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Bibliography on: CRISPR-Cas

The Electronic Scholarly Publishing Project: Providing world-wide, free access to classic scientific papers and other scholarly materials, since 1993.


ESP: PubMed Auto Bibliography 26 Mar 2019 at 01:36 Created: 


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: 2019-03-25

Brandt K, R Barrangou (2019)

Applications of CRISPR Technologies Across the Food Supply Chain.

Annual review of food science and technology, 10:133-150.

The food industry faces a 2050 deadline for the advancement and expansion of the food supply chain to support the world's growing population. Improvements are needed across crops, livestock, and microbes to achieve this goal. Since 2005, researchers have been attempting to make the necessary strides to reach this milestone, but attempts have fallen short. With the introduction of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins, the food production field is now able to achieve some of its most exciting advancements since the Green Revolution. This review introduces the concept of applying CRISPR-Cas technology as a genome-editing tool for use in the food supply chain, focusing on its implementation to date in crop, livestock, and microbe production, advancement of products to market, and regulatory and societal hurdles that need to be overcome.

RevDate: 2019-03-25

Xiao G, Yi Y, Che R, et al (2019)

Characterization of CRISPR-Cas systems in Leptospira reveals potential application of CRISPR in genotyping of Leptospira interrogans.

APMIS : acta pathologica, microbiologica, et immunologica Scandinavica, 127(4):202-216.

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira. However, understanding of the pathogenic mechanism of Leptospira is still elusive due to the limited number of genetic tools available for this microorganism. Currently, the reason for the genetic inaccessibility of Leptospira is still unknown. It is well known that as an acquired immunity of bacteria, Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR-associated gene (CRISPR-Cas) systems can help bacteria against invading mobile genetic elements. In this study, the occurrence and diversity of CRISPR-Cas systems in 41 genomes of Leptospira strains were investigated. Three subtypes (subtype I-B, subtype I-C and subtype I-E) of CRISPR-Cas systems were identified in both pathogenic and intermediate Leptospira species but not in saprophytic species. Noteworthy, the majority of pathogenic species harbor two different types of CRISPR-Cas systems (subtype I-B and subtype I-E). Furthermore, Cas2 protein of subtype I-C in L. interrogans exhibited a metal-dependent DNase activity in a nonspecific manner. CRISPR spacers in subtype I-B are highly conserved within the same serovars and hypervariable across different serovars of L. interrogans. Based on the subtype I-B CRISPR arrays, the serotypes of different L. interrogans strains were easily identified. Investigation of the origin of CRISPR spacers showed that 192 spacers (23.5%) matched to mobile genetic elements, indicating CRISPR-Cas systems may play an important role in the defense of foreign invading DNA.

RevDate: 2019-03-25

Kim JH (2018)

Genetics of Alzheimer's Disease.

Dementia and neurocognitive disorders, 17(4):131-136.

Alzheimer's disease (AD) related genes have been elucidated by advanced genetic techniques. Familial autosomal dominant AD genes founded by linkage analyses are APP, PSEN1, PSEN2, ABCA7, and SORL1. Genome-wide association studies have found risk genes such as ABCA7, BIN1, CASS4, CD33, CD2AP, CELF1, CLU, CR1, DSG2, EPHA1, FERMT2, HLA-DRB5-HLA-DRB1, INPP5D, MEF2C, MS4A6A/MS4A4E, NME8, PICALM, PTK2B, SLC24A4, SORL1, and ZCWPW1. ABCA7, SORL1, TREM2, and APOE are proved to have high odds ratio (>2) in risk of AD using next generation sequencing studies. Thanks to the promising genetic techniques such as CRISPR-CAS9 and single-cell RNA sequencing opened a new era in genetics. CRISPR-CAS9 can directly link genetic knowledge to future treatment. Single-cell RNA sequencing are providing useful information on cell biology and pathogenesis of diverse diseases.

RevDate: 2019-03-25

Zhang F (2019)

Exploration of Microbial Diversity to Discover Novel Molecular Technologies.

The Keio journal of medicine, 68(1):26.

Many powerful molecular biology tools have their origin in nature. From restriction enzymes to CRISPR-Cas9, microbes utilize a diverse array of systems to get ahead evolutionarily. We are exploring this natural diversity through bioinformatics, biochemical, and molecular work to better understand the fundamental ways in which microbes and other living organisms sense and respond to their environment and as possible to develop these natural systems as molecular tools and to improve human health. Building on our demonstration that Cas9 can be repurposed for precision genome editing in mammalian cells, we look for novel CRISPR-Cas systems that are different and may have other useful properties. This led to the discovery of several new CRISPR systems, including the CRISPR-Cas13 family that target RNA, rather than DNA. We have developed a toolbox for RNA modulation based on Cas13, including methods for precision base editing, adding to our robust toolbox for DNA based on Cas9 and Cas12. We are expanding our biodiscovery efforts to search for new microbial proteins that may be adapted for applications beyond genome and transcriptome modulation, capitalizing on the growing volume of microbial genomic sequences. We are particularly interested in identifying new therapeutic modalities and vehicles for delivering them into patients. We hope that additional robust tools and delivery options will further accelerate research into human disease and open up new therapeutic possibilities.

RevDate: 2019-03-25

de Graeff N, Jongsma KR, Johnston J, et al (2019)

The ethics of genome editing in non-human animals: a systematic review of reasons reported in the academic literature.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180106.

In recent years, new genome editing technologies have emerged that can edit the genome of non-human animals with progressively increasing efficiency. Despite ongoing academic debate about the ethical implications of these technologies, no comprehensive overview of this debate exists. To address this gap in the literature, we conducted a systematic review of the reasons reported in the academic literature for and against the development and use of genome editing technologies in animals. Most included articles were written by academics from the biomedical or animal sciences. The reported reasons related to seven themes: human health, efficiency, risks and uncertainty, animal welfare, animal dignity, environmental considerations and public acceptability. Our findings illuminate several key considerations about the academic debate, including a low disciplinary diversity in the contributing academics, a scarcity of systematic comparisons of potential consequences of using these technologies, an underrepresentation of animal interests, and a disjunction between the public and academic debate on this topic. As such, this article can be considered a call for a broad range of academics to get increasingly involved in the discussion about genome editing, to incorporate animal interests and systematic comparisons, and to further discuss the aims and methods of public involvement. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Buchthal J, Evans SW, Lunshof J, et al (2019)

Mice Against Ticks: an experimental community-guided effort to prevent tick-borne disease by altering the shared environment.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180105.

Mice Against Ticks is a community-guided ecological engineering project that aims to prevent tick-borne disease by using CRISPR-based genome editing to heritably immunize the white-footed mice (Peromyscus leucopus) responsible for infecting many ticks in eastern North America. Introducing antibody-encoding resistance alleles into the local mouse population is anticipated to disrupt the disease transmission cycle for decades. Technology development is shaped by engagement with community members and visitors to the islands of Nantucket and Martha's Vineyard, including decisions at project inception about which types of disease resistance to pursue. This engagement process has prompted the researchers to use only white-footed mouse DNA if possible, meaning the current project will not involve gene drive. Instead, engineered mice would be released in the spring when the natural population is low, a plan unlikely to increase total numbers above the normal maximum in autumn. Community members are continually asked to share their suggestions and concerns, a process that has already identified potential ecological consequences unanticipated by the research team that will likely affect implementation. As an early example of CRISPR-based ecological engineering, Mice Against Ticks aims to start small and simple by working with island communities whose mouse populations can be lastingly immunized without gene drive. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Ramachandran G, D Bikard (2019)

Editing the microbiome the CRISPR way.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180103.

