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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 17 Jun 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: 2019-06-15

Yang J, X Wang (2019)

Role of long non-coding RNAs in lymphoma: A systematic review and clinical perspectives.

Critical reviews in oncology/hematology, 141:13-22 pii:S1040-8428(18)30432-3 [Epub ahead of print].

Long non-coding RNAs (lncRNAs), are over 200 nucleotides in length, and they rarely act as templates for protein synthesis. Mounting studies have shown that lncRNAs play a crucial regulatory role in various processes that sustain life, such as epigenetic regulation, cell cycle control, splicing, and post-transcriptional regulation. LncRNAs were aberrantly expressed in most hematological malignancies including lymphoma, participating in tumor suppression or promoting oncogenesis and modulating key genes in different pathways. The specific expression patterns of lncRNAs in lymphoma make them good candidates to be used as diagnostic biomarkers or as therapeutic targets. LncRNAs can be targeted by multiple approaches including nucleic acid therapeutics, CRISPR/Cas genome editing techniques, small molecule inhibitors, and gene therapy. Efforts are made to develop therapeutic strategies aimed at targeting lncRNAs, but there are still some avenues to be covered before they can be applied to the clinical treatment of lymphoma.

RevDate: 2019-06-15

Artyukhova MA, Tyurina YY, Chu CT, et al (2019)

Interrogating Parkinson's disease associated redox targets: Potential application of CRISPR editing.

Free radical biology & medicine pii:S0891-5849(19)30251-5 [Epub ahead of print].

Loss of dopaminergic neurons in the substantia nigra is one of the pathogenic hallmarks of Parkinson's disease, yet the underlying molecular mechanisms remain enigmatic. While aberrant redox metabolism strongly associated with iron dysregulation and accumulation of dysfunctional mitochondria is considered as one of the major contributors to neurodegeneration and death of dopaminergic cells, the specific anomalies in the molecular machinery and pathways leading to the PD development and progression have not been identified. The high efficiency and relative simplicity of a new genome editing tool, CRISPR/Cas9, make its applications attractive for deciphering molecular changes driving PD-related impairments of redox metabolism and lipid peroxidation in relation to mishandling of iron, aggregation and oligomerization of alpha-synuclein and mitochondrial injury as well as in mechanisms of mitophagy and programs of regulated cell death (apoptosis and ferroptosis). These insights into the mechanisms of PD pathology may be used for the identification of new targets for therapeutic interventions and innovative approaches to genome editing, including CRISPR/Cas9.

RevDate: 2019-06-14

Luo J, Chen W, Xue L, et al (2019)

Prediction of activity and specificity of CRISPR-Cpf1 using convolutional deep learning neural networks.

BMC bioinformatics, 20(1):332 pii:10.1186/s12859-019-2939-6.

BACKGROUND: CRISPR-Cpf1 has recently been reported as another RNA-guided endonuclease of class 2 CRISPR-Cas system, which expands the molecular biology toolkit for genome editing. However, most of the online tools and applications to date have been developed primarily for the Cas9. There are a limited number of tools available for the Cpf1.

RESULTS: We present DeepCpf1, a deep convolution neural networks (CNN) approach to predict Cpf1 guide RNAs on-target activity and off-target effects using their matched and mismatched DNA sequences. Trained on published data sets, DeepCpf1 is superior to other machine learning algorithms and reliably predicts the most efficient and less off-target effects guide RNAs for a given gene. Combined with a permutation importance analysis, the key features of guide RNA sequences are identified, which determine the activity and specificity of genome editing.

CONCLUSIONS: DeepCpf1 can significantly improve the accuracy of Cpf1-based genome editing and facilitates the generation of optimized guide RNAs libraries.

RevDate: 2019-06-13

Long J, Xu Y, Ou L, et al (2019)

Polymorphism of Type I-F CRISPR/Cas system in Escherichia coli of phylogenetic group B2 and its application in genotyping.

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases pii:S1567-1348(19)30137-6 [Epub ahead of print].

E. coli of phylogenetic group B2 is responsible for many extraintestinal infections, posing a great threat to health. The relatively polymorphic nature of CRISPR in phylogenetically related E. coli strains makes them potential markers for bacterial typing and evolutionary studies. In the current work, we investigated the occurrence and diversity of CRISPR/Cas system and explored its potential for genotyping. Type I-F CRISPR/Cas systems were found in 413 of 1190 strains of E. coli and exhibited the clustering within certain CCs and STs. And CRISPR spacer contents correlated well with MLST types. The divergence analysis of CRISPR showed stronger discriminatory power than MLST, and CRISPR polymorphism was instrumental for differentiating highly closely related strains. The timeline of spacer acquisition and deletion provided important information for inferring the evolution model between distinct serotypes. Identical spacer sequences were shared by strains with the same H-antigen type but not strains with the same O-antigen type. The homology between spacers and antibiotic-resistant plasmids demonstrated the role of Type I-F system in limiting the acquisition of antimicrobial resistance. Collectively, our data presents the dynamic nature of Type I-F CRISPR in E. coli of phylogenetic group B2 and provides new insights into the application of CRISPR-based typing in the species.

RevDate: 2019-06-13

O'Meara D, L Nunney (2019)

A phylogenetic test of the role of CRISPR-Cas in limiting plasmid acquisition and prophage integration in bacteria.

Plasmid pii:S0147-619X(19)30025-3 [Epub ahead of print].

CRISPR-Cas is a prokaryotic defense system capable of protecting the cell from damaging foreign genetic elements. However, some genetic elements can be beneficial, which suggests the hypothesis that bacteria with CRISPR-Cas incur a cost of reduced intake of mutualistic plasmids and prophage. Here we present the first robust test of this hypothesis that controls for phylogenic and ecological biases in the distribution of CRISPR-Cas. We filtered the available genomic data (~7000 strains from ~2100 species) by first selecting all pairs of conspecific strains, one with and one without CRISPR-Cas (controlling ecological bias), and second by retaining only one such pair per bacterial family (controlling phylogenetic bias), resulting in pairs representing 38 bacterial families. Analysis of these pairs of bacterial strains showed that on average the CRISPR-Cas strain of each pair contained significantly fewer plasmids than its CRISPR-Cas negative partner (0.86 vs. 1.86). It also showed that the CRISPR-Cas positive strains had 31% fewer intact prophage (1.17 vs. 1.75), but the effect was highly variable and not significant. These results support the hypothesis that CRISPR-Cas reduces the rate of plasmid-mediated HGT and, given the abundant evidence of beneficial genes carried by plasmids, provide a clear example of a cost associated with the CRISPR-Cas system.

RevDate: 2019-06-13

Hodges CA, RA Conlon (2019)

Delivering on the promise of gene editing for cystic fibrosis.

Genes & diseases, 6(2):97-108 pii:S2352-3042(18)30136-3.

In this review, we describe a path for translation of gene editing into therapy for cystic fibrosis (CF). Cystic fibrosis results from mutations in the CFTR gene, with one allele predominant in patient populations. This simple, genetic etiology makes gene editing appealing for treatment of this disease. There already have been success in applying this approach to cystic fibrosis in cell and animal models, although these advances have been modest in comparison to advances for other disease. Less than six years after its first demonstration in animals, CRISPR/Cas gene editing is in early clinical trials for several disorders. Most clinical trials, thus far, attempt to edit genes in cells of the blood lineages. The advantage of the blood is that the stem cells are known, can be isolated, edited, selected, expanded, and returned to the body. The likely next trials will be in the liver, which is accessible to many delivery methods. For cystic fibrosis, the biggest hurdle is to deliver editors to other, less accessible organs. We outline a path by which delivery can be improved. The translation of new therapies doesn't occur in isolation, and the development of gene editors is occurring as advances in gene therapy and small molecule therapeutics are being made. The advances made in gene therapy may help develop delivery vehicles for gene editing, although major improvements are needed. Conversely, the approval of effective small molecule therapies for many patients with cystic fibrosis will raise the bar for translation of gene editing.

RevDate: 2019-06-14
CmpDate: 2019-06-14

Gu Y, Gao J, Cao M, et al (2019)

Construction of a series of episomal plasmids and their application in the development of an efficient CRISPR/Cas9 system in Pichia pastoris.

World journal of microbiology & biotechnology, 35(6):79 pii:10.1007/s11274-019-2654-5.

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.

RevDate: 2019-06-13
CmpDate: 2019-06-12

Zhu X, Lv MM, Liu JW, et al (2019)

DNAzyme activated protein-scaffolded CRISPR-Cas9 nanoassembly for genome editing.

Chemical communications (Cambridge, England), 55(46):6511-6514.

A novel self-assembled protein-scaffolded CRISPR-Cas9 nanosystem for facile and efficient gene editing in a DNAzyme-controlled manner has been developed.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Baumann V, Wiesbeck M, Breunig CT, et al (2019)

Targeted removal of epigenetic barriers during transcriptional reprogramming.

Nature communications, 10(1):2119 pii:10.1038/s41467-019-10146-8.

Master transcription factors have the ability to direct and reverse cellular identities, and consequently their genes must be subject to particular transcriptional control. However, it is unclear which molecular processes are responsible for impeding their activation and safeguarding cellular identities. Here we show that the targeting of dCas9-VP64 to the promoter of the master transcription factor Sox1 results in strong transcript and protein up-regulation in neural progenitor cells (NPCs). This gene activation restores lost neuronal differentiation potential, which substantiates the role of Sox1 as a master transcription factor. However, despite efficient transactivator binding, major proportions of progenitor cells are unresponsive to the transactivating stimulus. By combining the transactivation domain with epigenome editing we find that among a series of euchromatic processes, the removal of DNA methylation (by dCas9-Tet1) has the highest potential to increase the proportion of cells activating foreign master transcription factors and thus breaking down cell identity barriers.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Kundert K, Lucas JE, Watters KE, et al (2019)

Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs.

Nature communications, 10(1):2127 pii:10.1038/s41467-019-09985-2.

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Meltzer H, Marom E, Alyagor I, et al (2019)

Tissue-specific (ts)CRISPR as an efficient strategy for in vivo screening in Drosophila.

Nature communications, 10(1):2113 pii:10.1038/s41467-019-10140-0.

Gene editing by CRISPR/Cas9 is commonly used to generate germline mutations or perform in vitro screens, but applicability for in vivo screening has so far been limited. Recently, it was shown that in Drosophila, Cas9 expression could be limited to a desired group of cells, allowing tissue-specific mutagenesis. Here, we thoroughly characterize tissue-specific (ts)CRISPR within the complex neuronal system of the Drosophila mushroom body. We report the generation of a library of gRNA-expressing plasmids and fly lines using optimized tools, which provides a valuable resource to the fly community. We demonstrate the application of our library in a large-scale in vivo screen, which reveals insights into developmental neuronal remodeling.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Soyk S, Lemmon ZH, Sedlazeck FJ, et al (2019)

Duplication of a domestication locus neutralized a cryptic variant that caused a breeding barrier in tomato.

Nature plants, 5(5):471-479.

Genome editing technologies are being widely adopted in plant breeding1. However, a looming challenge of engineering desirable genetic variation in diverse genotypes is poor predictability of phenotypic outcomes due to unforeseen interactions with pre-existing cryptic mutations2-4. In tomato, breeding with a classical MADS-box gene mutation that improves harvesting by eliminating fruit stem abscission frequently results in excessive inflorescence branching, flowering and reduced fertility due to interaction with a cryptic variant that causes partial mis-splicing in a homologous gene5-8. Here, we show that a recently evolved tandem duplication carrying the second-site variant achieves a threshold of functional transcripts to suppress branching, enabling breeders to neutralize negative epistasis on yield. By dissecting the dosage mechanisms by which this structural variant restored normal flowering and fertility, we devised strategies that use CRISPR-Cas9 genome editing to predictably improve harvesting. Our findings highlight the under-appreciated impact of epistasis in targeted trait breeding and underscore the need for a deeper characterization of cryptic variation to enable the full potential of genome editing in agriculture.

RevDate: 2019-06-13
CmpDate: 2019-06-10

De Giorgi M, WR Lagor (2019)

Gene Delivery in Lipid Research and Therapies.

Methodist DeBakey cardiovascular journal, 15(1):62-69.

Cardiovascular disease is the leading cause of death worldwide, and elevated lipid levels is a major contributor. Gene delivery, which involves controlled transfer of nucleic acids into cells and tissues, has been widely used in research to study lipid metabolism and physiology. Several technologies have been developed to somatically overexpress, silence, or disrupt genes in animal models and have greatly advanced our knowledge of metabolism. This is particularly true with regard to the liver, which plays a central role in lipoprotein metabolism and is amenable to many delivery approaches. In addition to basic science applications, many of these delivery technologies have potential as gene therapies for both common and rare lipid disorders. This review discusses three major gene delivery technologies used in lipid research-including adeno-associated viral vector overexpression, antisense oligonucleotides and small interfering RNAs, and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing system-and examines their potential therapeutic applications.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Wang X, Wang S, Troisi EC, et al (2019)

BRD9 defines a SWI/SNF sub-complex and constitutes a specific vulnerability in malignant rhabdoid tumors.

Nature communications, 10(1):1881 pii:10.1038/s41467-019-09891-7.

