hi II genotype. Detailed procedures will guide through the essential steps including gRNA design, CRISPR/Cas9 vector construction, Agrobacterium-mediated maize immature embryo transformation, and molecular analysis of the transgenic plants to identify desired mutant lines.

RevDate: 2019-01-04

Bayat H, Naderi F, Khan AH, et al (2018)

The Impact of CRISPR-Cas System on Antiviral Therapy.

Advanced pharmaceutical bulletin, 8(4):591-597.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein nuclease (Cas) is identified as an adaptive immune system in archaea and bacteria. Type II of this system, CRISPR-Cas9, is the most versatile form that has enabled facile and efficient targeted genome editing. Viral infections have serious impacts on global health and conventional antiviral therapies have not yielded a successful solution hitherto. The CRISPR-Cas9 system represents a promising tool for eliminating viral infections. In this review, we highlight 1) the recent progress of CRISPR-Cas technology in decoding and diagnosis of viral outbreaks, 2) its applications to eliminate viral infections in both pre-integration and provirus stages, and 3) various delivery systems that are employed to introduce the platform into target cells.

RevDate: 2019-01-04

Liu L, Yin M, Wang M, et al (2018)

Phage AcrIIA2 DNA Mimicry: Structural Basis of the CRISPR and Anti-CRISPR Arms Race.

Molecular cell pii:S1097-2765(18)30981-X [Epub ahead of print].

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems provide prokaryotic cells with adaptive immunity against invading bacteriophages. Bacteriophages counteract bacterial responses by encoding anti-CRISPR inhibitor proteins (Acr). However, the structural basis for their inhibitory actions remains largely unknown. Here, we report the crystal structure of the AcrIIA2-SpyCas9-sgRNA (single-guide RNA) complex at 3.3 Å resolution. We show that AcrIIA2 binds SpyCas9 at a position similar to the target DNA binding region. More specifically, AcrIIA2 interacts with the protospacer adjacent motif (PAM) recognition residues of Cas9, preventing target double-stranded DNA (dsDNA) detection. Thus, phage-encoded AcrIIA2 appears to act as a DNA mimic that blocks subsequent dsDNA binding by virtue of its highly acidic residues, disabling bacterial Cas9 by competing with target dsDNA binding with a binding motif distinct from AcrIIA4. Our study provides a more detailed mechanistic understanding of AcrIIA2-mediated inhibition of SpyCas9, the most widely used genome-editing tool, opening new avenues for improved regulatory precision during genome editing.

RevDate: 2019-01-04
CmpDate: 2019-01-04

Nagasaki A, Kato Y, Meguro K, et al (2018)

A genome editing vector that enables easy selection and identification of knockout cells.

Plasmid, 98:37-44.

The CRISPR/Cas9 system is a powerful genome editing tool for disrupting the expression of specific genes in a variety of cells. However, the genome editing procedure using currently available vectors is laborious, and there is room for improvement to obtain knockout cells more efficiently. Therefore, we constructed a novel vector for high efficiency genome editing, named pGedit, which contains EGFP-Bsr as a selection marker, expression units of Cas9, and sgRNA without a terminator sequence of the U6 promoter. EGFP-Bsr is a fusion protein of EGFP and blasticidin S deaminase, and enables rapid selection and monitoring of transformants, as well as confirmation that the vector has not been integrated into the genome. By using pGedit, we targeted human ACTB, ACTG1 and mouse Nes genes coding for β-actin, γ-actin and nestin, respectively. Knockout cell lines of each gene were easily and efficiently obtained in all three cases. In this report, we show that our novel vector, pGedit, significantly facilitates genome editing.

RevDate: 2019-01-04
CmpDate: 2019-01-04

Musunuru K (2018)

How genome editing could be used in the treatment of cardiovascular diseases.

Personalized medicine, 15(2):67-69.