Our bodies are colonized by a complex ecosystem of bacteria, unicellular eukaryotes and their viruses that together play a major role in our health. Over the past few years tools derived from the prokaryotic immune system known as CRISPR-Cas have empowered researchers to modify and study organisms with unprecedented ease and efficiency. Here we discuss how various types of CRISPR-Cas systems can be used to modify the genome of gut microorganisms and bacteriophages. CRISPR-Cas systems can also be delivered to bacterial population and programmed to specifically eliminate members of the microbiome. Finally, engineered CRISPR-Cas systems can be used to control gene expression and modulate the production of metabolites and proteins. Together these tools provide exciting opportunities to investigate the complex interplay between members of the microbiome and our bodies, and present new avenues for the development of drugs that target the microbiome. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Westra ER, van Houte S, Gandon S, et al (2019)

The ecology and evolution of microbial CRISPR-Cas adaptive immune systems.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20190101.

RevDate: 2019-03-25

Chevallereau A, Meaden S, van Houte S, et al (2019)

The effect of bacterial mutation rate on the evolution of CRISPR-Cas adaptive immunity.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180094.

CRISPR-Cas immune systems are present in around half of bacterial genomes. Given the specificity and adaptability of this immune mechanism, it is perhaps surprising that they are not more widespread. Recent insights into the requirement for specific host factors for the function of some CRISPR-Cas subtypes, as well as the negative epistasis between CRISPR-Cas and other host genes, have shed light on potential reasons for the partial distribution of this immune strategy in bacteria. In this study, we examined how mutations in the bacterial mismatch repair system, which are frequently observed in natural and clinical isolates and cause elevated host mutation rates, influence the evolution of CRISPR-Cas-mediated immunity. We found that hosts with a high mutation rate very rarely evolved CRISPR-based immunity to phage compared to wild-type hosts. We explored the reason for this effect and found that the higher frequency at which surface mutants pre-exist in the mutator host background causes them to rapidly become the dominant phenotype under phage infection. These findings suggest that natural variation in bacterial mutation rates may, therefore, influence the distribution of CRISPR-Cas adaptive immune systems. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Pauly MD, Bautista MA, Black JA, et al (2019)

Diversified local CRISPR-Cas immunity to viruses of Sulfolobus islandicus.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180093.

The population diversity and structure of CRISPR-Cas immunity provides key insights into virus-host interactions. Here, we examined two geographically and genetically distinct natural populations of the thermophilic crenarchaeon Sulfolobus islandicus and their interactions with Sulfolobus spindle-shaped viruses (SSVs) and S. islandicus rod-shaped viruses (SIRVs). We found that both virus families can be targeted with high population distributed immunity, whereby most immune strains target a virus using unique unshared CRISPR spacers. In Kamchatka, Russia, we observed high immunity to chronic SSVs that increases over time. In this context, we found that some SSVs had shortened genomes lacking genes that are highly targeted by the S. islandicus population, indicating a potential mechanism of immune evasion. By contrast, in Yellowstone National Park, we found high inter- and intra-strain immune diversity targeting lytic SIRVs and low immunity to chronic SSVs. In this population, we observed evidence of SIRVs evolving immunity through mutations concentrated in the first five bases of protospacers. These results indicate that diversity and structure of antiviral CRISPR-Cas immunity for a single microbial species can differ by both the population and virus type, and suggest that different virus families use different mechanisms to evade CRISPR-Cas immunity. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Lopatina A, Medvedeva S, Artamonova D, et al (2019)

Natural diversity of CRISPR spacers of Thermus: evidence of local spacer acquisition and global spacer exchange.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180092.

We investigated the diversity of CRISPR spacers of Thermus communities from two locations in Italy, two in Chile and one location in Russia. Among the five sampling sites, a total of more than 7200 unique spacers belonging to different CRISPR-Cas systems types and subtypes were identified. Most of these spacers are not found in CRISPR arrays of sequenced Thermus strains. Comparison of spacer sets revealed that samples within the same area (separated by few to hundreds of metres) have similar spacer sets, which appear to be largely stable at least over the course of several years. While at further distances (hundreds of kilometres and more) the similarity of spacer sets is decreased, there are still multiple common spacers in Thermus communities from different continents. The common spacers can be reconstructed in identical or similar CRISPR arrays, excluding their independent appearance and suggesting an extensive migration of thermophilic bacteria over long distances. Several new Thermus phages were isolated in the sampling sites. Mapping of spacers to bacteriophage sequences revealed examples of local acquisition of spacers from some phages and distinct patterns of targeting of phage genomes by different CRISPR-Cas systems. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Watson BNJ, Easingwood RA, Tong B, et al (2019)

Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180090.

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against genetic invaders, such as bacteriophages. The systems integrate short sequences from the phage genome into the bacterial CRISPR array. These 'spacers' provide sequence-specific immunity but drive natural selection of evolved phage mutants that escape the CRISPR-Cas defence. Spacer acquisition occurs by either naive or primed adaptation. Naive adaptation typically results in the incorporation of a single spacer. By contrast, priming is a positive feedback loop that often results in acquisition of multiple spacers, which occurs when a pre-existing spacer matches the invading phage. We predicted that single and multiple spacers, representative of naive and primed adaptation, respectively, would cause differing outcomes after phage infection. We investigated the response of two phages, ϕTE and ϕM1, to the Pectobacterium atrosepticum type I-F CRISPR-Cas system and observed that escape from single spacers typically occurred via point mutations. Alternatively, phages escaped multiple spacers through deletions, which can occur in genes encoding structural proteins. Cryo-EM analysis of the ϕTE structure revealed shortened tails in escape mutants with tape measure protein deletions. We conclude that CRISPR-Cas systems can drive phage genetic diversity, altering morphology and fitness, through selective pressures arising from naive and primed acquisition events. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Hoikkala V, Almeida GMF, Laanto E, et al (2019)

Aquaculture as a source of empirical evidence for coevolution between CRISPR-Cas and phage.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180100.

So far, studies on the bacterial immune system CRISPR-Cas and its ecological and evolutionary effects have been largely limited to laboratory conditions. While providing crucial information on the constituents of CRISPR-Cas, such studies may overlook fundamental components that affect bacterial immunity in natural habitats. Translating laboratory-derived predictions to nature is not a trivial task, owing partly to the instability of natural communities and difficulties in repeated sampling. To this end, we review how aquaculture, the farming of fishes and other aquatic species, may provide suitable semi-natural laboratories for examining the role of CRISPR-Cas in phage/bacterium coevolution. Existing data from disease surveillance conducted in aquaculture, coupled with growing interest towards phage therapy, may have already resulted in large collections of bacterium and phage isolates. These data, combined with premeditated efforts, can provide empirical evidence on phage-bacterium dynamics such as the bacteriophage adherence to mucus hypothesis, phage life cycles and their relationship with CRISPR-Cas and other immune defences. Typing of CRISPR spacer content in pathogenic bacteria can also provide practical information on diversity and origin of isolates during outbreaks. In addition to providing information of CRISPR functionality and phage-bacterium dynamics, aquaculture systems can significantly impact perspectives on design of phage-based disease treatment at the current era of increasing antibiotic resistance. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

McKitterick AC, LeGault KN, Angermeyer A, et al (2019)

Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180089.