Bromodomain-containing protein 9 (BRD9) is a recently identified subunit of SWI/SNF(BAF) chromatin remodeling complexes, yet its function is poorly understood. Here, using a genome-wide CRISPR-Cas9 screen, we show that BRD9 is a specific vulnerability in pediatric malignant rhabdoid tumors (RTs), which are driven by inactivation of the SMARCB1 subunit of SWI/SNF. We find that BRD9 exists in a unique SWI/SNF sub-complex that lacks SMARCB1, which has been considered a core subunit. While SMARCB1-containing SWI/SNF complexes are bound preferentially at enhancers, we show that BRD9-containing complexes exist at both promoters and enhancers. Mechanistically, we show that SMARCB1 loss causes increased BRD9 incorporation into SWI/SNF thus providing insight into BRD9 vulnerability in RTs. Underlying the dependency, while its bromodomain is dispensable, the DUF3512 domain of BRD9 is essential for SWI/SNF integrity in the absence of SMARCB1. Collectively, our results reveal a BRD9-containing SWI/SNF subcomplex is required for the survival of SMARCB1-mutant RTs.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Taemaitree L, Shivalingam A, El-Sagheer AH, et al (2019)

An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically modified CRISPR sgRNA.

Nature communications, 10(1):1610 pii:10.1038/s41467-019-09600-4.

As the applications of CRISPR-Cas9 technology diversify and spread beyond the laboratory to diagnostic and therapeutic use, the demands of gRNA synthesis have increased and access to tailored gRNAs is now restrictive. Enzymatic routes are time-consuming, difficult to scale-up and suffer from polymerase-bias while existing chemical routes are inefficient. Here, we describe a split-and-click convergent chemical route to individual or pools of sgRNAs. The synthetic burden is reduced by splitting the sgRNA into a variable DNA/genome-targeting 20-mer, produced on-demand and in high purity, and a fixed Cas9-binding chemically-modified 79-mer, produced cost-effectively on large-scale, a strategy that provides access to site-specific modifications that enhance sgRNA activity and in vivo stability. Click ligation of the two components generates an artificial triazole linkage that is tolerated in functionally critical regions of the sgRNA and allows efficient DNA cleavage in vitro as well as gene-editing in cells with no unexpected off-target effects.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Gisler S, Gonçalves JP, Akhtar W, et al (2019)

Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency.

Nature communications, 10(1):1598 pii:10.1038/s41467-019-09551-w.

Understanding the impact of guide RNA (gRNA) and genomic locus on CRISPR-Cas9 activity is crucial to design effective gene editing assays. However, it is challenging to profile Cas9 activity in the endogenous cellular environment. Here we leverage our TRIP technology to integrate ~ 1k barcoded reporter genes in the genomes of mouse embryonic stem cells. We target the integrated reporters (IRs) using RNA-guided Cas9 and characterize induced mutations by sequencing. We report that gRNA-sequence and IR locus explain most variation in mutation efficiency. Predominant insertions of a gRNA-specific nucleotide are consistent with template-dependent repair of staggered DNA ends with 1-bp 5' overhangs. We confirm that such staggered ends are induced by Cas9 in mouse pre-B cells. To explain observed insertions, we propose a model generating primarily blunt and occasionally staggered DNA ends. Mutation patterns indicate that gRNA-sequence controls the fraction of staggered ends, which could be used to optimize Cas9-based insertion efficiency.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Zou Y, Palte MJ, Deik AA, et al (2019)

A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis.

Nature communications, 10(1):1617 pii:10.1038/s41467-019-09277-9.

Clear-cell carcinomas (CCCs) are a histological group of highly aggressive malignancies commonly originating in the kidney and ovary. CCCs are distinguished by aberrant lipid and glycogen accumulation and are refractory to a broad range of anti-cancer therapies. Here we identify an intrinsic vulnerability to ferroptosis associated with the unique metabolic state in CCCs. This vulnerability transcends lineage and genetic landscape, and can be exploited by inhibiting glutathione peroxidase 4 (GPX4) with small-molecules. Using CRISPR screening and lipidomic profiling, we identify the hypoxia-inducible factor (HIF) pathway as a driver of this vulnerability. In renal CCCs, HIF-2α selectively enriches polyunsaturated lipids, the rate-limiting substrates for lipid peroxidation, by activating the expression of hypoxia-inducible, lipid droplet-associated protein (HILPDA). Our study suggests targeting GPX4 as a therapeutic opportunity in CCCs, and highlights that therapeutic approaches can be identified on the basis of cell states manifested by morphological and metabolic features in hard-to-treat cancers.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Toda E, Koiso N, Takebayashi A, et al (2019)

An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice.

Nature plants, 5(4):363-368.

Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants1,2. Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9-guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment3, CRISPR-Cas9 integration increases the chance of off-target modifications4, and foreign DNA sequences cause legislative concerns about genetically modified organisms5. Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol-calcium (PEG-Ca2+)-mediated transfection in tobacco, Arabidopsis, lettuce, rice6, Petunia7, grapevine, apple8 and potato9, or into embryo cells by biolistic bombardment in maize10 and wheat11. However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9-gRNA RNPs into plant zygotes. Cas9-gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes12 and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14-64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Zheng T, Li J, Ni Y, et al (2019)

Mining, analyzing, and integrating viral signals from metagenomic data.

Microbiome, 7(1):42 pii:10.1186/s40168-019-0657-y.

BACKGROUND: Viruses are important components of microbial communities modulating community structure and function; however, only a couple of tools are currently available for phage identification and analysis from metagenomic sequencing data. Here we employed the random forest algorithm to develop VirMiner, a web-based phage contig prediction tool especially sensitive for high-abundances phage contigs, trained and validated by paired metagenomic and phagenomic sequencing data from the human gut flora.

RESULTS: VirMiner achieved 41.06% ± 17.51% sensitivity and 81.91% ± 4.04% specificity in the prediction of phage contigs. In particular, for the high-abundance phage contigs, VirMiner outperformed other tools (VirFinder and VirSorter) with much higher sensitivity (65.23% ± 16.94%) than VirFinder (34.63% ± 17.96%) and VirSorter (18.75% ± 15.23%) at almost the same specificity. Moreover, VirMiner provides the most comprehensive phage analysis pipeline which is comprised of metagenomic raw reads processing, functional annotation, phage contig identification, and phage-host relationship prediction (CRISPR-spacer recognition) and supports two-group comparison when the input (metagenomic sequence data) includes different conditions (e.g., case and control). Application of VirMiner to an independent cohort of human gut metagenomes obtained from individuals treated with antibiotics revealed that 122 KEGG orthology and 118 Pfam groups had significantly differential abundance in the pre-treatment samples compared to samples at the end of antibiotic administration, including clustered regularly interspaced short palindromic repeats (CRISPR), multidrug resistance, and protein transport. The VirMiner webserver is available at http://sbb.hku.hk/VirMiner/ .

CONCLUSIONS: We developed a comprehensive tool for phage prediction and analysis for metagenomic samples. Compared to VirSorter and VirFinder-the most widely used tools-VirMiner is able to capture more high-abundance phage contigs which could play key roles in infecting bacteria and modulating microbial community dynamics.

TRIAL REGISTRATION: The European Union Clinical Trials Register, EudraCT Number: 2013-003378-28 . Registered on 9 April 2014.

RevDate: 2019-06-13
CmpDate: 2019-06-11

Guo M, Yang L, Du W, et al (2019)

[CRISPR/Cas9-mediated foreign gene targeted knock-in into the chicken EAV-HP genome].

Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 35(2):236-243.

The study aims to use CRISPR/Cas9 introducing foreign gene targeted knock-in into chicken EAV-HP genome. First, specific primers were designed for amplification of EAV-HP left, right homologous arms and enhanced green fluorescent protein (eGFP) expression cassette. PCR products of homologous arms were ligated to both sides of eGFP by overlap extension PCR, resulting in full-length donor DNA fragment designated as LER. Then LER fragments were cloned into pMD19-T to obtain donor vector pMDT-LER. Subsequently, the donor vector pMDT-LER was transfected into HEK293T cells to verify the expression of eGFP gene. Furthermore, co-transfection of CRISPR/Cas9 expression vector and pMDT-LER into chicken DF-1 cells was performed to achieve eGFP transgenic cells. Meanwhile, eGFP expression was observed in cells, and the event of eGFP integration into EAV-HP genome was detectable by amplification of target DNA. Finally, the transgenic DF-1 cells were passaged seven times, and the stable integration and expression of eGFP was checked by PCR and Western blotting. These results demonstrated that eGFP gene was knocked into the EAV-HP genome successfully, which provides a new integration site for research of transgenic chicken.

RevDate: 2019-06-13
CmpDate: 2019-06-12

Hannafon BN, Gin AL, Xu YF, et al (2019)

Metastasis-associated protein 1 (MTA1) is transferred by exosomes and contributes to the regulation of hypoxia and estrogen signaling in breast cancer cells.

Cell communication and signaling : CCS, 17(1):13 pii:10.1186/s12964-019-0325-7.

BACKGROUND: Exosomes are small membrane-bound vesicles that contribute to tumor progression and metastasis by mediating cell-to-cell communication and modifying the tumor microenvironment at both local and distant sites. However, little is known about the predominant factors in exosomes that contribute to breast cancer (BC) progression. MTA1 is a transcriptional co-regulator that can act as both a co-activator and co-repressor to regulate pathways that contribute to cancer development. MTA1 is also one of the most up-regulated proteins in cancer, whose expression correlates with cancer progression, poor prognosis and increased metastatic potential.

METHODS: We identified MTA1 in BC exosomes by antibody array and confirmed expression of exosome-MTA1 across five breast cancer cells lines. Ectopic expression of tdTomato-tagged MTA1 and exosome transfer were examined by fluorescent microscopy. CRISPR/Cas9 genetic engineering was implemented to knockout MTA1 in MCF7 and MDA-MB-231 breast cancer cells. Reporter assays were used to monitor hypoxia and estrogen receptor signaling regulation by exosome-MTA1 transfer.

RESULTS: Ectopic overexpression of tdTomato-MTA1 in BC cell lines demonstrated exosome transfer of MTA1 to BC and vascular endothelial cells. MTA1 knockout in BC cells reduced cell proliferation and attenuated the hypoxic response in these cells, presumably through its co-repressor function, which could be rescued by the addition of exosomes containing MTA1. On the other hand, consistent with its co-activator function, estrogen receptor signaling was enhanced in MTA1 knockout cells and could be reversed by addition of MTA1-exosomes. Importantly, MTA1 knockout sensitized hormone receptor negative cells to 4-hydroxy tamoxifen treatment, which could be reversed by the addition of MTA1-exosomes.

CONCLUSIONS: This is the first report showing that BC exosomes contain MTA1 and can transfer it to other cells resulting in changes to hypoxia and estrogen receptor signaling in the tumor microenvironment. These results, collectively, provide evidence suggesting that exosome-mediated transfer of MTA1 contributes to BC progression by modifying cellular responses to important signaling pathways and that exosome-MTA1 may be developed as a biomarker and therapeutic target for BC.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Chen YM, Ou BT, Chen CY, et al (2019)

Staufen1 Protein Participates Positively in the Viral RNA Replication of Enterovirus 71.

Viruses, 11(2): pii:v11020142.

The double-stranded RNA-binding protein Staufen1 (Stau1) has multiple functions during RNA virus infection. In this study, we investigated the role of Stau1 in viral translation by using a combination of enterovirus 71 (EV-A71) infection, RNA reporter transfection, and in vitro functional and biochemical assays. We demonstrated that Stau1 specifically binds to the 5'-untranslated region of EV-A71 viral RNA. The RNA-binding domain 2-3 of Stau1 is responsible for this binding ability. Subsequently, we created a Stau1 knockout cell line using the CRISPR/Cas9 approach to further characterize the functional role of Stau1's interaction with viral RNA in the EV-A71-infected cells. Both the viral RNA accumulation and viral protein expression were downregulated in the Stau1 knockout cells compared with the wild-type naïve cells. Moreover, dysregulation of viral RNA translation was observed in the Stau1 knockout cells using ribosome fractionation assay, and a reduced RNA stability of 5'-UTR of the EV-A71 was also identified using an RNA stability assay, which indicated that Stau1 has a role in facilitating viral translation during EV-A71 infection. In conclusion, we determined the functional relevance of Stau1 in the EV-A71 infection cycle and herein describe the mechanism of Stau1 participation in viral RNA translation through its interaction with viral RNA. Our results suggest that Stau1 is an important host factor involved in viral translation and influential early in the EV-A71 replication cycle.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Jin H, Sophocleous A, Azfer A, et al (2019)

Analysis of Transcriptional Regulation in Bone Cells.

Methods in molecular biology (Clifton, N.J.), 1914:145-167.

Transcription is a process by which the rate of RNA synthesis is regulated. Here we describe the techniques for carrying out promoter-reporter assays, electrophoretic mobility shift assays, chromosome conformation capture (3C) assays, chromatin immunoprecipitation assays, and CRISPR-Cas9 assay, five commonly used methods for studying and altering gene transcription.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Kc M, CJ Steer (2019)

A new era of gene editing for the treatment of human diseases.

Swiss medical weekly, 149:w20021 pii:Swiss Med Wkly. 2019;149:w20021.

The treatment of human diseases using gene-editing technology has been envisioned for several decades with the realisation that so many were associated with mutations in DNA. The Human Genome Project opened new doors for identifying the genetic bases for human suffering. Research on gene editing has been active since the 1970s, but the technology has seen substantial growth and application just within the past decade. Simply stated, CRISPR technology has become a phenomenon in both biomedical and therapeutics research. Concurrently, cell therapies and pluripotent stem cell research have also been refined and now interfaced with CRISPR technology to enhance and maximise their potential in modelling as well as treatment of human diseases. In this review, we discuss the novel and revolutionary modality of gene editing, as this marks a new era in research and medicine. We also discuss gene-modifying technologies leading to CRISPR, as they are still being used for a wide variety of genomic applications. The modes and challenges for delivery of gene editing components are also discussed. Lastly, we review examples of human diseases that are not only amenable to gene editing techniques, but also show true promise of cure in the early 21st century of genetic correction and gene repair.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Fessenden M (2019)

Technologies to watch in 2019.