RevDate: 2019-01-04
CmpDate: 2019-01-04

Zhang Y, Shi Y, Song D, et al (2018)

[Establishment and characterization of A549 tumor monoclonal cell line with UCHL1 gene deletion].

Sheng li xue bao : [Acta physiologica Sinica], 70(2):184-192.

The purpose of this study was to investigate the effects of ubiquitin C-terminal hydrolase-L1 (UCHL1) on non-small cell lung cancer cell line A549. UCHL1 gene knockout A549 cell line was constructed by CRISPR-CAS9 gene editing technique. The mRNA and protein levels of UCHL1 were examined by RT-PCR and Western blot, respectively. Cell proliferation and cycles were analyzed by CCK-8 method and flow cytometry, respectively. The sensitivity of A549 cells to cisplatin was detected by CCK-8 method. Migration ability of A549 cells was detected by scratch assay and Transwell test, and p-Erk expression level was assessed by Western blot. The results showed that UCHL1 gene knockout A549 cells were successfully constructed by CRISPR-CAS9 gene editing technique. After UCHL1 gene knockout, there was no significant change in cell proliferation and cell cycle ratios in A549 cells. UCHL1 gene knockout A549 cells exhibited decreased sensitivity to cisplatin and migration activity, as well as increased p-Erk expression level. These results suggest that the loss of UCHL1 gene function may reduce the sensitivity and migration ability of A549 cells, and this effect may be related to the activation of Erk1/2 signaling pathway.

RevDate: 2019-01-04
CmpDate: 2019-01-04

Schenkwein D, S Ylä-Herttuala (2018)

Gene Editing of Human Embryos with CRISPR/Cas9: Great Promise Coupled with Important Caveats.

Molecular therapy : the journal of the American Society of Gene Therapy, 26(3):659-660.

RevDate: 2019-01-04
CmpDate: 2019-01-04

Zhao D, Badur MG, Luebeck J, et al (2018)

Combinatorial CRISPR-Cas9 Metabolic Screens Reveal Critical Redox Control Points Dependent on the KEAP1-NRF2 Regulatory Axis.

Molecular cell, 69(4):699-708.e7.

The metabolic pathways fueling tumor growth have been well characterized, but the specific impact of transforming events on network topology and enzyme essentiality remains poorly understood. To this end, we performed combinatorial CRISPR-Cas9 screens on a set of 51 carbohydrate metabolism genes that represent glycolysis and the pentose phosphate pathway (PPP). This high-throughput methodology enabled systems-level interrogation of metabolic gene dispensability, interactions, and compensation across multiple cell types. The metabolic impact of specific combinatorial knockouts was validated using 13C and 2H isotope tracing, and these assays together revealed key nodes controlling redox homeostasis along the KEAP-NRF2 signaling axis. Specifically, targeting KEAP1 in combination with oxidative PPP genes mitigated the deleterious effects of these knockouts on growth rates. These results demonstrate how our integrated framework, combining genetic, transcriptomic, and flux measurements, can improve elucidation of metabolic network alterations and guide precision targeting of metabolic vulnerabilities based on tumor genetics.

RevDate: 2019-01-04
CmpDate: 2019-01-04

Minton DR, Nam M, McLaughlin DJ, et al (2018)

Serine Catabolism by SHMT2 Is Required for Proper Mitochondrial Translation Initiation and Maintenance of Formylmethionyl-tRNAs.

Molecular cell, 69(4):610-621.e5.