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Bernheim A, Bikard D, Touchon M, et al (2019)

A matter of background: DNA repair pathways as a possible cause for the sparse distribution of CRISPR-Cas systems in bacteria.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180088.

The absence of CRISPR-Cas systems in more than half of the sequenced bacterial genomes is intriguing, because their role in adaptive immunity and their frequent transfer between species should have made them almost ubiquitous, as is the case in Archaea. Here, we investigate the possibility that the success of CRISPR-Cas acquisition by horizontal gene transfer is affected by the interactions of these systems with the host genetic background and especially with components of double-strand break repair systems (DSB-RS). We first described the distribution of systems specialized in the repair of double-strand breaks in Bacteria: homologous recombination and non-homologous end joining. This allowed us to show that such systems are more often positively or negatively correlated with the frequency of CRISPR-Cas systems than random genes of similar frequency. The detailed analysis of these co-occurrence patterns shows that our method identifies previously known cases of mechanistic interactions between these systems. It also reveals other positive and negative patterns of co-occurrence between DSB-RS and CRISPR-Cas systems. Notably, it shows that the patterns of distribution of CRISPR-Cas systems in Proteobacteria are strongly dependent on the epistatic groups including RecBCD and AddAB. Our results suggest that the genetic background plays an important role in the success of adaptive immunity in different bacterial clades and provide insights to guide further experimental research on the interactions between CRISPR-Cas and DSB-RS. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Shehreen S, Chyou TY, Fineran PC, et al (2019)

Genome-wide correlation analysis suggests different roles of CRISPR-Cas systems in the acquisition of antibiotic resistance genes in diverse species.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180384.

CRISPR-Cas systems are widespread in bacterial and archaeal genomes, and in their canonical role in phage defence they confer a fitness advantage. However, CRISPR-Cas may also hinder the uptake of potentially beneficial genes. This is particularly true under antibiotic selection, where preventing the uptake of antibiotic resistance genes could be detrimental. Newly discovered features within these evolutionary dynamics are anti-CRISPR genes, which inhibit specific CRISPR-Cas systems. We hypothesized that selection for antibiotic resistance might have resulted in an accumulation of anti-CRISPR genes in genomes that harbour CRISPR-Cas systems and horizontally acquired antibiotic resistance genes. To assess that question, we analysed correlations between the CRISPR-Cas, anti-CRISPR and antibiotic resistance gene content of 104 947 reference genomes, including 5677 different species. In most species, the presence of CRISPR-Cas systems did not correlate with the presence of antibiotic resistance genes. However, in some clinically important species, we observed either a positive or negative correlation of CRISPR-Cas with antibiotic resistance genes. Anti-CRISPR genes were common enough in four species to be analysed. In Pseudomonas aeruginosa, the presence of anti-CRISPRs was associated with antibiotic resistance genes. This analysis indicates that the role of CRISPR-Cas and anti-CRISPRs in the spread of antibiotic resistance is likely to be very different in particular pathogenic species and clinical environments. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Common J, Morley D, Westra ER, et al (2019)

CRISPR-Cas immunity leads to a coevolutionary arms race between Streptococcus thermophilus and lytic phage.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180098.

CRISPR-Cas is an adaptive prokaryotic immune system that prevents phage infection. By incorporating phage-derived 'spacer' sequences into CRISPR loci on the host genome, future infections from the same phage genotype can be recognized and the phage genome cleaved. However, the phage can escape CRISPR degradation by mutating the sequence targeted by the spacer, allowing them to re-infect previously CRISPR-immune hosts, and theoretically leading to coevolution. Previous studies have shown that phage can persist over long periods in populations of Streptococcus thermophilus that can acquire CRISPR-Cas immunity, but it has remained less clear whether this coexistence was owing to coevolution, and if so, what type of coevolutionary dynamics were involved. In this study, we performed highly replicated serial transfer experiments over 30 days with S. thermophilus and a lytic phage. Using a combination of phenotypic and genotypic data, we show that CRISPR-mediated resistance and phage infectivity coevolved over time following an arms race dynamic, and that asymmetry between phage infectivity and host resistance within this system eventually causes phage extinction. This work provides further insight into the way CRISPR-Cas systems shape the population and coevolutionary dynamics of bacteria-phage interactions. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Koonin EV, KS Makarova (2019)

Origins and evolution of CRISPR-Cas systems.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180087.

CRISPR-Cas, the bacterial and archaeal adaptive immunity systems, encompass a complex machinery that integrates fragments of foreign nucleic acids, mostly from mobile genetic elements (MGE), into CRISPR arrays embedded in microbial genomes. Transcripts of the inserted segments (spacers) are employed by CRISPR-Cas systems as guide (g)RNAs for recognition and inactivation of the cognate targets. The CRISPR-Cas systems consist of distinct adaptation and effector modules whose evolutionary trajectories appear to be at least partially independent. Comparative genome analysis reveals the origin of the adaptation module from casposons, a distinct type of transposons, which employ a homologue of Cas1 protein, the integrase responsible for the spacer incorporation into CRISPR arrays, as the transposase. The origin of the effector module(s) is far less clear. The CRISPR-Cas systems are partitioned into two classes, class 1 with multisubunit effectors, and class 2 in which the effector consists of a single, large protein. The class 2 effectors originate from nucleases encoded by different MGE, whereas the origin of the class 1 effector complexes remains murky. However, the recent discovery of a signalling pathway built into the type III systems of class 1 might offer a clue, suggesting that type III effector modules could have evolved from a signal transduction system involved in stress-induced programmed cell death. The subsequent evolution of the class 1 effector complexes through serial gene duplication and displacement, primarily of genes for proteins containing RNA recognition motif domains, can be hypothetically reconstructed. In addition to the multiple contributions of MGE to the evolution of CRISPR-Cas, the reverse flow of information is notable, namely, recruitment of minimalist variants of CRISPR-Cas systems by MGE for functions that remain to be elucidated. Here, we attempt a synthesis of the diverse threads that shed light on CRISPR-Cas origins and evolution. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Chabas H, Nicot A, Meaden S, et al (2019)

Variability in the durability of CRISPR-Cas immunity.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180097.

The durability of host resistance is challenged by the ability of pathogens to escape the defence of their hosts. Understanding the variability in the durability of host resistance is of paramount importance for designing more effective control strategies against infectious diseases. Here, we study the durability of various clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) alleles of the bacteria Streptococcus thermophilus against lytic phages. We found substantial variability in durability among different resistant bacteria. Since the escape of the phage is driven by a mutation in the phage sequence targeted by CRISPR-Cas, we explored the fitness costs associated with these escape mutations. We found that, on average, escape mutations decrease the fitness of the phage. Yet, the magnitude of this fitness cost does not predict the durability of CRISPR-Cas immunity. We contend that this variability in the durability of resistance may be because of variations in phage mutation rate or in the proportion of lethal mutations across the phage genome. These results have important implications on the coevolutionary dynamics between bacteria and phages and for the optimal deployment of resistance strategies against pathogens and pests. Understanding the durability of CRISPR-Cas immunity may also help develop more effective gene-drive strategies based on CRISPR-Cas9 technology. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Gurney J, Pleška M, BR Levin (2019)

Why put up with immunity when there is resistance: an excursion into the population and evolutionary dynamics of restriction-modification and CRISPR-Cas.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180096.