Nature, 565(7740):521-523.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Allen F, Behan F, Khodak A, et al (2019)

JACKS: joint analysis of CRISPR/Cas9 knockout screens.

Genome research, 29(3):464-471.

Genome-wide CRISPR/Cas9 knockout screens are revolutionizing mammalian functional genomics. However, their range of applications remains limited by signal variability from different guide RNAs that target the same gene, which confounds gene effect estimation and dictates large experiment sizes. To address this problem, we report JACKS, a Bayesian method that jointly analyzes screens performed with the same guide RNA library. Modeling the variable guide efficacies greatly improves hit identification over processing a single screen at a time and outperforms existing methods. This more efficient analysis gives additional hits and allows designing libraries with a 2.5-fold reduction in required cell numbers without sacrificing performance compared to current analysis standards.

RevDate: 2019-06-14
CmpDate: 2019-06-14

Hong H, MM Daadi (2019)

Generating Neural Stem Cells from iPSCs with Dopaminergic Neurons Reporter Gene.

Methods in molecular biology (Clifton, N.J.), 1919:119-128.

Genetic reporters offer attractive approaches to investigate gene expression, gene function, and spatiotemporal assessment in vitro and in vivo. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of the dopamine neurotransmitter, and thus it has been used as a reliable marker for dopaminergic neurons in vitro and in vivo. Herein we describe a method for making iPSC lines with TH-green fluorescent protein reporter gene using CRISPR/Cas9 technique.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Bao A, Burritt DJ, Chen H, et al (2019)

The CRISPR/Cas9 system and its applications in crop genome editing.

Critical reviews in biotechnology, 39(3):321-336.

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein9) system is an RNA-guided genome editing tool that consists of a Cas9 nuclease and a single-guide RNA (sgRNA). By base-pairing with a DNA target sequence, the sgRNA enables Cas9 to recognize and cut a specific target DNA sequence, generating double strand breaks (DSBs) that trigger cell repair mechanisms and mutations at or near the DSBs sites. Since its discovery, the CRISPR/Cas9 system has revolutionized genome editing and is now becoming widely utilized to edit the genomes of a diverse range of crop plants. In this review, we present an overview of the CRISPR/Cas9 system itself, including its mechanism of action, system construction strategies, and the screening methods used to identify mutants containing edited genes. We evaluate recent examples of the use of CRISPR/Cas9 for crop plant improvement, and research into the function(s) of genes involved in determining crop yields, quality, environmental stress tolerance/resistance, regulation of gene transcription and translation, and the construction of mutant libraries and production of transgene-free genome-edited crops. In addition, challenges and future opportunities for the use of the CRISPR/Cas9 system in crop breeding are discussed.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Jang Y, Choi J, Park N, et al (2019)

Development of immunocompatible pluripotent stem cells via CRISPR-based human leukocyte antigen engineering.

Experimental & molecular medicine, 51(1):3 pii:10.1038/s12276-018-0190-2.

Pluripotent stem cell transplantation is a promising regenerative strategy for treating intractable diseases. However, securing human leukocyte antigen (HLA)-matched donor stem cells is extremely difficult. The traditional approach for generating such cells is to establish homozygous pluripotent stem cell lines. Unfortunately, because of HLA diversity, this strategy is too time-consuming to be of practical use. HLA engineering of donor stem cells has been proposed recently as a means to evade graft-versus-host rejection in stem cell allotransplantation. This approach would be advantageous in both time and cost to the traditional method, but its feasibility must be investigated. In this study, we used CRISPR/Cas9 to knockout HLA-B from inducible pluripotent stem cells (iPSCs) with heterogenous HLA-B and showed that the HLA-B knockout iPSCs resulted in less immunogenicity in HLA-B antisera than that in the control. Our results support the feasibility of HLA-engineered iPSCs in stem cell allotransplantation.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Zhang YQ, Pei JH, Shi SS, et al (2019)

CRISPR/Cas9-mediated knockout of the PDEF gene inhibits migration and invasion of human gastric cancer AGS cells.

Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 111:76-85.

Gastric cancer is one of the most common malignant tumors worldwide and has the second highest incidence and mortality rate among malignant tumors in China. Prostate-derived Ets factor (PDEF) is a member of the Ets family of transcription factors. Although PDEF plays an important role in tumorigenesis, its biological function in gastric cancer is still unclear. Here, we evaluated PDEF expression in 30 cases of human gastric carcinoma and the corresponding peritumoral tissues, using immunohistochemistry and immunofluorescence. Significantly higher levels of PDEF were detected in tumors compared to peritumoral tissues. We then investigated PDEF expression in the gastric cancer cell lines SGC and AGS and the normal gastric epithelial cell line GES; The CRISPR/Cas9 genome-editing system was used to knockout PDEF in AGS cells as a model for gastric cancer. Cell proliferation, apoptosis, migration, and invasion of PDEF-knockout AGS cells were evaluated using CCK-8, flow cytometry, scratch wound, and transwell assays, respectively. The results illustrated that PDEF-knockout inhibited AGS cell proliferation, migration, and invasion. Taken together, the results imply that PDEF plays important roles in the proliferation, migration, and invasion of AGS cells and may serve as a new treatment target in gastric cancer.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Li B, Cao Y, Meng G, et al (2019)

Targeting glutaminase 1 attenuates stemness properties in hepatocellular carcinoma by increasing reactive oxygen species and suppressing Wnt/beta-catenin pathway.

EBioMedicine, 39:239-254.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive malignant disease with poor prognosis. Recent advances suggest the existence of cancer stem cells (CSCs) within liver cancer, which are considered to be responsible for tumor relapse, metastasis, and chemoresistance. However, novel therapeutic approaches for eradicating CSCs are yet to be established. Here, we aimed to identify the role of glutaminase 1 (GLS1) in stemness, and the feasibility that GLS1 serves as a therapeutic target for elimination CSCs as well as the possible mechanism.

METHODS: Publicly-available data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) was mined to unearth the association between GLS1 and stemness phenotype. Using big data, human tissues and multiple cell lines, we gained a general picture of GLS1 expression in HCC progression. We generated stable cell lines by lentiviral-mediated overexpression or CRISPR/Cas9-based knockout. Sphere formation assays and colony formation assays were employed to analyze the relationship between GLS1 and stemness. A series of bioinformatics analyses and molecular experiments including qRT-PCR, immunoblotting, flow cytometry, and immunofluorescence were employed to investigate the role of GLS1 in regulating stemness in vitro and in vivo.

FINDINGS: We observed GLS1 (both KGA and GAC isoform) is highly expressed in HCC, and that high expression of GAC predicts a poor prognosis. GLS1 is exclusively expressed in the mitochondrial matrix. Upregulation of GLS1 is positively associated with advanced clinicopathological features and stemness phenotype. Targeting GLS1 reduced the expression of stemness-related genes and suppressed CSC properties in vitro. We further found GLS1 regulates stemness properties via ROS/Wnt/β-catenin signaling and that GLS1 knockout inhibits tumorigenicity in vivo.

INTERPRETATION: Targeting GLS1 attenuates stemness properties in HCC by increasing ROS accumulation and suppressing Wnt/β-catenin pathway, which implied that GLS1 could serve as a therapeutic target for elimination of CSCs.

RevDate: 2019-06-13
CmpDate: 2019-06-11

Li C, B Zhang (2019)

Genome Editing in Cotton Using CRISPR/Cas9 System.

Methods in molecular biology (Clifton, N.J.), 1902:95-104.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as effective strategy for generating site-specific mutations. Recently, CRISPR/Cas9-mediated genome editing system have been rapidly optimized and applied in crop genetic improvement. In this chapter, we present a simple and high-efficiency method for crating targeted gene mutation in allotetraploid cotton genome using CRISPR/Cas9 system. This chapter will employ GhMYB25-like A and GhMYB25-like D that derived from upland cotton (Gossypium hirsutum) A subgenome and the D subgenome, respectively, as an example to introduce the procedure of how to generate effective mutations in cotton genome using CRISPR/Cas9-based biotechnology. Based on our previous results, this CRISPR/Cas9 system can induce a proportion of 14.2-21.4% fragment truncation events in GhMYB25-like A and GhMYB25-like D genome sites. In addition, PCR product sequencing results suggest that the mutation frequencies that occurred in GhMYB25-like A and GhMYB25-like D DNA sites are 100% and 98.8%, respectively. More important, the off-target-caused mutation events have not been detected in our transgenic plants, even one of the putative off-target site only have one nucleotide mismatch with the designed GhMYB25 sgRNA. Thus, this CRISPR/Cas9 method might be an effective approach for targeted mutagenesis in cotton genome.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Mallapaty S (2018)

Engineering a biomedical revolution.

Nature, 564(7735):S66-S68.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Rattanapornsompong K, Ngamkham J, Chavalit T, et al (2019)

Generation of Human Pyruvate Carboxylase Knockout Cell Lines Using Retrovirus Expressing Short Hairpin RNA and CRISPR-Cas9 as Models to Study Its Metabolic Role in Cancer Research.

Methods in molecular biology (Clifton, N.J.), 1916:273-288.

We report two protocols to generate human pyruvate carboxylase knockdown and knockout cell lines using short hairpin RNA (shRNA) and CRISPR-Cas9 technologies. The first protocol involved cloning of a shRNA cassette targeted to human pyruvate carboxylase (PC) under the control of a U6 promoter in a retrovirus-based vector. The stable knockdown cells were achieved following infection of retroviruses expressing shRNA in target cells followed by selecting these in medium containing puromycin. The second protocol describes a CRISPR Cas9-knockout cell constructed by cloning of single guide RNA (gRNA) targeted to the human pyruvate carboxylase gene placed adjacent to Cas 9 in the pSpCas9(BB)-2A-GFP vector. The knockout cells can be selected by sorting the cells expressing GFP. We also describe protocols for detecting the level of PC mRNA and protein in the knockdown or knockout cells using qPCR and Western blot analyses, respectively. The above protocols allow investigators to create PC deficient cell lines as a tool to study role of this enzyme in cancer research.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Endo M, Mikami M, Endo A, et al (2019)

Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM.

Nature plants, 5(1):14-17.

Streptococcus pyogenes Cas9 (SpCas9) is widely used for genome editing and requires NGG as a protospacer adjacent motif (PAM). Here, we show that the engineered SpCas9 (SpCas9-NGv1) can efficiently mutagenize endogenous target sites with NG PAMs in the rice and Arabidopsis genomes. Furthermore, we demonstrate that the SpCas9-NGv1 nickase fused to cytidine deaminase mediates C-to-T substitutions near the 5' end of the target sequence.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Pei W, SM Burgess (2019)

Microinjection in Zebrafish for Genome Editing and Functional Studies.

Methods in molecular biology (Clifton, N.J.), 1874:459-474.

A major strength of zebrafish as a model organism is their rapid, in vitro development. The easy access to embryos compared to mammals, allows larval molecular and cellular composition to be manipulated by microinjection, providing a powerful avenue for biological and translational studies. Here, we describe the essential steps and different applications of microinjection in zebrafish for genome editing and functional studies, along with some experimental tips that are critical for microinjection success.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Iyer J, DeVaul N, Hansen T, et al (2019)

Using Microinjection to Generate Genetically Modified Caenorhabditis elegans by CRISPR/Cas9 Editing.

Methods in molecular biology (Clifton, N.J.), 1874:431-457.

In this chapter, we describe the procedure for generating genetically modified Caenorhabditis elegans using microinjection via the Cas9-mediated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing technique. Specifically, we describe the detailed method of performing CRISPR editing by microinjection using the Cloning-free Co-CRISPR method described by the Seydoux lab. This microinjection protocol can also be used for CRISPR editing with protocols from other labs as well as for a variety of other editing techniques including Mos1-mediated single-copy transgene insertions (MosSCI), transcriptional activator-like nucleases (TALENs), and zinc-finger nucleases (ZFNs). Further, this microinjection protocol can also be used for injecting plasmid DNA to generate heritable extrachromosomal arrays for gene expression and mosaic analysis, performing RNAi by injection and delivering RNA, dyes or other molecules into the C. elegans germline.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Fan Z, Yang M, Regouski M, et al (2019)

Gene Knockouts in Goats Using CRISPR/Cas9 System and Somatic Cell Nuclear Transfer.

Methods in molecular biology (Clifton, N.J.), 1874:373-390.

The combination of CRISPR/Cas9 and SCNT techniques greatly facilitates the production of gene-edited livestock. Here, we describe the detailed procedure to create gene knockout goats using this strategy starting from the construction of CRISPR/Cas9 targeting vectors to the transfer of cloned embryos into recipient females. In this procedure, the transfection conditions for goat fibroblasts were optimized due to their high sensitivity to electrotransfection, which enabled the isolation of single-cell colonies carrying simultaneous disruption of multiple genes for SCNT with a single co-transfection of pooled CRISPR/Cas9 targeting vectors.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Li R, Miao J, Z Wang (2019)

Production of Genetically Engineered Porcine Embryos by Handmade Cloning.

Methods in molecular biology (Clifton, N.J.), 1874:347-360.

Genetic engineering is essential to realize the full potentials of pigs both as livestock and as animal models of human disease. With the development of new genetic engineering technologies, such as the clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system, the porcine genome can be engineered with high efficiency. In this chapter, we describe a protocol in employing the CRISPR/Cas9 system to genetically engineer the porcine genome in fibroblast cells, the procedures to establish single-cell-derived porcine fibroblast cell colonies carrying the desired genetic modifications, and the handmade cloning (HMC) technique to generate cloned embryos ready for embryo transfer.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Yang D, Xu J, YE Chen (2019)

Generation of Rabbit Models by Gene Editing Nucleases.