Upon glucose restriction, eukaryotic cells upregulate oxidative metabolism to maintain homeostasis. Using genetic screens, we find that the mitochondrial serine hydroxymethyltransferase (SHMT2) is required for robust mitochondrial oxygen consumption and low glucose proliferation. SHMT2 catalyzes the first step in mitochondrial one-carbon metabolism, which, particularly in proliferating cells, produces tetrahydrofolate (THF)-conjugated one-carbon units used in cytoplasmic reactions despite the presence of a parallel cytoplasmic pathway. Impairing cytoplasmic one-carbon metabolism or blocking efflux of one-carbon units from mitochondria does not phenocopy SHMT2 loss, indicating that a mitochondrial THF cofactor is responsible for the observed phenotype. The enzyme MTFMT utilizes one such cofactor, 10-formyl THF, producing formylmethionyl-tRNAs, specialized initiator tRNAs necessary for proper translation of mitochondrially encoded proteins. Accordingly, SHMT2 null cells specifically fail to maintain formylmethionyl-tRNA pools and mitochondrially encoded proteins, phenotypes similar to those observed in MTFMT-deficient patients. These findings provide a rationale for maintaining a compartmentalized one-carbon pathway in mitochondria.

RevDate: 2019-01-04
CmpDate: 2019-01-04

Meyer C, Garzia A, Mazzola M, et al (2018)

The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated Stress Response and Cell Cycle Progression.

Molecular cell, 69(4):622-635.e6.

TIA1 and TIAL1 encode a family of U-rich element mRNA-binding proteins ubiquitously expressed and conserved in metazoans. Using PAR-CLIP, we determined that both proteins bind target sites with identical specificity in 3' UTRs and introns proximal to 5' as well as 3' splice sites. Double knockout (DKO) of TIA1 and TIAL1 increased target mRNA abundance proportional to the number of binding sites and also caused accumulation of aberrantly spliced mRNAs, most of which are subject to nonsense-mediated decay. Loss of PRKRA by mis-splicing triggered the activation of the double-stranded RNA (dsRNA)-activated protein kinase EIF2AK2/PKR and stress granule formation. Ectopic expression of PRKRA cDNA or knockout of EIF2AK2 in DKO cells rescued this phenotype. Perturbation of maturation and/or stability of additional targets further compromised cell cycle progression. Our study reveals the essential contributions of the TIA1 protein family to the fidelity of mRNA maturation, translation, and RNA-stress-sensing pathways in human cells.

RevDate: 2019-01-04
CmpDate: 2019-01-04

Imtiaz A, Belyantseva IA, Beirl AJ, et al (2018)

CDC14A phosphatase is essential for hearing and male fertility in mouse and human.

Human molecular genetics, 27(5):780-798.

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.

RevDate: 2019-01-04
CmpDate: 2019-01-04

Giannelli SG, Luoni M, Castoldi V, et al (2018)

Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery.

Human molecular genetics, 27(5):761-779.

P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro. We, then, translated this approach in vivo by delivering the CRISPR/Cas9 components in murine Rho+/P23H mutant retinae. Targeted retinae presented a high rate of cleavage in the P23H but not WT Rho allele. This gene manipulation was sufficient to slow photoreceptor degeneration and improve retinal functions. To improve the translational potential of our approach, we tested intravitreal delivery of this system by means of adeno-associated viruses (AAVs). To this purpose, the employment of the AAV9-PHP.B resulted the most effective in disrupting the P23H Rho mutant. Finally, this approach was translated successfully in human cells engineered with the homozygous P23H RHO gene mutation. Overall, this is a significant proof-of-concept that gene allele specific targeting by CRISPR/Cas9 technology is specific and efficient and represents an unprecedented tool for treating RP and more broadly dominant genetic human disorders affecting the eye, as well as other tissues.

RevDate: 2019-01-04
CmpDate: 2019-01-04

Boëte C (2018)

Public engagement and communication: who is in charge?.

EMBO reports, 19(1):1-2.

RevDate: 2019-01-04
CmpDate: 2019-01-04

Reichardt I, Bonnay F, Steinmann V, et al (2018)

The tumor suppressor Brat controls neuronal stem cell lineages by inhibiting Deadpan and Zelda.

EMBO reports, 19(1):102-117.