Bacteria can readily generate mutations that prevent bacteriophage (phage) adsorption and thus make bacteria resistant to infections with these viruses. Nevertheless, the majority of bacteria carry complex innate and/or adaptive immune systems: restriction-modification (RM) and CRISPR-Cas, respectively. Both RM and CRISPR-Cas are commonly assumed to have evolved and be maintained to protect bacteria from succumbing to infections with lytic phage. Using mathematical models and computer simulations, we explore the conditions under which selection mediated by lytic phage will favour such complex innate and adaptive immune systems, as opposed to simple envelope resistance. The results of our analysis suggest that when populations of bacteria are confronted with lytic phage: (i) In the absence of immunity, resistance to even multiple bacteriophage species with independent receptors can evolve readily. (ii) RM immunity can benefit bacteria by preventing phage from invading established bacterial populations and particularly so when there are multiple bacteriophage species adsorbing to different receptors. (iii) Whether CRISPR-Cas immunity will prevail over envelope resistance depends critically on the number of steps in the coevolutionary arms race between the bacteria-acquiring spacers and the phage-generating CRISPR-escape mutants. We discuss the implications of these results in the context of the evolution and maintenance of RM and CRISPR-Cas and highlight fundamental questions that remain unanswered. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25

Bradde S, Mora T, AM Walczak (2019)

Cost and benefits of clustered regularly interspaced short palindromic repeats spacer acquisition.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772):20180095.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-mediated immunity in bacteria allows bacterial populations to protect themselves against pathogens. However, it also exposes them to the dangers of auto-immunity by developing protection that targets its own genome. Using a simple model of the coupled dynamics of phage and bacterial populations, we explore how acquisition rates affect the probability of the bacterial colony going extinct. We find that the optimal strategy depends on the initial population sizes of both viruses and bacteria. Additionally, certain combinations of acquisition and dynamical rates and initial population sizes guarantee protection, owing to a dynamical balance between the evolving population sizes, without relying on acquisition of viral spacers. Outside this regime, the high cost of auto-immunity limits the acquisition rate. We discuss these optimal strategies that minimize the probability of the colony going extinct in terms of recent experiments. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

RevDate: 2019-03-25
CmpDate: 2019-03-25

Feng Z, Zhang Z, Hua K, et al (2018)

A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis.

International journal of molecular sciences, 19(12): pii:ijms19123925.

The CRISPR/Cas9 system has been widely used for targeted genome editing in numerous plant species. In Arabidopsis, constitutive promoters usually result in a low efficiency of heritable mutation in the T1 generation. In this work, CRISPR/Cas9 gene editing efficiencies using different promoters to drive Cas9 expression were evaluated. Expression of Cas9 under the constitutive CaMV 35S promoter resulted in a 2.3% mutation rate in T1 plants and failed to produce homozygous mutations in the T1 and T2 generations. In contrast, expression of Cas9 under two cell division-specific promoters, YAO and CDC45, produced mutation rates of 80.9% to 100% in the T1 generation with nonchimeric mutations in the T1 (4.4⁻10%) and T2 (32.5⁻46.1%) generations. The pCDC45 promoter was used to modify a previously reported multiplex CRISPR/Cas9 system, replacing the original constitutive ubiquitin promoter. The multi-pCDC45-Cas9 system produced higher mutation efficiencies than the multi-pUBQ-Cas9 system in the T1 generation (60.17% vs. 43.71%) as well as higher efficiency of heritable mutations (11.30% vs. 4.31%). Sextuple T2 homozygous mutants were identified from a construct targeting seven individual loci. Our results demonstrate the advantage of using cell division promoters for CRISPR/Cas9 gene editing applications in Arabidopsis, especially in multiplex applications.

RevDate: 2019-03-25
CmpDate: 2019-03-25

Cai Y, Chen L, Sun S, et al (2018)

CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean.

International journal of molecular sciences, 19(12): pii:ijms19123835.

At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 'transgene-free' homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean.

RevDate: 2019-03-25
CmpDate: 2019-03-25

Malik S, Oyaghire S, R Bahal (2019)

Applications of PNA-laden nanoparticles for hematological disorders.

Cellular and molecular life sciences : CMLS, 76(6):1057-1065.

Safe and efficient genome editing has been an unmitigated goal for biomedical researchers since its inception. The most prevalent strategy for gene editing is the use of engineered nucleases that induce DNA damage and take advantage of cellular DNA repair machinery. This includes meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) systems. However, the clinical viability of these nucleases is marred by their off-target cleavage activity (≥ 50% in RNA-guided endonucleases). In addition, in vivo applications of CRISPR require systemic administration of Cas9 protein, mRNA, or DNA, which presents a significant delivery challenge. The development of nucleic acid probes that can recognize specific double-stranded DNA (dsDNA) regions and activate endogenous DNA repair machinery holds great promise for gene editing applications. Triplex-forming oligonucleotides (TFOs), which were introduced more than 25 years ago, are among the most extensively studied oligomeric dsDNA-targeting agents. TFOs bind duplex DNA to create a distorted helical structure, which can stimulate DNA repair and the exchange of a nearby mutated region-otherwise leading to an undesired phenotype-for a short single-stranded donor DNA that contains the corrective nucleotide sequence. Recombination can be induced within several hundred base-pairs of the TFO binding site and has been shown to depend on triplex-induced initiation of the nucleotide excision repair pathway and engagement of the homology-dependent repair pathway. Since TFOs do not possess any direct nuclease activity, their off-target effects are minimal when compared to engineered nucleases. This review comprehensively covers the advances made in peptide nucleic acid-based TFOs for site-specific gene editing and their therapeutic applications.

RevDate: 2019-03-25
CmpDate: 2019-03-25

Kory N, Wyant GA, Prakash G, et al (2018)

SFXN1 is a mitochondrial serine transporter required for one-carbon metabolism.

Science (New York, N.Y.), 362(6416):.

One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism.

RevDate: 2019-03-25
CmpDate: 2019-03-25

Nguyen BH, Ishii M, Maxson RE, et al (2018)

Culturing and Manipulation of O9-1 Neural Crest Cells.

Journal of visualized experiments : JoVE.

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

RevDate: 2019-03-25
CmpDate: 2019-03-25

Mollanoori H, Shahraki H, Rahmati Y, et al (2018)

CRISPR/Cas9 and CAR-T cell, collaboration of two revolutionary technologies in cancer immunotherapy, an instruction for successful cancer treatment.

Human immunology, 79(12):876-882.

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease9 (CRISPR/Cas9) technology, an acquired immune system in bacteria and archaea, has provided a new tool for accurately genome editing. Using only a single nuclease protein in complex with 2 short RNA as a site-specific endonuclease made it a simple and flexible genome editing tool to target nearly any genomic locus. Due to recent developments in therapeutic engineered T cell and effective responses of CD19-directed chimeric antigen receptor T cells (CART19) in patients with B-cell leukemia and lymphoma, adoptive T cell immunotherapy, particularly CAR-T cell therapy became a rapidly growing field in cancer therapy and recently Kymriah and Yescarta (CD19-directed CAR-T cells) were approved by FDA. Therefore, the combination of CRISPR/Cas9 technology as a genome engineering tool and CAR-T cell therapy (engineered T cells that express chimeric antigen receptors) may lead to further improvement in efficiency and safety of CAR-T cells. This article reviews mechanism and therapeutic application of CRISPR/Cas9 technology, accuracy of this technology, cancer immunotherapy by CAR T cells, the application of CRISPR technology for the production of universal CAR T cells, improving their antitumor efficacy, and biotech companies that invested in CRISPR technology for CAR-T cell therapy.

RevDate: 2019-03-25
CmpDate: 2019-03-25

Gehrke JM, Cervantes O, Clement MK, et al (2018)

An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities.