Methods in molecular biology (Clifton, N.J.), 1874:327-345.

Due to the lack of germline transmitting pluripotent stem cells (PSCs) cell lines and the extreme difficulty of somatic cell nuclear transfer (SCNT) in rabbit, the gene targeting technology in rabbit was lagging far behind those in rodents and in farm animals. As a result, the development and application of genetically engineered rabbit model are much limited. With the advent of gene editing nucleases, including ZFN, TALEN, and CRISPR/Cas9, it is now possible to produce gene targeting (i.e., knockout, knockin) rabbits with high success rates. In this chapter, we describe a comprehensive, step-by-step protocol for rabbit genome editing based on gene editing nucleases with specific emphasis of CRISPR/Cas9.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Alcantar TM, XY Rairdan (2019)

Microinjection and Oviduct Transfer Procedures for Rat Model Generation with CRISPR-Cas9 Technology.

Methods in molecular biology (Clifton, N.J.), 1874:273-294.

Since the first knockout rat model was generated with zinc-finger nucleases (ZFNs) by Geurt's group in 2009, the demand for making targeted rat models has increased tremendously. The advent of the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system provides researchers with a more efficient method for producing modified animals, which has since then been developed and applied in rat. Since we established a rat model production system at our facility in 2014, we have consistently generated rat models. Due to differences in physiology and embryology between mouse and rat, species-specific protocols for superovulation conditions, microinjection, and embryo transfer (among others) are required. There are over 100 rat strains, and Sprague Dawley is one of the commonly used outbred strains in biomedical research. In this chapter, we describe in detail a range of topics including donor and recipient preparation, microinjection setup, CRISPR reagent preparation, and oviduct transfer procedures for making rat models in the Sprague Dawley background.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Sakurai T, Kamiyoshi A, Ohtsuka M, et al (2019)

Isolation and Analysis of a Genome-Edited Single-Hepatocyte from a Cas9 Transgenic Mouse Line.

Methods in molecular biology (Clifton, N.J.), 1874:257-271.

The primary cells isolated from the freshly dissected organ are thought to be different from those cultured for a long time in vitro. For instance, hepatocytes isolated in situ from the liver, display the ability to produce albumin, cultured for about a week often tend to cease production of albumin, including loss of proliferation capability. Thus, it is difficult to perform genome editing (i.e., production of genome-edited hepatocytes by in vitro gene delivery) in such cultured cells. Furthermore, hepatic cell lines available so far do not produce albumin and they would also have lost several characteristics of native liver cells. This poses a serious disadvantage when researchers want to study gene expression profiles under specific experimental settings, for example before and after genome editing. However, this demerit can be overcome if genome-editing is performed in situ in liver and single hepatocytes (both genome-edited and wild-type) can be isolated for analysis immediately following transient gene editing. Previously, we demonstrated successful isolation of genome-edited single hepatocytes, using mice expressing systemic Cas9 transgene (called "sCAT" mouse) and by tail-vein-mediated hydrodynamics-based gene delivery of gRNA targeted to Albumin gene (Sakurai et al., Sci Rep 6:20011, 2016). Here, we describe the detailed protocols for collection and analysis of single genome-edited hepatocytes, which will be useful for many types of hepatocyte functional studies.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Liu Y, Du Y, Xie W, et al (2019)

Generation of Conditional Knockout Mice by Sequential Insertion of Two loxP Sites In Cis Using CRISPR/Cas9 and Single-Stranded DNA Oligonucleotides.

Methods in molecular biology (Clifton, N.J.), 1874:191-210.

Conditional knockout (cKO) mice are extremely valuable for biomedical research because they enable detailed analyses of gene functions in a tissue- or temporally-specific fashion. The conventional method for generating cKO mice is time consuming and labor intensive, which involves making a large gene-targeting construct, transfecting and screening many embryonic stem (ES) cell clones, injecting positive ES clones into blastocysts to produce chimeric mice, and breeding the chimeras to transmit the targeted gene through the germline. Recently developed CRISPR technology has revolutionized the way genetically engineered organisms are created. Knockout and knockin mice can now be made by directly injecting zygotes with Cas9, sgRNA, and donor DNA. In theory, cKO mice can be generated by simultaneously inserting two loxP sites using two sgRNAs and two oligonucleotides as donors, but in practice the probability of obtaining cKO mice in one step is still very low, partly because the efficiency of oligo-mediated knockin is much lower than non-homologous end joining (NHEJ) and partly because co-cutting juxtaposed sites in one allele at the same time often leads to the deletion of the entire fragment between the two cutting sites. Therefore, many laboratories prefer to insert the two loxP sites sequentially, i.e., generating mice with one loxP first and then use embryos collected from these mice to insert the second loxP site. In this chapter, we describe our procedures and timeline using this sequential method to make a Six6 cKO mouse line as a demonstration of its feasibility.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Qin W, H Wang (2019)

Delivery of CRISPR-Cas9 into Mouse Zygotes by Electroporation.

Methods in molecular biology (Clifton, N.J.), 1874:179-190.

The CRISPR-Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including the mice. In this scheme, components of the CRISPR-Cas9 system are delivered into the mouse zygote and mutant mice carrying genetic modifications derived. Although microinjection has been the technology of choice, electroporation has also emerged and been proven to be effective delivering CRISPR-Cas9 reagents into the mouse zygote. Here, we describe the experimental protocol employing electroporation to deliver CRISPR-Cas9 reagents into mouse embryos and derive gene-edited mutant mice.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Scott MA, YC Hu (2019)

Generation of CRISPR-Edited Rodents Using a Piezo-Driven Zygote Injection Technique.

Methods in molecular biology (Clifton, N.J.), 1874:169-178.

Direct modification of the genome of the zygotes (i.e., one-cell embryos) by the CRISPR/Cas9-editing reagents, followed by embryo transfer to pseudopregnant females for live birth, has been the most effective method to generate laboratory rodent models for research. The method relies on proper delivery of the editing reagents into zygotes, which is commonly achieved by a standard or slightly modified pronuclear microinjection technique. In this chapter, we describe in detail an alternative delivery method, named piezo-driven cytoplasmic microinjection, which offers a superior embryo survival and birth rate. Because this method uses a much wider injection needle than that in pronuclear injection, it allows a larger volume of the editing materials to be transported into the zygotes, leading to an increase in the targeting efficiency. This also eliminates the clogging issues seen regularly in pronuclear injection. Moreover, Cytochalasin B that is used to soften zygotes during piezo-driven microinjection has been suggested a role in improving the knockin efficiency, which provides an additional benefit to use this injection method.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Du Y, Xie W, Zhang F, et al (2019)

Using CRISPR/Cas9 for Gene Knockout in Immunodeficient NSG Mice.

Methods in molecular biology (Clifton, N.J.), 1874:139-168.

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice are an immunodeficient strain that enables human cell xenografts. However, NSG mice possess a complex genetic background that would complicate cross-breeding with other inbred transgenic or knockout mouse strains to establish a congenic strain with a desired genetic modification in the NSG background. Newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology enables modification of the mouse genome at the zygote stage without the need for extensive cross-breeding or the use of embryonic stem cells. In this chapter, we use the knockout of the X-linked Cybb gene as an example to describe our procedures for genetically modifying NSG mice using the CRISPR/Cas9 method. Briefly, two sgRNAs were designed and made to target exon 1 and exon 3 of the Cybb gene, and either sgRNA was then microinjected together with Cas9 mRNA into fertilized eggs collected from NSG mice. The injected embryos are subsequently transferred into the oviducts of pseudopregnant surrogate mothers. Offspring born to the foster mothers were genotyped by PCR and DNA sequencing. In this chapter, we describe our experiment procedures in detail and report our genotyping results for demonstrating that NSG mice can be genetically modified using the CRISPR/Cas9 technology in a highly efficient manner.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Liu P, Li Y, Lei J, et al (2019)

Creating Knockin Alleles in Mouse Embryonic Stem Cells by CRISPR/Cas9-Mediated Homologous Recombination Without Drug Selection.

Methods in molecular biology (Clifton, N.J.), 1874:115-137.

The rapidly evolving CRISPR/Cas9-mediated genome editing provides the convenience of genome manipulation directly in mouse zygotes for a number of genomic manipulations; but knockins of large insertions prove to be relatively inefficient at least with double-strand DNA as targeting constructs. Here, we describe an alternative approach to the direct genome editing in mouse zygotes by generating knockin alleles in mouse embryonic stem cells first with CRIPSR-mediated homologous recombination. Our results show this approach is efficient and requires no drug selection in mouse embryonic stem cells as in classic gene targeting. As the result, knockin alleles across many target loci are created in mouse embryonic stem cells and readily transmitted through germline. The knockin alleles created in ES cells can also serve as valuable tools for in vitro stem cell differentiation.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Xu W (2019)

Microinjection and Micromanipulation: A Historical Perspective.

Methods in molecular biology (Clifton, N.J.), 1874:1-16.

Microinjection/micromanipulation is more than 100 years old. It is a technique that is instrumental in biomedical research and healthcare. Its longevity lies in its preciseness in mechanical retrieval, or delivery of biological materials, which in some cases is simply necessary or more effective than other retrieval/delivery means. Microinjection is favored for its straightforwardness in transferring contents from micromolecules to macromolecules and from organelles to cells. Microinjection/micromanipulation has been practiced over the century like an art form. Variations in handlings and instruments can be tolerated to a surprising degree with satisfactory outcomes. Throughout the century, microinjection developed as an indispensable tool along with the evolution of biomedical fields: from transgenics to gene targeting, from animal cloning to human infertility treatment, from nuclease-guided genetic engineering to RNA-guided genome editing (Fig. 1). The birth of the CRISPRology rejuvenated microinjection. For microinjection/micromanipulation, the second century has already begun with the early arrival of computerized instrumentation and lately of the high-throughput nanomanipulators potentially operable by artificial intelligence. As we yin-yang both systemic and precision approaches in research and medicine, microinjection will no doubt continue to find its unique place in the future.

RevDate: 2019-06-14
CmpDate: 2019-06-14

Sharon E, Chen SA, Khosla NM, et al (2018)

Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing.

Cell, 175(2):544-557.e16.

A major challenge in genetics is to identify genetic variants driving natural phenotypic variation. However, current methods of genetic mapping have limited resolution. To address this challenge, we developed a CRISPR-Cas9-based high-throughput genome editing approach that can introduce thousands of specific genetic variants in a single experiment. This enabled us to study the fitness consequences of 16,006 natural genetic variants in yeast. We identified 572 variants with significant fitness differences in glucose media; these are highly enriched in promoters, particularly in transcription factor binding sites, while only 19.2% affect amino acid sequences. Strikingly, nearby variants nearly always favor the same parent's alleles, suggesting that lineage-specific selection is often driven by multiple clustered variants. In sum, our genome editing approach reveals the genetic architecture of fitness variation at single-base resolution and could be adapted to measure the effects of genome-wide genetic variation in any screen for cell survival or cell-sortable markers.

RevDate: 2019-06-14
CmpDate: 2019-06-14

Leenay RT, Vento JM, Shah M, et al (2019)

Genome Editing with CRISPR-Cas9 in Lactobacillus plantarum Revealed That Editing Outcomes Can Vary Across Strains and Between Methods.

Biotechnology journal, 14(3):e1700583.

Lactic-acid bacteria such as Lactobacillus plantarum are commonly used for fermenting foods and as probiotics, where increasingly sophisticated genome-editing tools are employed to elucidate and enhance these microbes' beneficial properties. The most advanced tools to date utilize an oligonucleotide or double-stranded DNA donor for recombineering and Cas9 for targeted DNA cleavage. As the associated methods are often developed in isolation for one strain, it remains unclear how different Cas9-based editing methods compare across strains. Here, this work directly compares two methods in different strains of L. plantarum: one utilizing a plasmid-encoded recombineering template and another utilizing an oligonucleotide donor and an inducible DNA recombinase. This comparison reveals one instance in which only the recombineering-template method generates desired edits and another instance in which only the oligo method generates desired edits. It is further found that both methods exhibit highly variable success editing the same site across multiple L. plantarum strains. Finally, failure modes are identified for the recombineering-template method, including a consistent genomic deletion and reversion of a point mutation in the recombineering template. This study therefore highlights surprising differences for Cas9-mediated genome editing between methods and related strains, arguing for the need for multiple, distinct methods when performing CRISPR-based editing in bacteria.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Tibayrenc M, de Meeûs T, S Morand (2018)

Modern research tools to combat the infectious peril: just at the beginning.

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 63:291.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Liu R, Liang L, Choudhury A, et al (2019)

Multiplex navigation of global regulatory networks (MINR) in yeast for improved ethanol tolerance and production.

Metabolic engineering, 51:50-58.

Multiplex navigation of global regulatory networks (MINR) is an approach for combinatorially reprogramming gene expression to manipulate complex phenotypes. We designed, constructed, and mapped MINR libraries containing 43,020 specific mutations in 25 regulatory genes expected to perturb the yeast regulatory network. We selected growth competition experiments for library mutants conferring increased ethanol and/or glucose tolerance. We identified specific mutants that not only possessed improved ethanol and/or glucose tolerance but also produced ethanol at concentrations up to 2-fold higher than those produced by the wild-type strain. We further determined that mutations increasing ethanol tolerance were transferable to a diploid industrial yeast strain. The facile construction and mapping of 43,020 designer regulatory mutations provide a roadmap for how to access and engineer complex phenotypes in future synthetic biology and broader efforts.