The TRIM-NHL protein Brain tumor (Brat) acts as a tumor suppressor in the Drosophila brain, but how it suppresses tumor formation is not completely understood. Here, we combine temperature-controlled brat RNAi with transcriptome analysis to identify the immediate Brat targets in Drosophila neuroblasts. Besides the known target Deadpan (Dpn), our experiments identify the transcription factor Zelda (Zld) as a critical target of Brat. Our data show that Zld is expressed in neuroblasts and required to allow re-expression of Dpn in transit-amplifying intermediate neural progenitors. Upon neuroblast division, Brat is enriched in one daughter cell where its NHL domain directly binds to specific motifs in the 3'UTR of dpn and zld mRNA to mediate their degradation. In brat mutants, both Dpn and Zld continue to be expressed, but inhibition of either transcription factor prevents tumorigenesis. Our genetic and biochemical data indicate that Dpn inhibition requires higher Brat levels than Zld inhibition and suggest a model where stepwise post-transcriptional inhibition of distinct factors ensures sequential generation of fates in a stem cell lineage.

RevDate: 2019-01-03
CmpDate: 2019-01-03

Huang Y, Gu L, GM Li (2018)

H3K36me3-mediated mismatch repair preferentially protects actively transcribed genes from mutation.

The Journal of biological chemistry, 293(20):7811-7823.

Histone H3 trimethylation at lysine 36 (H3K36me3) is an important histone mark involved in both transcription elongation and DNA mismatch repair (MMR). It is known that H3K36me3 recruits the mismatch-recognition protein MutSα to replicating chromatin via its physical interaction with MutSα's PWWP domain, but the exact role of H3K36me3 in transcription is undefined. Using ChIP combined with whole-genome DNA sequencing analysis, we demonstrate here that H3K36me3, together with MutSα, is involved in protecting against mutation, preferentially in actively transcribed genomic regions. We found that H3K36me3 and MutSα are much more co-enriched in exons and actively transcribed regions than in introns and nontranscribed regions. The H3K36me3-MutSα co-enrichment correlated with a much lower mutation frequency in exons and actively transcribed regions than in introns and nontranscribed regions. Correspondingly, depleting H3K36me3 or disrupting the H3K36me3-MutSα interaction elevated the spontaneous mutation frequency in actively transcribed genes, but it had little influence on the mutation frequency in nontranscribed or transcriptionally inactive regions. Similarly, H2O2-induced mutations, which mainly cause base oxidations, preferentially occurred in actively transcribed genes in MMR-deficient cells. The data presented here suggest that H3K36me3-mediated MMR preferentially safeguards actively transcribed genes not only during replication by efficiently correcting mispairs in early replicating chromatin but also during transcription by directly or indirectly removing DNA lesions associated with a persistently open chromatin structure.

RevDate: 2019-01-03
CmpDate: 2019-01-03

Zhang Y, Guan Q, Liu Y, et al (2018)

Regulation of hepatic gluconeogenesis by nuclear factor Y transcription factor in mice.

The Journal of biological chemistry, 293(20):7894-7904.

Hepatic gluconeogenesis is essential to maintain blood glucose levels, and its abnormal activation leads to hyperglycemia and type 2 diabetes. However, the molecular mechanisms in the regulation of hepatic gluconeogenesis remain to be fully defined. In this study, using murine hepatocytes and a liver-specific knockout mouse model, we explored the physiological role of nuclear factor Y (NF-Y) in regulating hepatic glucose metabolism and the underlying mechanism. We found that NF-Y targets the gluconeogenesis pathway in the liver. Hepatic NF-Y expression was effectively induced by cAMP, glucagon, and fasting in vivo Lentivirus-mediated NF-Y overexpression in Hepa1-6 hepatocytes markedly raised the gluconeogenic gene expression and cellular glucose production compared with empty vector control cells. Conversely, CRISPR/Cas9-mediated knockdown of NF-Y subunit A (NF-YA) attenuated gluconeogenic gene expression and glucose production. We also provide evidence indicating that CRE-loxP-mediated, liver-specific NF-YA knockout compromises hepatic glucose production. Mechanistically, luciferase reporter gene assays and ChIP analysis indicated that NF-Y activates transcription of the gluconeogenic genes Pck1 and G6pc, by encoding phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase), respectively, via directly binding to the CCAAT regulatory sequence motif in their promoters. Of note, NF-Y enhanced gluconeogenesis by interacting with cAMP-responsive element-binding protein (CREB). Overall, our results reveal a previously unrecognized physiological function of NF-Y in controlling glucose metabolism by up-regulating the gluconeogenic genes Pck1 and G6pc Modulation of hepatic NF-Y expression may therefore offer an attractive therapeutic approach to manage type 2 diabetes.