Nature biotechnology, 36(10):977-982.

Base editor technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine DNA alterations. However, existing base editors create unwanted C-to-T alterations when more than one C is present in the enzyme's five-base-pair editing window. Here we describe a strategy for reducing bystander mutations using an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN hierarchy. In direct comparisons with the widely used base editor 3 (BE3) fusion in human cells, our eA3A-BE3 fusion exhibits similar activities on cytidines in TC motifs but greatly reduced editing on cytidines in other sequence contexts. eA3A-BE3 corrects a human β-thalassemia promoter mutation with much higher (>40-fold) precision than BE3. We also demonstrate that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites.

RevDate: 2019-03-25
CmpDate: 2019-03-25

Sung K, Park J, Kim Y, et al (2018)

Target Specificity of Cas9 Nuclease via DNA Rearrangement Regulated by the REC2 Domain.

Journal of the American Chemical Society, 140(25):7778-7781.

Understanding the underlying principles for the target-specific nuclease activity of CRISPR/Cas9 is a prerequisite to minimize its off-target DNA cleavage for genome engineering applications. Here, we show that the noncatalytic REC2 domain of Cas9 nuclease plays a crucial role in off-target discrimination. Using single-molecule fluorescence methods, we investigate conformational dynamics of the non-target strand (NTS) of DNA interacting with Cas9 and find that REC2 regulates the NTS rearrangement for cleavage reaction with the help of positively charged residues on its surface. This mechanistic model for the target specificity of Cas9 provides molecular insights for the rational approach to Cas9 engineering for highly specific genome editing.

RevDate: 2019-03-22

Mehnert M, Li W, Wu C, et al (2019)

Combining Rapid Data Independent Acquisition and CRISPR Gene Deletion for Studying Potential Protein Functions: A Case of HMGN1.

Proteomics [Epub ahead of print].

CRISPR-Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non-histone chromosomal protein HMG-14 (HMGN1) in regulating chromatin structure and tumor immunity, we performed gene knockout of HMGN1 by CRISPR in cancer cells and studied the following proteomic regulation events. In particular, we utilized DIA mass spectrometry (DIA-MS) and reproducibly measured more than 6200 proteins (protein- FDR 1%) and more than 82,000 peptide precursors in the single MS shots of two hours. HMGN1 protein deletion was confidently verified by DIA-MS in all of the clone- and dish- replicates following CRISPR. Statistical analysis revealed 147 proteins changed their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces the histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. We suggest that DIA-MS can be reliably used as a rapid, robust, and cost-effective proteomic-screening tool to assess the outcome of the CRISPR experiments. This article is protected by copyright. All rights reserved.

RevDate: 2019-03-22

Schmidt C, Pacher M, H Puchta (2019)

Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system.

The Plant journal : for cell and molecular biology [Epub ahead of print].

During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Thus, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from S. aureus (SaCas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana. Via deep sequencing, we defined the patterns of junction formation in wild-type and in the non-homologous end joining (NHEJ) mutant ku70-1. Surprisingly, in plants deficient of KU70, inversion induction is enhanced, indicating that KU70 is required for tethering the local broken ends together during repair. However, in contrast to wild-type, most junctions are formed by microhomology-mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg cell specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will. This article is protected by copyright. All rights reserved.

RevDate: 2019-03-22

Choquet K, Forget D, Meloche E, et al (2019)

Leukodystrophy-associated POLR3A mutations down-regulate the RNA polymerase III transcript and important regulatory RNA BC200.

The Journal of biological chemistry pii:RA118.006271 [Epub ahead of print].

RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small non-coding RNAs, a number of which are involved in mRNA translation. Recessive mutations in POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has been so far elusive. Here, we used CRISPR-Cas9 to introduce the POLR3A mutation c.2554A>G (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. Transcriptomic profiling uncovered a subset of transcripts vulnerable to Pol III hypofunction, including a global reduction in tRNA levels. The brain cytoplasmic BC200 RNA (BCYRN1), involved in translation regulation, was consistently affected in all our cellular models, including patient-derived fibroblasts. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Upon differentiation, mRNA levels of the MBP gene, encoding myelin basic protein, were significantly decreased in POLR3A-mutant cells. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3-HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease.

RevDate: 2019-03-22
CmpDate: 2019-03-22

Xu ZS, Feng K, AS Xiong (2019)

CRISPR/Cas9-Mediated Multiply Targeted Mutagenesis in Orange and Purple Carrot Plants.

Molecular biotechnology, 61(3):191-199.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system has been successfully used for precise genome editing in many plant species, including in carrot cells, very recently. However, no stable gene-editing carrot plants were obtained with CRISPR/Cas9 system to date. In the present study, four sgRNA expression cassettes, individually driven by four different promoters and assembled in a single CRISPR/Cas9 vector, were transformed into carrots using Agrobacterium-mediated genetic transformation. Four sites of DcPDS and DcMYB113-like genes were chosen as targets. Knockout of DcPDS in orange carrot 'Kurodagosun' resulted in the generation of albino carrot plantlets, with about 35.3% editing efficiency. DcMYB113-like was also successfully edited in purple carrot 'Deep purple', resulting in purple depigmented carrot plants, with about 36.4% rate of mutation. Sequencing analyses showed that insertion, deletion, and substitution occurred in the target sites, generating heterozygous, biallelic, and chimeric mutations. The highest efficiency of mutagenesis was observed in the sites targeted by AtU6-29-driven sgRNAs in both DcPDS- and DcMYB113-like-knockout T0 plants, which always induced double-strand breaks in the target sites. Our results proved that CRISPR/Cas9 system could be for generating stable gene-editing carrot plants.

RevDate: 2019-03-22
CmpDate: 2019-03-22

Kyrou K, Hammond AM, Galizi R, et al (2018)

A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.

Nature biotechnology, 36(11):1062-1066.

In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR-Cas9-targeted disruption of the intron 4-exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR-Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7-11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.

RevDate: 2019-03-22
CmpDate: 2019-03-22

Cao J, Wei J, Yang P, et al (2018)

Genome-scale CRISPR-Cas9 knockout screening in gastrointestinal stromal tumor with Imatinib resistance.

Molecular cancer, 17(1):121.

Genome-scale CRISPR-Cas9 Knockout Screening was applied to investigate novel targets in imatinib-resistant gastrointestinal stromal tumor (GIST). 20 genes and 2 miRNAs have been selected by total reads of sgRNA and sgRNA diversity, which has been further validated in imatinib-resistant GIST cells by CCK8 and qPCR analysis. Our study has finally revealed 9 genes (DBP, NR3C1, TCF12, TP53, ZNF12, SOCS6, ZFP36, ACYP1, and DRD1) involved in imatinib-resistant GIST-T1 cells. TP53 and SOCS6 may be the most promising candidate genes for imatinib-resistance due to the possible signaling pathway, such as apoptosis pathway and Wnt signaling pathway, JAK-STAT signaling pathway. It is necessary to perform more studies to discover novel targets in imatinib-resistant GIST, including DBP, NR3C1, TCF12, ZNF12, ZFP36, ACYP1 and DRD1.

RevDate: 2019-03-22
CmpDate: 2019-03-22

Yamazaki T, Souquere S, Chujo T, et al (2018)

Functional Domains of NEAT1 Architectural lncRNA Induce Paraspeckle Assembly through Phase Separation.

Molecular cell, 70(6):1038-1053.e7.