RevDate: 2019-06-14
CmpDate: 2019-06-14

Stein T, Wollschlegel A, Te H, et al (2018)

Interferon regulatory factor 5 and nuclear factor kappa-B exhibit cooperating but also divergent roles in the regulation of pro-inflammatory cytokines important for the development of TH1 and TH17 responses.

The FEBS journal, 285(16):3097-3113.

A large body of data demonstrates that interferon regulatory factor 5 (IRF5) and nuclear factor kappa B (NF-κB) are the two major transcription factors in classically activated macrophages responsible for the transcriptional control of proinflammatory genes. Although recent evidence suggests that IRF5 interacts with certain members of the nuclear factor kappa B pathway, the extent of cooperation and its implications in disease are ambiguous. Since both pathways are known for their strong contributions in TLR8 signaling we used the human monocytic cell line THP-1.Dual, featuring gene reporters for NF-κB and IRFs, to simultaneously study the roles of IRF5 and the NF-κB subunit p65 in TLR8-mediated gene reporter activities. Furthermore, we profiled from these cells the proinflammatory cytokines involved in the differentiation of TH1 and TH17 cells. After ablation of IRF5 and/or p65 we activated the resultant cells with the TLR8 agonists R848 or the psoriasis-associated antimicrobial peptide LL-37 complexed with ssRNA and demonstrate that IRF5 deficiency drastically impairs the secretion of IL-1β, IL-6, IL-12, IL-23 and TNFα. In contrast, the lack of p65 impaired only IL-6, IL-12, and IL-23 secretion. Furthermore, we discovered that upon TLR8 stimulation, IRF5 but not NF-κB signaling is essential to provide a cytokine milieu supporting TH1 responses. Additionally, we demonstrate that IRF5 and NF-κB cooperate to provide a cytokine milieu supporting TH17 responses. Therefore, the distinct role of IRF5 in the intricate signaling network downstream of TLR8 may open new treatment options interfering with but not disrupting NF-κB signaling in human diseases.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Elison GL, M Acar (2018)

Scarless genome editing: progress towards understanding genotype-phenotype relationships.

Current genetics, 64(6):1229-1238.

The ability to predict phenotype from genotype has been an elusive goal for the biological sciences for several decades. Progress decoding genotype-phenotype relationships has been hampered by the challenge of introducing precise genetic changes to specific genomic locations. Here we provide a comparative review of the major techniques that have been historically used to make genetic changes in cells as well as the development of the CRISPR technology which enabled the ability to make marker-free disruptions in endogenous genomic locations. We also discuss how the achievement of truly scarless genome editing has required further adjustments of the original CRISPR method. We conclude by examining recently developed genome editing methods which are not reliant on the induction of a DNA double strand break and discuss the future of both genome engineering and the study of genotype-phenotype relationships.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Anonymous (2018)

CRISPR still needs microbiologists.

Nature microbiology, 3(6):641.

RevDate: 2019-06-14
CmpDate: 2019-06-14

Liu YM, Liu W, Jia JS, et al (2018)

Abnormalities of hair structure and skin histology derived from CRISPR/Cas9-based knockout of phospholipase C-delta 1 in mice.

Journal of translational medicine, 16(1):141.

BACKGROUND: Hairless mice have been widely applied in skin-related researches, while hairless pigs will be an ideal model for skin-related study and other biomedical researches because of the similarity of skin structure with humans. The previous study revealed that hairlessness phenotype in nude mice is caused by insufficient expression of phospholipase C-delta 1 (PLCD1), an essential molecule downstream of Foxn1, which encouraged us to generate PLCD1-deficient pigs. In this study, we plan to firstly produce PLCD1 knockout (KO) mice by CRISPR/Cas9 technology, which will lay a solid foundation for the generation of hairless PLCD1 KO pigs.

METHODS: Generation of PLCD1 sgRNAs and Cas 9 mRNA was performed as described (Shao in Nat Protoc 9:2493-2512, 2014). PLCD1-modified mice (F0) were generated via co-microinjection of PLCD1-sgRNA and Cas9 mRNA into the cytoplasm of C57BL/6J zygotes. Homozygous PLCD1-deficient mice (F1) were obtained by intercrossing of F0 mice with the similar mutation.

RESULTS: PLCD1-modified mice (F0) showed progressive hair loss after birth and the genotype of CRISPR/Cas9-induced mutations in exon 2 of PLCD1 locus, suggesting the sgRNA is effective to cause mutations that lead to hair growth defect. Homozygous PLCD1-deficient mice (F1) displayed baldness in abdomen and hair sparse in dorsa. Histological abnormalities of the reduced number of hair follicles, irregularly arranged and curved hair follicles, epidermal hyperplasia and disturbed differentiation of epidermis were observed in the PLCD1-deficient mice. Moreover, the expression level of PLCD1 was significantly decreased, while the expression levels of other genes (i.e., Krt1, Krt5, Krt13, loricrin and involucrin) involved in the differentiation of hair follicle were remarkerably increased in skin tissues of PLCD1-deficient mice.

CONCLUSIONS: In conclusion, we achieve PLCD1 KO mice by CRISPR/Cas9 technology, which provide a new animal model for hair development research, although homozygotes don't display completely hairless phenotype as expected.

RevDate: 2019-06-14
CmpDate: 2019-06-14

Lu Y, Zhou Q, Han Q, et al (2018)

Inactivation of deubiquitinase CYLD enhances therapeutic antibody production in Chinese hamster ovary cells.

Applied microbiology and biotechnology, 102(14):6081-6093.

Chinese hamster ovary (CHO) cells are promising host engineering cells for industry manufacturing of therapeutic antibodies. However, cell death due to apoptosis remains a huge challenge to augment antibody production, and developing CHO cells with enhanced anti-apoptosis and proliferation ability is fundamental for cell line development and high-yielding bioprocesses. Deubiquitinase cylindromatosis (CYLD) has been proved to be a tumor suppressor by negatively regulating NF-κB and Wnt/β-catenin signaling pathways. Its mutation or deletion is a common chromosome variation in several types of cancers. Here, we engineered CHO CYLD-/- cells by CRISPR-Cas9 editing technology. These cells displayed stronger cell proliferation and anti-apoptosis ability compared to parental cells. Three antibody expression plasmid kits were transiently transfected into these cells. Our data showed that inactivation of CYLD increased the highest titers of rituximab, Herceptin, and one bispecific antibody by 105, 63, and 228%, respectively. Reversely, overexpression of CYLD could promote cell apoptosis, whereas inhibiting cell proliferation and antibody production. Furthermore, inhibition of CYLD in CHO cells stably expressing an IgG antibody (CHO-IgG) achieved about 50% increase in product titer compared to parental cells. Meanwhile, inhibition of CYLD did not affect the quality of antibody. Thus, our data demonstrated that inactivation of CYLD could promote CHO cell proliferation, anti-apoptosis ability, and subsequent antibody production, suggesting that CYLD is a potential functional target for CHO cell engineering.

RevDate: 2019-06-13
CmpDate: 2019-06-11

Zhang QQ, Li Y, Fu ZY, et al (2018)

Intact Arabidopsis RPB1 functions in stem cell niches maintenance and cell cycling control.

The Plant journal : for cell and molecular biology, 95(1):150-167.

Plant meristem activity depends on accurate execution of transcriptional networks required for establishing optimum functioning of stem cell niches. An Arabidopsis mutant card1-1 (constitutive auxin response with DR5:GFP) that encodes a truncated RPB1 (RNA Polymerase II's largest subunit) with shortened C-terminal domain (CTD) was identified. Phosphorylation of the CTD repeats of RPB1 is coupled to transcription in eukaryotes. Here we uncover that the truncated CTD of RPB1 disturbed cell cycling and enlarged the size of shoot and root meristem. The defects in patterning of root stem cell niche in card1-1 indicates that intact CTD of RPB1 is necessary for fine-tuning the specific expression of genes responsible for cell-fate determination. The gene-edited plants with different CTD length of RPB1, created by CRISPR-CAS9 technology, confirmed that both the full length and the DK-rich tail of RPB1's CTD play roles in the accurate transcription of CYCB1;1 encoding a cell-cycle marker protein in root meristem and hence participate in maintaining root meristem size. Our experiment proves that the intact RPB1 CTD is necessary for stem cell niche maintenance, which is mediated by transcriptional regulation of cell cycling genes.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Tromp AT, Van Gent M, Abrial P, et al (2018)

Human CD45 is an F-component-specific receptor for the staphylococcal toxin Panton-Valentine leukocidin.

Nature microbiology, 3(6):708-717.

The staphylococcal bi-component leukocidins Panton-Valentine leukocidin (PVL) and γ-haemolysin CB (HlgCB) target human phagocytes. Binding of the toxins' S-components to human complement C5a receptor 1 (C5aR1) contributes to cellular tropism and human specificity of PVL and HlgCB. To investigate the role of both leukocidins during infection, we developed a human C5aR1 knock-in (hC5aR1KI) mouse model. HlgCB, but unexpectedly not PVL, contributed to increased bacterial loads in tissues of hC5aR1KI mice. Compared to humans, murine hC5aR1KI neutrophils showed a reduced sensitivity to PVL, which was mediated by the toxin's F-component LukF-PV. By performing a genome-wide CRISPR-Cas9 screen, we identified CD45 as a receptor for LukF-PV. The human-specific interaction between LukF-PV and CD45 provides a molecular explanation for resistance of hC5aR1KI mouse neutrophils to PVL and probably contributes to the lack of a PVL-mediated phenotype during infection in these mice. This study demonstrates an unsuspected role of the F-component in driving the sensitivity of human phagocytes to PVL.

RevDate: 2019-06-13
CmpDate: 2019-06-11

Koo T, Lu-Nguyen NB, Malerba A, et al (2018)

Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9.

Molecular therapy : the journal of the American Society of Gene Therapy, 26(6):1529-1538.

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle-wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the EGFP gene in the tibialis anterior muscle of the Dmd knockout mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out of frame to in frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9, has great potential for the treatment of DMD and other neuromuscular diseases.

RevDate: 2019-06-14
CmpDate: 2019-06-14

Arranz-Solís D, Regidor-Cerrillo J, Lourido S, et al (2018)

Toxoplasma CRISPR/Cas9 constructs are functional for gene disruption in Neospora caninum.

International journal for parasitology, 48(8):597-600.

Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite.

RevDate: 2019-06-14
CmpDate: 2019-06-14

Burle-Caldas GA, Soares-Simões M, Lemos-Pechnicki L, et al (2018)

Assessment of two CRISPR-Cas9 genome editing protocols for rapid generation of Trypanosoma cruzi gene knockout mutants.

International journal for parasitology, 48(8):591-596.

CRISPR/Cas9 technology has been used to edit genomes in a variety of organisms. Using the GP72 gene as a target sequence, we tested two distinct approaches to generate Trypanosoma cruzi knockout mutants using the Cas9 nuclease and in vitro transcribed single guide RNA. Highly efficient rates of disruption of GP72 were achieved either by transfecting parasites stably expressing Streptococcus pyogenes Cas9 with single guide RNA or by transfecting wild type parasites with recombinant Staphylococcus aureus Cas9 previously associated with single guide RNA. In both protocols, we used single-stranded oligonucleotides as a repair template for homologous recombination and insertion of stop codons in the target gene.

RevDate: 2019-06-13
CmpDate: 2019-06-13

Jacob F, Alam S, Konantz M, et al (2018)

Transition of Mesenchymal and Epithelial Cancer Cells Depends on α1-4 Galactosyltransferase-Mediated Glycosphingolipids.

Cancer research, 78(11):2952-2965.

The reversible transitions of cancer cells between epithelial and mesenchymal states comprise cellular and molecular processes essential for local tumor growth and respective dissemination. We report here that globoside glycosphingolipid (GSL) glycosyltransferase-encoding genes are elevated in epithelial cells and correlate with characteristic EMT signatures predictive of disease outcome. Depletion of globosides through CRISPR-Cas9-mediated deletion of the key enzyme A4GALT induces EMT, enhances chemoresistance, and increased CD24low/CD44high cells. The cholera toxin-induced mesenchymal-to-epithelial transition occurred only in cells with functional A4GALT. Cells undergoing EMT lost E-cadherin expression through epigenetic silencing at the promoter region of CDH1 However, in ΔA4GALT cells, demethylation was able to rescue E-cadherin-mediated cell-cell adhesion only in the presence of exogenous A4GALT. Overall, our data suggest another class of biomolecules vital for epithelial cancer cells and for maintaining cell integrity and function.Significance: This study highlights the essential role of glycosphingolipids in the maintenance of epithelial cancer cell properties. Cancer Res; 78(11); 2952-65. ©2018 AACR.

RevDate: 2019-06-13
CmpDate: 2019-06-12

Yu G, Cheng CJ, Lin SC, et al (2018)

Organelle-Derived Acetyl-CoA Promotes Prostate Cancer Cell Survival, Migration, and Metastasis via Activation of Calmodulin Kinase II.

Cancer research, 78(10):2490-2502.