RevDate: 2019-01-03
CmpDate: 2019-01-03

Zhong C, Chen Z, Luo X, et al (2018)

Barhl1 is required for the differentiation of inner ear hair cell-like cells from mouse embryonic stem cells.

The international journal of biochemistry & cell biology, 96:79-89.

Inner ear hair cells are mechanoreceptors responsible for hearing. Pathogenic defects of hair cell-specific genes are one of the major causes of deafness. The BarH class homeobox gene Barhl1 is a deafness gene expressed in developing hair cells, yet the role of Barhl1 during hair cell development remains poorly understood. In the present study, we first established an in vitro differentiation system to efficiently obtain mouse embryonic stem cell (mESC)-derived hair cell-like cells. Subsequently, a mESC line carrying a targeted disruption of Barhl1 was generated using CRISPR/Cas9 technology and subjected to the established in vitro hair cell differentiation protocol. Targeted disruption of Barhl1 does not affect the induction of mESCs toward early primitive ectoderm-like (EPL) cells and otic progenitors but strongly inhibits the differentiation of hair cell-like cells. Using RNA-sequencing and bioinformatics, we further unravel the molecular mechanism underlying Barhl1-mediated hair cell development. Our data demonstrate the essential role of Barhl1 during hair cell development and provide a basis for the treatment of Barhl1 mutation-based deafness.

RevDate: 2019-01-03
CmpDate: 2019-01-03

Rosenbluh J, Mercer J, Shrestha Y, et al (2016)

Genetic and Proteomic Interrogation of Lower Confidence Candidate Genes Reveals Signaling Networks in β-Catenin-Active Cancers.

Cell systems, 3(3):302-316.e4.

Genome-scale expression studies and comprehensive loss-of-function genetic screens have focused almost exclusively on the highest confidence candidate genes. Here, we describe a strategy for characterizing the lower confidence candidates identified by such approaches. We interrogated 177 genes that we classified as essential for the proliferation of cancer cells exhibiting constitutive β-catenin activity and integrated data for each of the candidates, derived from orthogonal short hairpin RNA (shRNA) knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene editing knockout screens, to yield 69 validated genes. We then characterized the relationships between sets of these genes using complementary assays: medium-throughput stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry, yielding 3,639 protein-protein interactions, and a CRISPR-mediated pairwise double knockout screen, yielding 375 combinations exhibiting greater- or lesser-than-additive phenotypic effects indicating genetic interactions. These studies identify previously unreported regulators of β-catenin, define functional networks required for the survival of β-catenin-active cancers, and provide an experimental strategy that may be applied to define other signaling networks.

RevDate: 2019-01-02
CmpDate: 2019-01-02

Yamaguchi H, Hopf FW, Li SB, et al (2018)

In vivo cell type-specific CRISPR knockdown of dopamine beta hydroxylase reduces locus coeruleus evoked wakefulness.

Nature communications, 9(1):5211.