A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.

RevDate: 2019-03-22
CmpDate: 2019-03-22

Wu M, Liu S, Gao Y, et al (2018)

Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system.

Stem cell research, 29:6-14.

Precise genome editing in human induced pluripotent stem cells (iPSCs) significantly enhances our capability to use human iPSCs for disease modeling, drug testing and screening as well as investigation of human cell biology. In this study, we seek to achieve conditional expression of the CD55 gene in order to interrogate its functions. We used two human iPSC lines that have unique genotypes, and constructed an inducible Cas9 gene expression system that is integrated at the AAVS1 safe harbor site in the human genome. Using paired guide RNAs, we observed efficient knock-out with an intended deletion in the coding region of several genes including CD55 and ETV6 genes. This paired guide RNA approach enabled us to efficiently identify homozygous iPSC clones with an intended deletion. Once an iPSC clone lacking CD55 expression was identified and characterized, we were able to use the same doxycycline system to induce expression of a CD55 transgene from a piggyBac vector, in both undifferentiated and differentiated iPSCs. This single cell line of gene knock-out complemented with an inducible transgene is sufficient to achieve conditional expression of the CD55 gene. The methodology described here is broadly applicable to other genes in order to interrogate their functions.

RevDate: 2019-03-22
CmpDate: 2019-03-22

Hyun I, C Osborn (2017)

Query the merits of embryo editing for reproductive research now.

Nature biotechnology, 35(11):1023-1025.

RevDate: 2019-03-21

Karpov DS, Karpov VL, Klimova RR, et al (2019)

[A Plasmid-Expressed CRISPR/Cas9 System Suppresses Replication of HSV Type I in a Vero Cell Culture].

Molekuliarnaia biologiia, 53(1):91-100.

Herpesviruses are widespread in the human population. Herpes simplex virus type 1 (HSV1) alone infects more than 3.7 billion people. In most of these, the virus establishes a latent form resistant to the action of all antiviral drugs. Moreover, completely drug-resistant strains of herpesviruses are known, which has prompted the search for alternative approaches to the treatment of herpesviruses, including genome editing with prokaryotic CRISPR/Cas. The CRISPR/Cas9 system of Streptococcus pyogens effectively suppresses HSV1 infection when expressed from genome-integrated lentiviral vectors. However, there are concerns about the safety of this approach. Here we describe the system built upon the plasmid-encoded CRISPR/Cas9 targeted against UL52 and UL29 genes of the HSV1 primase-helicase complex. The construct was transfected into Vero cells with no significant cytotoxic effects detected. Complete suppression of HSV1 infection within two days was observed, raising the possibility that the proposed plasmid-expressed CRISPR/Cas9 system may be used for the screening of genes important for the HSV1 life cycle and for development of novel strategies for targeted therapy of herpesvirus infections.

RevDate: 2019-03-21

Castelli A, Susani L, Menale C, et al (2019)

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse iPSCs.

Stem cells (Dayton, Ohio) [Epub ahead of print].

In spite of the progresses in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed Chromosomal Transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the Chronic Granulomatous Disease (CGD), an X-linked severe immunodeficiency due to abnormalities in Cybb (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the HAT selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer (MMCT). X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. SIGNIFICANCE STATEMENT: A lot of genetic diseases involving chromosome abnormalities can not be treated by conventional gene therapy approaches. Here we applied Chromosome Transplantation, a new genomic therapy method that we have recently developed, to induced pluripotent stem cells (iPSCs) derived from an X-linked mouse disease model. As a proof of principle we demonstrated that X-chromosome transplanted corrected cells restored the normal function to differentiated cells. Although all the strategies based on iPSCs are still far away from the clinic, we believe that our results establish an important step for the usefulness of this kind of genomic therapy in selected patients. © AlphaMed Press 2019.

RevDate: 2019-03-21

Hendriks S, Giesbertz NAA, Bredenoord AL, et al (2018)

Reasons for being in favour of or against genome modification: a survey of the Dutch general public.

Human reproduction open, 2018(3):hoy008 pii:hoy008.

STUDY QUESTION: What are the general public's reasons for being in favour of or against the use of genome modification for five potential applications?

SUMMARY ANSWER: Overall, 43 reasons for being in favour, 45 reasons for being against as well as 26 conditional reasons for the use of genome modification were identified.

WHAT IS KNOWN ALREADY: Various applications of somatic genome modification are progressing towards clinical introduction and several recent studies have reported on germline genome modification. This has incited a debate on ethical and legal implications and acceptability. There is a growing plea to involve the general public earlier on in the developmental process of science and (bio)technology including genome modification.

STUDY DESIGN SIZE DURATION: In April 2016, a cross-sectional survey was launched online among the Dutch general public. A documentary on genome modification on public television and calls in social media invited viewers and non-viewers, respectively, to participate.

The questionnaire introduced five potential future applications of genome modification: modified wheat for individuals with gluten intolerance; somatic modification for individuals with neuromuscular diseases; germline modification to prevent passing on a neuromuscular disease; germline modification to introduce resistance to HIV; and germline modification to increase intelligence. Participants were asked to indicate whether and why they would make use of genome modification in these scenarios. The reasons mentioned were analysed through content analysis by two researchers independently. The proportion of respondents that was willing to modify was described per scenario and associations with respondent characteristics were analysed.

The survey was completed by 1013 participants. Forty-three reasons for being in favour, 45 reasons for being against as well as 26 conditional reasons for the use of genome modification were identified. These could be categorized into 14 domains: safety of the individuals concerned; effectiveness; quality of life of the individuals concerned; existence of a clinical need or an alternative; biodiversity and ecosystems; animal homo sapiens (i.e. relating to effects on humans as a species); human life and dignity; trust in regulation; justice; costs; slippery slope; argument of nature; parental rights and duties; and (reproductive) autonomy. Participants' willingness to use genome modification was dependent on the application: most participants would eat modified wheat if gluten intolerant (74%), would use genome modification to cure his/her own neuromuscular disease (85%) and would apply germline modification to prevent passing on this neuromuscular disease (66%). A minority would apply germline modification to introduce resistance to HIV (30%) or increase intelligence (16%). Being young (odds ratio (OR) = 0.98 per year increase), being male (OR = 2.38), and having watched the documentary (OR = 1.82) were associated with being willing to apply genome modification in more scenarios.

Inquiring for reasons through open questions in a survey allowed for a larger sample size and intuitive responses but resulted in less depth than traditional face-to-face interviews. As the survey was disseminated through social media, the sample is not representative of the overall Dutch population, and hence the quantitative results should not be interpreted as such.

Further public consultation and a more in-depth ethical and societal debate on principles and conditions for responsible use of (germline) genome modification is required prior to future clinical introduction.

Funded by the University of Amsterdam and University Medical Centre Utrecht. No conflict of interest.


RevDate: 2019-03-21

Najah S, Saulnier C, Pernodet JL, et al (2019)

Design of a generic CRISPR-Cas9 approach using the same sgRNA to perform gene editing at distinct loci.

BMC biotechnology, 19(1):18 pii:10.1186/s12896-019-0509-7.

BACKGROUND: The CRISPR/Cas (clustered regularly interspaced short palindromic repeat and CRISPR-associated nucleases) based technologies have revolutionized genome engineering. While their use for prokaryotic genome editing is expanding, some limitations remain such as possible off-target effects and design constraints. These are compounded when performing systematic genome editing at distinct loci or when targeting repeated sequences (e.g. multicopy genes or mobile genetic elements). To overcome these limitations, we designed an approach using the same sgRNA and CRISPR-Cas9 system to independently perform gene editing at different loci.