Although emerging evidence suggests a potential role of calcium/calmodulin-dependent kinase II (CaMKII) in prostate cancer, its role in prostate cancer tumorigenesis is largely unknown. Here, we examine whether the acetyl CoA-CaMKII pathway, first described in frog oocytes, promotes prostate cancer tumorigenesis. In human prostate cancer specimens, metastatic prostate cancer expressed higher levels of active CaMKII compared with localized prostate cancer. Correspondingly, basal CaMKII activity was significantly higher in the more tumorigenic PC3 and PC3-mm2 cells relative to the less tumorigenic LNCaP and C4-2B4 cells. Deletion of CaMKII by CRISPR/Cas9 in PC3-mm2 cells abrogated cell survival under low-serum conditions, anchorage-independent growth and cell migration; overexpression of constitutively active CaMKII in C4-2B4 cells promoted these phenotypes. In an animal model of prostate cancer metastasis, genetic ablation of CaMKII reduced PC3-mm2 cell metastasis from the prostate to the lymph nodes. Knockdown of the acetyl-CoA transporter carnitine acetyltransferase abolished CaMKII activation, providing evidence that acetyl-CoA generated from organelles is a major activator of CaMKII. Genetic deletion of the β-oxidation rate-limiting enzyme ACOX family proteins decreased CaMKII activation, whereas overexpression of ACOXI increased CaMKII activation. Overall, our studies identify active CaMKII as a novel connection between organelle β-oxidation and acetyl-CoA transport with cell survival, migration, and prostate cancer metastasis.Significance: This study identifies a cell metabolic pathway that promotes prostate cancer metastasis and suggests prostate cancer may be susceptible to β-oxidation inhibitors. Cancer Res; 78(10); 2490-502. ©2018 AACR.

RevDate: 2019-06-13
CmpDate: 2019-06-12

Gershkovitz M, Caspi Y, Fainsod-Levi T, et al (2018)

TRPM2 Mediates Neutrophil Killing of Disseminated Tumor Cells.

Cancer research, 78(10):2680-2690.

Neutrophils play a critical role in cancer, with both protumor and antitumor neutrophil subpopulations reported. The antitumor neutrophil subpopulation has the capacity to kill tumor cells and limit metastatic spread, yet not all tumor cells are equally susceptible to neutrophil cytotoxicity. Because cells that evade neutrophils have greater chances of forming metastases, we explored the mechanism neutrophils use to kill tumor cells. Neutrophil cytotoxicity was previously shown to be mediated by secretion of H2O2 We report here that neutrophil cytotoxicity is Ca2+ dependent and is mediated by TRPM2, a ubiquitously expressed H2O2-dependent Ca2+ channel. Perturbing TRPM2 expression limited tumor cell proliferation, leading to attenuated tumor growth. Concomitantly, cells expressing reduced levels of TRPM2 were protected from neutrophil cytotoxicity and seeded more efficiently in the premetastatic lung.Significance: These findings identify the mechanism utilized by neutrophils to kill disseminated tumor cells and to limit metastatic spread. Cancer Res; 78(10); 2680-90. ©2018 AACR.

RevDate: 2019-06-13
CmpDate: 2019-06-12

Sorrentino C, Ciummo SL, Cipollone G, et al (2018)

Interleukin-30/IL27p28 Shapes Prostate Cancer Stem-like Cell Behavior and Is Critical for Tumor Onset and Metastasization.

Cancer research, 78(10):2654-2668.

Prostate cancer stem-like cells (PCSLC) are believed to be responsible for prostate cancer onset and metastasis. Autocrine and microenvironmental signals dictate PCSLC behavior and patient outcome. In prostate cancer patients, IL30/IL27p28 has been linked with tumor progression, but the mechanisms underlying this link remain mostly elusive. Here, we asked whether IL30 may favor prostate cancer progression by conditioning PCSLCs and assessed the value of blocking IL30 to suppress tumor growth. IL30 was produced by PCSLCs in human and murine prostatic intraepithelial neoplasia and displayed significant autocrine and paracrine effects. PCSLC-derived IL30 supported PCSLC viability, self-renewal and tumorigenicity, expression of inflammatory mediators and growth factors, tumor immune evasion, and regulated chemokine and chemokine receptor genes, primarily via STAT1/STAT3 signaling. IL30 overproduction by PCSLCs promoted tumor onset and development associated with increased proliferation, vascularization, and myeloid cell recruitment. Furthermore, it promoted PCSLC dissemination to lymph nodes and bone marrow by upregulating the CXCR5/CXCL13 axis, and drove metastasis to lungs through the CXCR4/CXCL12 axis. These mechanisms were drastically hindered by IL30 knockdown or knockout in PCSLCs. Collectively, these results mark IL30 as a key driver of PCSLC behavior. Targeting IL30 signaling may be a potential therapeutic strategy against prostate cancer progression and recurrence.Significance: IL30 plays an important role in regulating prostate cancer stem-like cell behavior and metastatic potential, therefore targeting this cytokine could hamper prostate cancer progression or recurrence. Cancer Res; 78(10); 2654-68. ©2018 AACR.

RevDate: 2019-06-13
CmpDate: 2019-06-10

Ross MJ, PT Coates (2018)

Using CRISPR to inactivate endogenous retroviruses in pigs: an important step toward safe xenotransplantation?.

Kidney international, 93(1):4-6.

Xenotransplantation could theoretically provide an unlimited supply of organs for patients living with end-stage kidney disease and other end-stage organ failure, but severe rejection and concerns about possible transmission of zoonotic infections remain important obstacles. In a recent study, investigators used CRISPR-cas9 to generate genetically modified pigs in which all endogenous retroviruses were inactivated. This approach may address one important barrier to the feasibility of clinical trials of xenotransplantation.

RevDate: 2019-06-12

Klompe SE, Vo PLH, Halpin-Healy TS, et al (2019)

Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration.

Nature pii:10.1038/s41586-019-1323-z [Epub ahead of print].

Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a remarkable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyze RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in E. coli requires CRISPR- and transposon-associated molecular machineries, including a novel co-complex between Cascade and the transposition protein TniQ. Donor DNA integration occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep sequencing experiments reveal highly specific, genome-wide DNA integration across dozens of unique target sites. This work provides the first example of a fully programmable, RNA-guided integrase and lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.

RevDate: 2019-06-12

Rao MC (2019)

Physiology of Electrolyte Transport in the Gut: Implications for Disease.

Comprehensive Physiology, 9(3):947-1023.

We now have an increased understanding of the genetics, cell biology, and physiology of electrolyte transport processes in the mammalian intestine, due to the availability of sophisticated methodologies ranging from genome wide association studies to CRISPR-CAS technology, stem cell-derived organoids, 3D microscopy, electron cryomicroscopy, single cell RNA sequencing, transgenic methodologies, and tools to manipulate cellular processes at a molecular level. This knowledge has simultaneously underscored the complexity of biological systems and the interdependence of multiple regulatory systems. In addition to the plethora of mammalian neurohumoral factors and their cross talk, advances in pyrosequencing and metagenomic analyses have highlighted the relevance of the microbiome to intestinal regulation. This article provides an overview of our current understanding of electrolyte transport processes in the small and large intestine, their regulation in health and how dysregulation at multiple levels can result in disease. Intestinal electrolyte transport is a balance of ion secretory and ion absorptive processes, all exquisitely dependent on the basolateral Na+ /K+ ATPase; when this balance goes awry, it can result in diarrhea or in constipation. The key transporters involved in secretion are the apical membrane Cl- channels and the basolateral Na+ -K+ -2Cl- cotransporter, NKCC1 and K+ channels. Absorption chiefly involves apical membrane Na+ /H+ exchangers and Cl- /HCO3- exchangers in the small intestine and proximal colon and Na+ channels in the distal colon. Key examples of our current understanding of infectious, inflammatory, and genetic diarrheal diseases and of constipation are provided. © 2019 American Physiological Society. Compr Physiol 9:947-1023, 2019.

RevDate: 2019-06-12

Zhang B, Ye Y, Ye W, et al (2019)

Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d.

Nature communications, 10(1):2544 pii:10.1038/s41467-019-10507-3.

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3'-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.

RevDate: 2019-06-12

Śmiga M, Stępień P, Olczak M, et al (2019)

PgFur participates differentially in expression of virulence factors in more virulent A7436 and less virulent ATCC 33277 Porphyromonas gingivalis strains.

BMC microbiology, 19(1):127 pii:10.1186/s12866-019-1511-x.

BACKGROUND: Porphyromonas gingivalis is considered a keystone pathogen responsible for chronic periodontitis. Although several virulence factors produced by this bacterium are quite well characterized, very little is known about regulatory mechanisms that allow different strains of P. gingivalis to efficiently survive in the hostile environment of the oral cavity, a typical habitat characterized by low iron and heme concentrations. The aim of this study was to characterize P. gingivalis Fur homolog (PgFur) in terms of its role in production of virulence factors in more (A7436) and less (ATCC 33277) virulent strains.

RESULTS: Expression of a pgfur depends on the growth phase and iron/heme concentration. To better understand the role played by the PgFur protein in P. gingivalis virulence under low- and high-iron/heme conditions, a pgfur-deficient ATCC 33277 strain (TO16) was constructed and its phenotype compared with that of a pgfur A7436-derived mutant strain (TO6). In contrast to the TO6 strain, the TO16 strain did not differ in the growth rate and hemolytic activity compared with the ATCC 33277 strain. However, both mutant strains were more sensitive to oxidative stress and they demonstrated changes in the production of lysine- (Kgp) and arginine-specific (Rgp) gingipains. In contrast to the wild-type strains, TO6 and TO16 mutant strains produced larger amounts of HmuY protein under high iron/heme conditions. We also demonstrated differences in production of glycoconjugates between the A7436 and ATCC 33277 strains and we found evidence that PgFur protein might regulate glycosylation process. Moreover, we revealed that PgFur protein plays a role in interactions with other periodontopathogens and is important for P. gingivalis infection of THP-1-derived macrophages and survival inside the cells. Deletion of the pgfur gene influences expression of many transcription factors, including two not yet characterized transcription factors from the Crp/Fnr family. We also observed lower expression of the CRISPR/Cas genes.

CONCLUSIONS: We show here for the first time that inactivation of the pgfur gene exerts a different influence on the phenotype of the A7436 and ATCC 33277 strains. Our findings further support the hypothesis that PgFur regulates expression of genes encoding surface virulence factors and/or genes involved in their maturation.

RevDate: 2019-06-10

Mougari S, Abrahao J, Oliveira GP, et al (2019)

Role of the R349 Gene and Its Repeats in the MIMIVIRE Defense System.

Frontiers in microbiology, 10:1147.

MIMIVIRE is a defense system described in lineage A Mimivirus (Mimiviridae family) that mediates resistance against Zamilon virophage. It is composed of putative helicase and nuclease associated with a gene of unknown function called R349, which contains four 15 bp repeats homologous to the virophage sequence. In a previous study, the silencing of such genes restored virophage susceptibility. Moreover, the CRISPR Cas-4 like activity of the nuclease has recently been characterized. In this study, a recently isolated Mimivirus of lineage A with R349 gene lacking 3 of 4 repeats was demonstrated to be susceptible to Zamilon. To reinforce the importance of the R349 gene in the MIMIVIRE system, we developed and presented, for the first time to our knowledge, a protocol for Mimivirus genomic editing. By knocking out R349 gene in a Mimivirus lineage A, we observed the replication of Zamilon, indicating that this gene is critical in the resistance against this specific group of virophages.

RevDate: 2019-06-09

Williams MA, O'Grady J, Ball B, et al (2019)

The application of CRISPR-Cas for single species identification from environmental DNA.

Molecular ecology resources [Epub ahead of print].

We report the first application of CRISPR-Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows non-invasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR-based techniques to detect a given species from eDNA samples, posing a logistical challenge for on-site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR-Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof-of-concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 h). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity. This article is protected by copyright. All rights reserved.

RevDate: 2019-06-08

Qu D, Lu S, Wang P, et al (2019)

Analysis of CRISPR/Cas system of Proteus and the factors affected the functional mechanism.

Life sciences pii:S0024-3205(19)30443-6 [Epub ahead of print].

BACKGROUND: The Proteus is one of the most common human and animal pathogens. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) are inheritable genetic elements found in a variety of archaea and bacteria in the evolution, providing immune function against foreign invasion.

OBJECTIVES: To analyze the characteristics and functions of the CRISPR/Cas system in Proteus genomes, as well as the internal and external factors affecting the system.

METHODS: CRISPR loci were identified and divided into groups based on the repeat sequence in 96 Proteus strains by identification. Compared the RNA secondary structure and minimum free energy of CRISPR loci through bioinformatics, the evolution of cas genes, and the effects of related elements were also discussed.

RESULTS: 85 CRISPR loci were identified and divided into six groups based on the sequence of repeats, and the more stable the secondary structure of RNA, the smaller the minimum free energy, the fewer base mutations in the repeat, the more stable the CRISPR and the more complete the evolution of the system. In addition, Cas1 gene can be a symbol to distinguish species to some extent. Of all the influencing factors, CRISPR/Cas had the greatest impact on plasmids.

CONCLUSIONS: This study examined the diversity of CRISPR/Cas system in Proteus and found statistically significant positive/negative correlations between variety factors (the RNA stability, free energy, etc.) and the CRISPR locus, which played a vital role in regulating the CRISPR/Cas system.

RevDate: 2019-06-07

Plateau P, Moch C, S Blanquet (2019)

Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1-Cas2 integrase.

The Journal of biological chemistry pii:RA119.007619 [Epub ahead of print].

Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro However, this nonspecific integration activity is rare in vivo Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1-Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mM spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor (IHF), spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1-Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.

RevDate: 2019-06-07

Strecker J, Ladha A, Gardner Z, et al (2019)

RNA-guided DNA insertion with CRISPR-associated transposases.

Science (New York, N.Y.) pii:science.aax9181 [Epub ahead of print].

CRISPR-Cas nucleases are powerful tools to manipulate nucleic acids; however, targeted insertion of DNA remains a challenge as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase (CAST) from cyanobacteria Scytonema hofmanni which consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60-66 bp downstream of the protospacer. ShCAST integrates DNA into unique sites in the E. coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.

RevDate: 2019-06-05

Gong T, Lu M, Zhou X, et al (2019)

CRISPR-Cas Systems in Streptococci.