Locus coeruleus (LC) neurons in the brainstem have long been associated with attention and arousal. Optogenetic stimulation of LC-NE neurons induces immediate sleep-to-wake transitions. However, LC neurons also secrete other neurotransmitters in addition to NE. To interrogate the role of NE derived from the LC in regulating wakefulness, we applied in vivo cell type-specific CRISPR/Cas9 technology to disrupt the dopamine beta hydroxylase (dbh) gene selectively in adult LC-NE neurons. Unilateral dbh gene disruption abolished immediate arousal following optogenetic stimulation of LC. Bilateral LC-specific dbh disruption significantly reduced NE concentration in LC projection areas and reduced wake length even in the presence of salient stimuli. These results suggest that NE may be crucial for the awakening effect of LC stimulation and serve as proof-of-principle that CRISPR gene editing in adult neurons can be used to interrogate gene function within genetically-defined neuronal circuitry associated with complex behaviors.

RevDate: 2019-01-02
CmpDate: 2019-01-02

Rice CM, Davies LC, Subleski JJ, et al (2018)

Tumour-elicited neutrophils engage mitochondrial metabolism to circumvent nutrient limitations and maintain immune suppression.

Nature communications, 9(1):5099 pii:10.1038/s41467-018-07505-2.

Neutrophils are a vital component of immune protection, yet in cancer they may promote tumour progression, partly by generating reactive oxygen species (ROS) that disrupts lymphocyte functions. Metabolically, neutrophils are often discounted as purely glycolytic. Here we show that immature, c-Kit+ neutrophils subsets can engage in oxidative mitochondrial metabolism. With limited glucose supply, oxidative neutrophils use mitochondrial fatty acid oxidation to support NADPH oxidase-dependent ROS production. In 4T1 tumour-bearing mice, mitochondrial fitness is enhanced in splenic neutrophils and is driven by c-Kit signalling. Concordantly, tumour-elicited oxidative neutrophils are able to maintain ROS production and T cell suppression when glucose utilisation is restricted. Consistent with these findings, peripheral blood neutrophils from patients with cancer also display increased immaturity, mitochondrial content and oxidative phosphorylation. Together, our data suggest that the glucose-restricted tumour microenvironment induces metabolically adapted, oxidative neutrophils to maintain local immune suppression.

RevDate: 2019-01-02
CmpDate: 2019-01-02

Jansson S (2018)

Gene-edited plants: What is happening now?.

Physiologia plantarum, 164(4):370-371.

RevDate: 2019-01-02
CmpDate: 2019-01-02

Daou S, Barbour H, Ahmed O, et al (2018)

Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1.

Nature communications, 9(1):4385.

The tumor suppressor and deubiquitinase (DUB) BAP1 and its Drosophila ortholog Calypso assemble DUB complexes with the transcription regulators Additional sex combs-like (ASXL1, ASXL2, ASXL3) and Asx respectively. ASXLs and Asx use their DEUBiquitinase ADaptor (DEUBAD) domain to stimulate BAP1/Calypso DUB activity. Here we report that monoubiquitination of the DEUBAD is a general feature of ASXLs and Asx. BAP1 promotes DEUBAD monoubiquitination resulting in an increased stability of ASXL2, which in turn stimulates BAP1 DUB activity. ASXL2 monoubiquitination is directly catalyzed by UBE2E family of Ubiquitin-conjugating enzymes and regulates mammalian cell proliferation. Remarkably, Calypso also regulates Asx monoubiquitination and transgenic flies expressing monoubiquitination-defective Asx mutant exhibit developmental defects. Finally, the protein levels of ASXL2, BAP1 and UBE2E enzymes are highly correlated in mesothelioma tumors suggesting the importance of this signaling axis for tumor suppression. We propose that monoubiquitination orchestrates a molecular symbiosis relationship between ASXLs and BAP1.

RevDate: 2019-01-02
CmpDate: 2019-01-02

Ho BX, Loh SJH, Chan WK, et al (2018)

In Vivo Genome Editing as a Therapeutic Approach.

International journal of molecular sciences, 19(9):.