RESULTS: We developed a two-step procedure based on the introduction by homologous recombination of 'bait' DNA at the vicinity of a gene copy of interest before inducing CRISPR-Cas9 activity. The introduction of a genetic tool encoding a CRISPR-Cas9 complex targeting this 'bait' DNA induces a double strand break near the copy of interest. Its repair by homologous recombination can lead either to reversion or gene copy-specific editing. The relative frequencies of these events are linked to the impact of gene editing on cell fitness. In our study, we used this technology to successfully delete the native copies of two xenogeneic silencers lsr2 paralogs in Streptomyces ambofaciens. We observed that one of these paralogs is a candidate-essential gene since its native locus can be deleted only in the presence of an extra copy.

CONCLUSION: By targeting 'bait' DNA, we designed a 'generic' CRISPR-Cas9 toolkit that can be used to edit different loci. The differential action of this CRISPR-Cas9 system is exclusively based on the specific recombination between regions surrounding the gene copy of interest. This approach is suitable to edit multicopy genes. One such particular example corresponds to the mutagenesis of candidate-essential genes that requires the presence of an extra copy of the gene before gene disruption. This opens new insights to explore gene essentiality in bacteria and to limit off-target effects during systematic CRISPR-Cas9 based approaches.

RevDate: 2019-03-21
CmpDate: 2019-03-21

Bando H, Lee Y, Sakaguchi N, et al (2018)

Inducible Nitric Oxide Synthase Is a Key Host Factor for Toxoplasma GRA15-Dependent Disruption of the Gamma Interferon-Induced Antiparasitic Human Response.

mBio, 9(5):.

Although Toxoplasma virulence mechanisms targeting gamma interferon (IFN-γ)-induced cell-autonomous antiparasitic immunity have been extensively characterized in mice, the virulence mechanisms in humans remain uncertain, partly because cell-autonomous immune responses against Toxoplasma differ markedly between mice and humans. Despite the identification of inducible nitric oxide synthase (iNOS) as an anti-Toxoplasma host factor in mice, here we show that iNOS in humans is a pro-Toxoplasma host factor that promotes the growth of the parasite. The GRA15 Toxoplasma effector-dependent disarmament of IFN-γ-induced parasite growth inhibition was evident when parasite-infected monocytes were cocultured with hepatocytes. Interleukin-1β (IL-1β), produced from monocytes in a manner dependent on GRA15 and the host's NLRP3 inflammasome, combined with IFN-γ to strongly stimulate iNOS expression in hepatocytes; this dramatically reduced the levels of indole 2,3-dioxygenase 1 (IDO1), a critically important IFN-γ-inducible anti-Toxoplasma protein in humans, thus allowing parasite growth. Taking the data together, Toxoplasma utilizes human iNOS to antagonize IFN-γ-induced IDO1-mediated cell-autonomous immunity via its GRA15 virulence factor.IMPORTANCEToxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Gamma interferon (IFN-γ) is produced in the host to inhibit the proliferation of this parasite and eventually cause its death. Unlike mouse disease models, which involve well-characterized virulence strategies that are used by Toxoplasma to suppress IFN-γ-dependent immunity, the strategies used by Toxoplasma in humans remain unclear. Here, we show that GRA15, a Toxoplasma effector protein, suppresses the IFN-γ-induced indole-2,3-dioxygenase 1-dependent antiparasite immune response in human cells. Because NLRP3-dependent production of IL-1β and nitric oxide (NO) in Toxoplasma-infected human cells is involved in the GRA15-dependent virulence mechanism, blocking NO or IL-1β production in the host could represent a novel therapeutic approach for treating human toxoplasmosis.

RevDate: 2019-03-21
CmpDate: 2019-03-21

Foronda M, LE Dow (2018)

CRISPR: Stressed about p53?.

Trends in molecular medicine, 24(9):731-733.

Two recent reports show that, in some contexts, CRISPR-mediated genome editing can lead to a p53-mediated stress response and cell-cycle arrest. These findings may help to explain why CRISPR-mediated genetic manipulation in different cell types leads to dissimilar outcomes, and highlights the need for a better understanding of the factors that influence effective genome editing in vitro and in vivo.

RevDate: 2019-03-21
CmpDate: 2019-03-21

Slotta C, Storm J, Pfisterer N, et al (2018)

IKK1/2 protect human cells from TNF-mediated RIPK1-dependent apoptosis in an NF-κB-independent manner.

Biochimica et biophysica acta. Molecular cell research, 1865(8):1025-1033.

TNF signaling is directly linked to cancer development and progression. A broad range of tumor cells is able to evade cell death induced by TNF impairing the potential anti-cancer value of TNF in therapy. Although sensitizing cells to TNF-induced death therefore has great clinical implications, detailed mechanistic insights into TNF-mediated human cell death still remain unknown. Here, we analyzed human cells by applying CRISPR/Cas9n to generate cells deficient of IKK1, IKK2, IKK1/2 and RELA. Despite stimulation with TNF resulted in impaired NF-κB activation in all genotypes compared to wildtype cells, increased cell death was observable only in IKK1/2-double-deficient cells. Cell death could be detected by Caspase-3 activation and binding of Annexin V. TNF-induced programmed cell death in IKK1/2-/- cells was further shown to be mediated via RIPK1 in a predominantly apoptotic manner. Our findings demonstrate the IKK complex to protect from TNF-induced cell death in human cells independently to NF-κB RelA suggesting IKK1/2 to be highly promising targets for cancer therapy.

RevDate: 2019-03-21
CmpDate: 2019-03-21

Sapio RT, Nezdyur AN, Krevetski M, et al (2017)

Inhibition of post-transcriptional steps in ribosome biogenesis confers cytoprotection against chemotherapeutic agents in a p53-dependent manner.

Scientific reports, 7(1):9041.

The p53-mediated nucleolar stress response associated with inhibition of ribosomal RNA transcription was previously shown to potentiate killing of tumor cells. Here, we asked whether targeting of ribosome biogenesis can be used as the basis for selective p53-dependent cytoprotection of nonmalignant cells. Temporary functional inactivation of the 60S ribosome assembly factor Bop1 in a 3T3 cell model markedly increased cell recovery after exposure to camptothecin or methotrexate. This was due, at least in part, to reversible pausing of the cell cycle preventing S phase associated DNA damage. Similar cytoprotective effects were observed after transient shRNA-mediated silencing of Rps19, but not several other tested ribosomal proteins, indicating distinct cellular responses to the inhibition of different steps in ribosome biogenesis. By temporarily inactivating Bop1 function, we further demonstrate selective killing of p53-deficient cells with camptothecin while sparing isogenic p53-positive cells. Thus, combining cytotoxic treatments with inhibition of select post-transcriptional steps of ribosome biogenesis holds potential for therapeutic targeting of cells that have lost p53.

RevDate: 2019-03-20

Eckerstorfer MF, Heissenberger A, Reichenbecher W, et al (2019)

An EU Perspective on Biosafety Considerations for Plants Developed by Genome Editing and Other New Genetic Modification Techniques (nGMs).

Frontiers in bioengineering and biotechnology, 7:31.