Current issues in molecular biology, 32:1-38 pii:v32/1 [Epub ahead of print].

Streptococci are one of the most important and common constituents of the host's microbiota and can colonize and live in the upper respiratory and urogenital tract of humans and animals. The CRISPR-Cas systems (i.e., clustered regularly interspaced short palindromic repeat, with CRISPR-associated proteins) found in bacteria and archaea provide sequence-based adaptive immunity against mobile genetic elements, especially in the streptococci. Here, recent research progress on CRISPR-Cas systems in the streptococci is reviewed, including their classification (mainly type I, type II, and type III), physiological function, defense mechanism (CRISPR adaptation, crRNA biogenesis, and target interference) and applications, which are useful for a better understanding of the functions of such systems. Finally, the advances that have been made in streptococci may help in the discovery of further novel CRISPR-Cas systems for use in new technologies and applications in other species.

RevDate: 2019-06-05

Faure G, Shmakov SA, Yan WX, et al (2019)

CRISPR-Cas in mobile genetic elements: counter-defence and beyond.

Nature reviews. Microbiology pii:10.1038/s41579-019-0204-7 [Epub ahead of print].

The principal function of CRISPR-Cas systems in archaea and bacteria is defence against mobile genetic elements (MGEs), including viruses, plasmids and transposons. However, the relationships between CRISPR-Cas and MGEs are far more complex. Several classes of MGE contributed to the origin and evolution of CRISPR-Cas, and, conversely, CRISPR-Cas systems and their components were recruited by various MGEs for functions that remain largely uncharacterized. In this Analysis article, we investigate and substantially expand the range of CRISPR-Cas components carried by MGEs. Three groups of Tn7-like transposable elements encode 'minimal' type I CRISPR-Cas derivatives capable of target recognition but not cleavage, and another group encodes an inactivated type V variant. These partially inactivated CRISPR-Cas variants might mediate guide RNA-dependent integration of the respective transposons. Numerous plasmids and some prophages encode type IV systems, with similar predicted properties, that appear to contribute to competition among plasmids and between plasmids and viruses. Many prokaryotic viruses also carry CRISPR mini-arrays, some of which recognize other viruses and are implicated in inter-virus conflicts, and solitary repeat units, which could inhibit host CRISPR-Cas systems.

RevDate: 2019-06-05

Lu S, Yang N, He J, et al (2019)

Generation of Cancer-Specific Cytotoxic PD-1- T Cells Using Liposome-Encapsulated CRISPR/Cas System with Dendritic/Tumor Fusion Cells.

Journal of biomedical nanotechnology, 15(3):593-601.

T-cell immunotherapy is showing great promise and therefore undergoing intensive developments for cancer treatment. In this study, we applied liposome-encapsulated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9) genome editing tool to specifically knock out the programmed death-1 (PD-1) gene from T cells (PD-1- T cells). We then activated these cells by dendritic/tumor fusion cells (FCs) and examined their anti-cancer potential. Results showed that, following the antigen presentation and activation by DC/HepG2 FCs, PD-1- T cells showed a significantly higher ability than PD-1+ T cells to proliferate, secrete pro-inflammatory cytokine IFN-γ, and kill HepG2 cells in vitro. Consistently, in vitro activated PD-1- T cells inhibited proliferation and induced apoptosis in HepG2 xenografts in vivo, leading to significantly suppressed tumor growth and improved mouse survival. Liposome-encapsulated CRISPR/Cas9 genome editing technology effectively knocked out PD-1 gene in T cells, stimulating T cell activation in response to DC/tumor FCs and affording T cell-mediated cancer immunotherapy. Our study provides evidence to target checkpoint receptors in adoptively transfected T cells, as a novel therapeutic modality for adoptive T cell transfer.

RevDate: 2019-06-10

Nussbaum L, Telenius JM, Hill S, et al (2018)

High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates.

Methods and protocols, 1(3): pii:mps1030029.

The emergence in recent years of DNA editing technologies-Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors-have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis.

RevDate: 2019-06-10

Lv J, Wu S, Wei R, et al (2019)

The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells.

Journal of veterinary science, 20(3):e23.

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.

RevDate: 2019-06-10

Ren F, Ren C, Zhang Z, et al (2019)

Efficiency Optimization of CRISPR/Cas9-Mediated Targeted Mutagenesis in Grape.

Frontiers in plant science, 10:612.

Clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system is an efficient targeted genome editing method. Although CRISPR/Cas9-mediated mutagenesis has been applied successfully in grape, few studies have examined the technique's efficiency. To optimize CRISPR/Cas9 editing efficiency in Vitis vinifera, we surveyed three key parameters: GC content of single guide RNA (sgRNA), variety of transformant cells used, and SpCas9 expression levels in transgenic cell mass. Four sgRNAs with differing GC content were designed to target exon sites of the V. vinifera phytoene desaturase gene. Suspension cells of 'Chardonnay' and '41B' varieties were used as the transgenic cell mass. Both T7EI and PCR/RE assays showed that CRISPR/Cas9 editing efficiency increases proportionally with sgRNA GC content with 65% GC content yielding highest editing efficiency in both varieties. Additionally, gene editing was more efficient in '41B' than in 'Chardonnay.' CRISPR/Cas9 systems with different editing efficiency showed different SpCas9 expression level, but compared with GC content of sgRNA, SpCas9 expression level has less influence on editing efficiency. Taken together, these results help optimize of CRISPR/Cas9 performance in grape.

RevDate: 2019-06-02

Li Y, Liu L, G Liu (2019)

CRISPR/Cas Multiplexed Biosensing: A Challenge or an Insurmountable Obstacle?.

Performing multiplex detection is still an elusive goal for molecular diagnostics. CRISPR/Cas-based biosensing has demonstrated potential for multiplex detection. Instead of being an insurmountable obstacle, CRISPR/Cas multiplexed biosensing is a realistic challenge with some recent successful applications. Strategic considerations are required to fully explore its potential in multiplex diagnostics.

RevDate: 2019-05-31

Harper JC, G Schatten (2019)

Are we ready for genome editing in human embryos for clinical purposes?.

European journal of medical genetics pii:S1769-7212(19)30074-6 [Epub ahead of print].

Perhaps the two most significant pioneering biomedical discoveries with immediate clinical implications during the past forty years have been the advent of assisted reproductive technologies (ART) and the genetics revolution. ART, including in vitro fertilization (IVF), intracytoplasmic sperm injection and preimplantation genetic testing, has resulted in the birth of more than 8 million children, and the pioneer of IVF, Professor Bob Edwards, was awarded the 2010 Nobel Prize. The genetics revolution has resulted in our genomes being sequenced and many of the molecular mechanisms understood, and technologies for genomic editing have been developed. With the combination of nearly routine ART protocols for healthy conceptions together with almost error-free, inexpensive and simple methods for genetic modification, the question "Are we ready for genome editing in human embryos for clinical purposes?" was debated at the 5th congress on controversies in preconception, preimplantation and Prenatal Genetic Diagnosis, in collaboration with the Ovarian Club Meeting, in November 2018 in Paris. The co-authors each presented scientific, medical and bioethical backgrounds, and the debate was chaired by Professor Alan Handyside. In this paper, we consider whether genome editing is safe and ethical. We conclude that we are currently not ready for genome editing to be used in human embryos for clinical purposes, and we call for a global debate to determine if and when this technology could be used in ART. ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬.

RevDate: 2019-06-10

Stachler AE, Schwarz TS, Schreiber S, et al (2019)

CRISPRi as an efficient tool for gene repression in archaea.

Methods (San Diego, Calif.) pii:S1046-2023(18)30472-9 [Epub ahead of print].

In the years following its discovery and characterization, the CRISPR-Cas system has been modified and converted into a multitude of applications for eukaryotes and bacteria, such as genome editing and gene regulation. Since no such method has been available for archaea, we developed a tool for gene repression in the haloarchaeon Haloferax volcanii by repurposing its endogenous type I-B CRISPR-Cas system. Here, we present the two possible approaches for gene repression as well as our workflow to achieve and assess gene knockdown, offer recommendations on protospacer selection and give some examples of genes we have successfully silenced.

RevDate: 2019-05-31

Filho DM, de Carvalho Ribeiro P, Oliveira LF, et al (2019)

Enhancing the Therapeutic Potential of Mesenchymal Stem Cells with the CRISPR-Cas System.

Stem cell reviews pii:10.1007/s12015-019-09897-0 [Epub ahead of print].

Mesenchymal stem cells (MSCs), also known as multipotent mesenchymal stromal stem cells, are found in the perivascular space of several tissues. These cells have been subject of intense research in the last decade due to their low teratogenicity, as well as their ability to differentiate into mature cells and to secrete immunomodulatory and trophic factors. However, they usually promote only a modest benefit when transplanted in experimental disease models, one of the limitations for their clinical application. The CRISPR-Cas system, in turn, is highlighted as a simple and effective tool for genetic engineering. This system was tested in clinical trials over a relatively short period of time after establishing its applicability to the edition of the mammalian cell genome. Similar to the research evolution in MSCs, the CRISPR-Cas system demonstrated inconsistencies that limited its clinical application. In this review, we outline the evolution of MSC research and its applicability, and the progress of the CRISPR-Cas system from its discovery to the most recent clinical trials. We also propose perspectives on how the CRISPR-Cas system may improve the therapeutic potential of MSCs, making it more beneficial and long lasting.

RevDate: 2019-05-31

Pickar-Oliver A, CA Gersbach (2019)

The next generation of CRISPR-Cas technologies and applications.

Nature reviews. Molecular cell biology pii:10.1038/s41580-019-0131-5 [Epub ahead of print].

The prokaryote-derived CRISPR-Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues. Genome editing by CRISPR-Cas can utilize non-homologous end joining and homology-directed repair for DNA repair, as well as single-base editing enzymes. In addition to targeting DNA, CRISPR-Cas-based RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, the new advances are considerably improving our understanding of biological processes and are propelling CRISPR-Cas-based tools towards clinical use in gene and cell therapies.

RevDate: 2019-05-30

Liu R, Mendez-Rios JD, Peng C, et al (2019)

SPI-1 is a missing host-range factor required for replication of the attenuated modified vaccinia Ankara (MVA) vaccine vector in human cells.

PLoS pathogens, 15(5):e1007710 pii:PPATHOGENS-D-19-00021.

Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements.

RevDate: 2019-05-29

Patel VK, Soni N, Prasad V, et al (2019)

CRISPR-Cas9 System for Genome Engineering of Photosynthetic Microalgae.

Molecular biotechnology pii:10.1007/s12033-019-00185-3 [Epub ahead of print].

Targeted genome editing using RNA-guided endonucleases is an emerging tool in algal biotechnology. Recently, CRISPR-Cas systems have been widely used to manipulate the genome of some freshwater and marine microalgae. Among two different classes, and six distinct types of CRISPR systems, Cas9-driven type II system has been widely used in most of the studies for targeted knock-in, knock-out and knock-down of desired genes in algae. CRISPR technology has been demonstrated in microalgae including diatoms to manifest the function of the particular gene (s) and developing industrial traits, such as improving lipid content and biomass productivity. Instead of these, there are a lot of gears to be defined about improving efficiency and specificity of targeted genome engineering of microalgae using CRISPR-Cas system. Optimization of tools and methods of CRISPR technology can undoubtedly transform the research toward the industrial-scale production of commodity chemicals, food and biofuels using photosynthetic cell factories. This review has been focused on the efforts made so far to targeted genome engineering of microalgae, identified scopes about the hurdles related to construction and delivery of CRISPR-Cas components, algae transformation toolbox, and outlined the future prospect toward developing the CRISPR platform for high-throughput genome-editing of microalgae.

RevDate: 2019-05-29

Lin DW, Chung BP, Huang JW, et al (2019)

Microhomology based CRISPR tagging tools for protein tracking, purification, and depletion.

The Journal of biological chemistry pii:RA119.008422 [Epub ahead of print].

Work in yeast models has tremendously benefited from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions. In contrast, work in mammalian cells largely relied on the overexpression of tagged proteins because high quality antibodies are only available for a fraction of the mammalian proteome. CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool, which can also be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins in most organisms, including mammals. Here we describe a versatile tool set for rapid tagging of endogenous proteins. The strategy utilizes CRISPR/Cas9 and microhomology-mediated end-joining (MMEJ) repair for efficient tagging. We provide tools to insert 3xHA (hemagglutinin), 6xHisFLAG, HBTH, mCherry, GFP, and the AID tag for compound induced protein depletion. This approach and the developed tools should greatly facilitate functional analysis of proteins in their native environment.

RevDate: 2019-05-29

Sebastian J, Hegde K, Kumar P, et al (2019)

Bioproduction of fumaric acid: an insight into microbial strain improvement strategies.

Critical reviews in biotechnology [Epub ahead of print].

Fumaric acid (FA), a metabolic intermediate, has been identified as an important carbohydrate derived platform chemical. Currently, it is commercially sourced from petrochemicals by chemical conversion. The shift to biochemical synthesis has become essential for sustainable development and for the transition to a biobased economy from a petroleum-based economy. The main limitation is that the concentrations of FA achieved during bioproduction are lower than that from a chemical process. Moreover, the high cost associated with bioproduction necessitates a higher yield to improve the feasibility of the process. To this effect, genetic modification of microorganism can be considered as an important tool to improve FA yield. This review discusses various genetic modifications strategies that have been studied in order to improve FA production. These strategies include the development of recombinant strains of Rhizopus oryzae, Escherichia coli, Saccharomyces cerevisiae, and Torulopsis glabrata as well as their mutants. The transformed strains were able to accumulate fumaric acid at a higher concentration than the corresponding wild strains but the fumaric acid titers obtained were lower than that reported with native fumaric acid producing R. oryzae strains. Moreover, one plausible adoption of gene editing tools, such as Agrobacterium-mediated transformation (AMT), CRISPR CAS-9 and RNA interference (RNAi) mediated knockout and silencing, have been proposed in order to improve fumaric acid yield. Additionally, the introduction of the glyoxylate pathway in R. oryzae to improve fumaric acid yield as well as the biosynthesis of fumarate esters have been proposed to improve the economic feasibility of the bioprocess. The adoption of some of these genetic engineering strategies may be essential to enable the development of a feasible bioproduction process.