Genome editing has been well established as a genome engineering tool that enables researchers to establish causal linkages between genetic mutation and biological phenotypes, providing further understanding of the genetic manifestation of many debilitating diseases. More recently, the paradigm of genome editing technologies has evolved to include the correction of mutations that cause diseases via the use of nucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and more recently, Cas9 nuclease. With the aim of reversing disease phenotypes, which arise from somatic gene mutations, current research focuses on the clinical translatability of correcting human genetic diseases in vivo, to provide long-term therapeutic benefits and potentially circumvent the limitations of in vivo cell replacement therapy. In this review, in addition to providing an overview of the various genome editing techniques available, we have also summarized several in vivo genome engineering strategies that have successfully demonstrated disease correction via in vivo genome editing. The various benefits and challenges faced in applying in vivo genome editing in humans will also be discussed.

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RJR Experience and Expertise

Researcher

Robbins holds BS, MS, and PhD degrees in the life sciences. He served as a tenured faculty member in the Zoology and Biological Science departments at Michigan State University. He is currently exploring the intersection between genomics, microbial ecology, and biodiversity — an area that promises to transform our understanding of the biosphere.

Educator

Robbins has extensive experience in college-level education: At MSU he taught introductory biology, genetics, and population genetics. At JHU, he was an instructor for a special course on biological database design. At FHCRC, he team-taught a graduate-level course on the history of genetics. At Bellevue College he taught medical informatics.

Administrator

Robbins has been involved in science administration at both the federal and the institutional levels. At NSF he was a program officer for database activities in the life sciences, at DOE he was a program officer for information infrastructure in the human genome project. At the Fred Hutchinson Cancer Research Center, he served as a vice president for fifteen years.

Technologist

Robbins has been involved with information technology since writing his first Fortran program as a college student. At NSF he was the first program officer for database activities in the life sciences. At JHU he held an appointment in the CS department and served as director of the informatics core for the Genome Data Base. At the FHCRC he was VP for Information Technology.

Publisher

While still at Michigan State, Robbins started his first publishing venture, founding a small company that addressed the short-run publishing needs of instructors in very large undergraduate classes. For more than 20 years, Robbins has been operating The Electronic Scholarly Publishing Project, a web site dedicated to the digital publishing of critical works in science, especially classical genetics.

Speaker

Robbins is well-known for his speaking abilities and is often called upon to provide keynote or plenary addresses at international meetings. For example, in July, 2012, he gave a well-received keynote address at the Global Biodiversity Informatics Congress, sponsored by GBIF and held in Copenhagen. The slides from that talk can be seen HERE.

Facilitator

Robbins is a skilled meeting facilitator. He prefers a participatory approach, with part of the meeting involving dynamic breakout groups, created by the participants in real time: (1) individuals propose breakout groups; (2) everyone signs up for one (or more) groups; (3) the groups with the most interested parties then meet, with reports from each group presented and discussed in a subsequent plenary session.

Designer

Robbins has been engaged with photography and design since the 1960s, when he worked for a professional photography laboratory. He now prefers digital photography and tools for their precision and reproducibility. He designed his first web site more than 20 years ago and he personally designed and implemented this web site. He engages in graphic design as a hobby.

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CRISPR-Cas

By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

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E-mail: RJR8222@gmail.com

Collection of publications by R J Robbins

Reprints and preprints of publications, slide presentations, instructional materials, and data compilations written or prepared by Robert Robbins. Most papers deal with computational biology, genome informatics, using information technology to support biomedical research, and related matters.

Research Gate page for R J Robbins

ResearchGate is a social networking site for scientists and researchers to share papers, ask and answer questions, and find collaborators. According to a study by Nature and an article in Times Higher Education , it is the largest academic social network in terms of active users.

Curriculum Vitae for R J Robbins

short personal version

Curriculum Vitae for R J Robbins

long standard version

RJR Picks from Around the Web (updated 11 MAY 2018 )