RevDate: 2019-06-06

Kim T, TK Lu (2019)

CRISPR/Cas-based devices for mammalian synthetic biology.

Current opinion in chemical biology, 52:23-30 pii:S1367-5931(19)30024-9 [Epub ahead of print].

Since its first demonstration for mammalian gene editing, CRISPR/Cas technology has been widely adopted in research, industry, and medicine. Beyond indel mutations induced by Cas9 activity, recent advances in CRISPR/Cas have enabled DNA or RNA base editing. In addition, multiple orthogonal methods for the spatiotemporal regulation of CRISPR/Cas activity and repurposed Cas proteins for the visualization and relocation of specific genomic loci in living cells have been described. By harnessing the versatility of CRISPR/Cas-based devices and gene circuits, synthetic biologists are developing memory devices for lineage tracing and technologies for unbiased, high-throughput interrogation of combinatorial gene perturbations. We envision that such approaches will enable researchers to gain deeper insights into the translation of genotypes to phenotypes in healthy and diseased states.

RevDate: 2019-05-28

Long J, Xu Y, Ou L, et al (2019)

Diversity of CRISPR/Cas system in Clostridium perfringens.

Molecular genetics and genomics : MGG pii:10.1007/s00438-019-01579-3 [Epub ahead of print].

Clostridium perfringens is an important pathogen of human and livestock infections, posing a threat to health. The horizontal gene transfer (HGT) of plasmids that carry toxin-related genes is involved in C. perfringens pathogenicity. The CRISPR/Cas system, which has been identified in a wide range of prokaryotes, provides acquired immunity against HGT. However, information about the CRISPR/Cas system in Clostridium perfringens is still limited. In this study, 111 C. perfringens strains with publicly available genomes were used to analyze the occurrence and diversity of CRISPR/Cas system and evaluate the potential of CRISPR-based genotyping in this multi-host pathogen. A total of 59 out of the 111 genomes harbored at least one confirmed CRISPR array. Four CRISPR/Cas system subtypes, including subtypes IB, IIA, IIC, and IIID systems, were identified in 32 strains. Subtype IB system was the most prevalent in this species, which was subdivided into four subgroups displaying subgroup specificity in terms of cas gene content, repeat sequence content, and PAM. We showed that the CRISPR spacer polymorphism can be used for evolutionary studies, and that it can provide discriminatory power for typing strains. Nevertheless, the application of this approach was largely limited to strains that contain the CRISPR/Cas system. Spacer origin analysis revealed that approximately one-fifth of spacers showed significant matches to plasmids and phages, thereby suggesting the implication of CRISPR/Cas systems in controlling HGT. Collectively, our results provide new insights into the diversity and evolution of CRISPR/Cas system in C. perfringens.

RevDate: 2019-06-10

Regmi A, EF Boyd (2019)

Carbohydrate metabolic systems present on genomic islands are lost and gained in Vibrio parahaemolyticus.

BMC microbiology, 19(1):112 pii:10.1186/s12866-019-1487-6.

BACKGROUND: Utilizing unique carbohydrates or utilizing them more efficiently help bacteria expand and colonize new niches. Horizontal gene transfer (HGT) of catabolic systems is a powerful mechanism by which bacteria can acquire new phenotypic traits that can increase survival and fitness in different niches. In this work, we examined carbon catabolism diversity among Vibrio parahaemolyticus, a marine species that is also an important human and fish pathogen.

RESULTS: Phenotypic differences in carbon utilization between Vibrio parahaemolyticus strains lead us to examine genotypic differences in this species and the family Vibrionaceae in general. Bioinformatics analysis showed that the ability to utilize D-galactose was present in all V. parahaemolyticus but at least two distinct transporters were present; a major facilitator superfamily (MFS) transporter and a sodium/galactose transporter (SGLT). Growth and genetic analyses demonstrated that SGLT was a more efficient transporter of D-galactose and was the predominant type among strains. Phylogenetic analysis showed that D-galactose gene galM was acquired multiples times within the family Vibrionaceae and was transferred between distantly related species. The ability to utilize D-gluconate was universal within the species. Deletion of eda (VP0065), which encodes aldolase, a key enzyme in the Entner-Doudoroff (ED) pathway, reached a similar biomass to wild type when grown on D-gluconate as a sole carbon source. Two additional eda genes were identified, VPA1708 (eda2) associated with a D-glucuronate cluster and VPA0083 (eda3) that clustered with an oligogalacturonide (OGA) metabolism cluster. EDA2 and EDA3 were variably distributed among the species. A metabolic island was identified that contained citrate fermentation, L-rhamnose and OGA metabolism clusters as well as a CRISPR-Cas system. Phylogenetic analysis showed that CitF and RhaA had a limited distribution among V. parahaemolyticus, and RhaA was acquired at least three times. Within V. parahaemolyticus, two different regions contained the gene for L-arabinose catabolism and most strains had the ability to catabolism this sugar.

CONCLUSION: Our data suggest that horizontal transfer of metabolic systems among Vibrionaceae is an important source of metabolic diversity. This work identified four EDA homologues suggesting that the ED pathway plays a significant role in metabolism. We describe previously uncharacterized metabolism islands that were hotspots for the gain and loss of functional modules likely mediated by transposons.

RevDate: 2019-06-10

Sharma D, Misba L, AU Khan (2019)

Antibiotics versus biofilm: an emerging battleground in microbial communities.

Antimicrobial resistance and infection control, 8:76 pii:533.

Biofilm is a complex structure of microbiome having different bacterial colonies or single type of cells in a group; adhere to the surface. These cells are embedded in extracellular polymeric substances, a matrix which is generally composed of eDNA, proteins and polysaccharides, showed high resistance to antibiotics. It is one of the major causes of infection persistence especially in nosocomial settings through indwelling devices. Quorum sensing plays an important role in regulating the biofilm formation. There are many approaches being used to control infections by suppressing its formation but CRISPR-CAS (gene editing technique) and photo dynamic therapy (PDT) are proposed to be used as therapeutic approaches to subside bacterial biofim infections, especially caused by deadly drug resistant bad bugs.

RevDate: 2019-06-10

Chen LX, Al-Shayeb B, Méheust R, et al (2019)

Candidate Phyla Radiation Roizmanbacteria From Hot Springs Have Novel and Unexpectedly Abundant CRISPR-Cas Systems.

Frontiers in microbiology, 10:928.

The Candidate Phyla Radiation (CPR) comprises a huge group of bacteria that have small genomes that rarely encode CRISPR-Cas systems for phage defense. Consequently, questions remain about their mechanisms of phage resistance and the nature of phage that infect them. The compact CRISPR-CasY system (Cas12d) with potential value in genome editing was first discovered in these organisms. Relatively few CasY sequences have been reported to date, and little is known about the function and activity of these systems in the natural environment. Here, we conducted a genome-resolved metagenomic investigation of hot spring microbiomes and recovered CRISPR systems mostly from Roizmanbacteria that involve CasY proteins that are divergent from published sequences. Within population diversity in the spacer set indicates current in situ diversification of most of the loci. In addition to CasY, some Roizmanbacteria genomes also encode large type I-B and/or III-A systems that, based on spacer targeting, are used in phage defense. CRISPR targeting identified three phage represented by complete genomes and a prophage, which are the first reported for bacteria of the Microgenomates superphylum. Interestingly, one phage encodes a Cas4-like protein, a scenario that has been suggested to drive acquisition of self-targeting spacers. Consistent with this, the Roizmanbacteria population that it infects has a CRISPR locus that includes self-targeting spacers and a fragmented CasY gene (fCasY). Despite gene fragmentation, the PAM sequence is the same as that of other CasY reported in this study. Fragmentation of CasY may avoid the lethality of self-targeting spacers. However, the spacers may still have some biological role, possibly in genome regulation. The findings expand our understanding of CasY diversity, and more broadly, CRISPR-Cas systems and phage of CPR bacteria.

RevDate: 2019-05-25

Mann CM, Martínez-Gálvez G, Welker JM, et al (2019)

The Gene Sculpt Suite: a set of tools for genome editing.

Nucleic acids research pii:5498621 [Epub ahead of print].

The discovery and development of DNA-editing nucleases (Zinc Finger Nucleases, TALENs, CRISPR/Cas systems) has given scientists the ability to precisely engineer or edit genomes as never before. Several different platforms, protocols and vectors for precision genome editing are now available, leading to the development of supporting web-based software. Here we present the Gene Sculpt Suite (GSS), which comprises three tools: (i) GTagHD, which automatically designs and generates oligonucleotides for use with the GeneWeld knock-in protocol; (ii) MEDJED, a machine learning method, which predicts the extent to which a double-stranded DNA break site will utilize the microhomology-mediated repair pathway; and (iii) MENTHU, a tool for identifying genomic locations likely to give rise to a single predominant microhomology-mediated end joining allele (PreMA) repair outcome. All tools in the GSS are freely available for download under the GPL v3.0 license and can be run locally on Windows, Mac and Linux systems capable of running R and/or Docker. The GSS is also freely available online at www.genesculpt.org.

RevDate: 2019-05-25

Sampson TR, Saroj SD, Llewellyn AC, et al (2019)

Author Correction: A CRISPR/Cas system mediates bacterial innate immune evasion and virulence.

Change history: We could not replicate the results in Fig. 2a and g of this Letter, and new information has revealed a flaw in the interpretation of Fig. 2h. As a result, we do not have evidence to support RNA degradation as the mechanism that underlies Cas9-mediated regulation of FTN_1103 mRNA expression; see accompanying Amendment. This has not been corrected online.

RevDate: 2019-05-25

Diakatou M, Manes G, Bocquet B, et al (2019)

Genome Editing as a Treatment for the Most Prevalent Causative Genes of Autosomal Dominant Retinitis Pigmentosa.

International journal of molecular sciences, 20(10): pii:ijms20102542.

: Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of diseases with more than 250 causative genes. The most common form is retinitis pigmentosa. IRDs lead to vision impairment for which there is no universal cure. Encouragingly, a first gene supplementation therapy has been approved for an autosomal recessive IRD. However, for autosomal dominant IRDs, gene supplementation therapy is not always pertinent because haploinsufficiency is not the only cause. Disease-causing mechanisms are often gain-of-function or dominant-negative, which usually require alternative therapeutic approaches. In such cases, genome-editing technology has raised hopes for treatment. Genome editing could be used to i) invalidate both alleles, followed by supplementation of the wild type gene, ii) specifically invalidate the mutant allele, with or without gene supplementation, or iii) to correct the mutant allele. We review here the most prevalent genes causing autosomal dominant retinitis pigmentosa and the most appropriate genome-editing strategy that could be used to target their different causative mutations.

RevDate: 2019-06-09

Hameed A, Shan-E-Ali Zaidi S, Sattar MN, et al (2019)

CRISPR technology to combat plant RNA viruses: A theoretical model for Potato virus Y (PVY) resistance.

Microbial pathogenesis, 133:103551 pii:S0882-4010(18)31566-3 [Epub ahead of print].

RNA viruses are the most diverse phytopathogens which cause severe epidemics in important agricultural crops and threaten the global food security. Being obligatory intracellular pathogens, these viruses have developed fine-tuned evading mechanisms and are non-responsive to most of the prophylactic treatments. Additionally, their sprint ability to overcome host defense demands a broad-spectrum and durable mechanism of resistance. In context of CRISPR-Cas discoveries, some variants of Cas effectors have been characterized as programmable RNA-guided RNases in the microbial genomes and could be reprogramed in mammalian and plant cells with guided RNase activity. Recently, the RNA variants of CRISPR-Cas systems have been successfully employed in plants to engineer resistance against RNA viruses. Some variants of CRISPR-Cas9 have been tamed either for directly targeting plant RNA viruses' genome or through targeting the host genes/factors assisting in viral proliferation. The new frontiers in CRISPR-Cas discoveries, and more importantly shifting towards RNA targeting will pyramid the opportunities in plant virus research. The current review highlights the probable implications of CRISPR-Cas system to confer the pathogen-derived or host-mediated resistance against phytopathogenic RNA viruses. Furthermore, a multiplexed CRISPR-Cas13a methodology is proposed here to combat Potato virus Y (PVY); a globally diverse phytopathogen infecting multiple crops.

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In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.

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In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.

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Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.

ESP Content

When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.

ESP Help

Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.

ESP Plans

With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.

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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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Papers in Classical Genetics

The ESP began as an effort to share a handful of key papers from the early days of classical genetics. Now the collection has grown to include hundreds of papers, in full-text format.

Digital Books

Along with papers on classical genetics, ESP offers a collection of full-text digital books, including many works by Darwin (and even a collection of poetry — Chicago Poems by Carl Sandburg).

Timelines

ESP now offers a much improved and expanded collection of timelines, designed to give the user choice over subject matter and dates.

Biographies

Biographical information about many key scientists.

Selected Bibliographies

Bibliographies on several topics of potential interest to the ESP community are now being automatically maintained and generated on the ESP site.

ESP Picks from Around the Web (updated 07 JUL 2018 )