The question whether new genetic modification techniques (nGM) in plant development might result in non-negligible negative effects for the environment and/or health is significant for the discussion concerning their regulation. However, current knowledge to address this issue is limited for most nGMs, particularly for recently developed nGMs, like genome editing, and their newly emerging variations, e.g., base editing. This leads to uncertainties regarding the risk/safety-status of plants which are developed with a broad range of different nGMs, especially genome editing, and other nGMs such as cisgenesis, transgrafting, haploid induction or reverse breeding. A literature survey was conducted to identify plants developed by nGMs which are relevant for future agricultural use. Such nGM plants were analyzed for hazards associated either (i) with their developed traits and their use or (ii) with unintended changes resulting from the nGMs or other methods applied during breeding. Several traits are likely to become particularly relevant in the future for nGM plants, namely herbicide resistance (HR), resistance to different plant pathogens as well as modified composition, morphology, fitness (e.g., increased resistance to cold/frost, drought, or salinity) or modified reproductive characteristics. Some traits such as resistance to certain herbicides are already known from existing GM crops and their previous assessments identified issues of concern and/or risks, such as the development of herbicide resistant weeds. Other traits in nGM plants are novel; meaning they are not present in agricultural plants currently cultivated with a history of safe use, and their underlying physiological mechanisms are not yet sufficiently elucidated. Characteristics of some genome editing applications, e.g., the small extent of genomic sequence change and their higher targeting efficiency, i.e., precision, cannot be considered an indication of safety per se, especially in relation to novel traits created by such modifications. All nGMs considered here can result in unintended changes of different types and frequencies. However, the rapid development of nGM plants can compromise the detection and elimination of unintended effects. Thus, a case-specific premarket risk assessment should be conducted for nGM plants, including an appropriate molecular characterization to identify unintended changes and/or confirm the absence of unwanted transgenic sequences.

RevDate: 2019-03-20

Gentile GM, Wetzel KS, Dedrick RM, et al (2019)

More Evidence of Collusion: a New Prophage-Mediated Viral Defense System Encoded by Mycobacteriophage Sbash.

mBio, 10(2): pii:mBio.00196-19.

The arms race between bacteria and their bacteriophages profoundly influences microbial evolution. With an estimated 1023 phage infections occurring per second, there is strong selection for both bacterial survival and phage coevolution for continued propagation. Many phage resistance systems, including restriction-modification systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) systems, a variety of abortive infection systems, and many others that are not yet mechanistically defined, have been described. Temperate bacteriophages are common and form stable lysogens that are immune to superinfection by the same or closely related phages. However, temperate phages collude with their hosts to confer defense against genomically distinct phages, to the mutual benefit of the bacterial host and the prophage. Prophage-mediated viral systems have been described in Mycobacterium phages and Pseudomonas phages but are predicted to be widespread throughout the microbial world. Here we describe a new viral defense system in which the mycobacteriophage Sbash prophage colludes with its Mycobacterium smegmatis host to confer highly specific defense against infection by the unrelated mycobacteriophage Crossroads. Sbash genes 30 and 31 are lysogenically expressed and are necessary and sufficient to confer defense against Crossroads but do not defend against any of the closely related phages grouped in subcluster L2. The mapping of Crossroads defense escape mutants shows that genes 132 and 141 are involved in recognition by the Sbash defense system and are proposed to activate a loss in membrane potential mediated by Sbash gp30 and gp31.IMPORTANCE Viral infection is an ongoing challenge to bacterial survival, and there is strong selection for development or acquisition of defense systems that promote survival when bacteria are attacked by bacteriophages. Temperate phages play central roles in these dynamics through lysogenic expression of genes that defend against phage attack, including those unrelated to the prophage. Few prophage-mediated viral defense systems have been characterized, but they are likely widespread both in phage genomes and in the prophages integrated in bacterial chromosomes.

RevDate: 2019-03-20
CmpDate: 2019-03-20

Lee K, Eggenberger AL, Banakar R, et al (2019)

CRISPR/Cas9-mediated targeted T-DNA integration in rice.

Plant molecular biology, 99(4-5):317-328.

KEY MESSAGE: Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome. Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50-80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Kandul NP, Liu J, Sanchez C HM, et al (2019)

Transforming insect population control with precision guided sterile males with demonstration in flies.

Nature communications, 10(1):84 pii:10.1038/s41467-018-07964-7.

The sterile insect technique (SIT) is an environmentally safe and proven technology to suppress wild populations. To further advance its utility, a novel CRISPR-based technology termed precision guided SIT (pgSIT) is described. PgSIT mechanistically relies on a dominant genetic technology that enables simultaneous sexing and sterilization, facilitating the release of eggs into the environment ensuring only sterile adult males emerge. Importantly, for field applications, the release of eggs will eliminate burdens of manually sexing and sterilizing males, thereby reducing overall effort and increasing scalability. Here, to demonstrate efficacy, we systematically engineer multiple pgSIT systems in Drosophila which consistently give rise to 100% sterile males. Importantly, we demonstrate that pgSIT-generated sterile males are fit and competitive. Using mathematical models, we predict pgSIT will induce substantially greater population suppression than can be achieved by currently-available self-limiting suppression technologies. Taken together, pgSIT offers to potentially transform our ability to control insect agricultural pests and disease vectors.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Balmus G, Pilger D, Coates J, et al (2019)

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks.

Nature communications, 10(1):87 pii:10.1038/s41467-018-07729-2.

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Sun J, Carlson-Stevermer J, Das U, et al (2019)

CRISPR/Cas9 editing of APP C-terminus attenuates β-cleavage and promotes α-cleavage.

Nature communications, 10(1):53 pii:10.1038/s41467-018-07971-8.

CRISPR/Cas9 guided gene-editing is a potential therapeutic tool, however application to neurodegenerative disease models has been limited. Moreover, conventional mutation correction by gene-editing would only be relevant for the small fraction of neurodegenerative cases that are inherited. Here we introduce a CRISPR/Cas9-based strategy in cell and animal models to edit endogenous amyloid precursor protein (APP) at the extreme C-terminus and reciprocally manipulate the amyloid pathway, attenuating APP-β-cleavage and Aβ production, while up-regulating neuroprotective APP-α-cleavage. APP N-terminus and compensatory APP-homologues remain intact, with no apparent effects on neurophysiology in vitro. Robust APP-editing is seen in human iPSC-derived neurons and mouse brains with no detectable off-target effects. Our strategy likely works by limiting APP and BACE-1 approximation, and we also delineate mechanistic events that abrogates APP/BACE-1 convergence in this setting. Our work offers conceptual proof for a selective APP silencing strategy.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Gdula MR, Nesterova TB, Pintacuda G, et al (2019)

The non-canonical SMC protein SmcHD1 antagonises TAD formation and compartmentalisation on the inactive X chromosome.

Nature communications, 10(1):30 pii:10.1038/s41467-018-07907-2.

The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), A/B compartments and formation of two mega-domains. Here we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 mutant cells reveals the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including A/B compartments and partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred following SmcHD1 knockout in a somatic cell model, but in this case, independent of Xi gene derepression. We conclude that SmcHD1 is a key factor in defining the unique chromosome architecture of Xi.

RevDate: 2019-03-20
CmpDate: 2019-03-20

Ng KP, Manjeri A, Lee LM, et al (2018)

The arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) induces apoptosis in leukemic cells specifically under hypoxic conditions but CRISPR/Cas9 excludes arginase 2 (ARG2) as the functional target.

PloS one, 13(10):e0205254.

Cancer cells, including in chronic myeloid leukemia (CML), depend on the hypoxic response to persist in hosts and evade therapy. Accordingly, there is significant interest in drugging cancer-specific hypoxic responses. However, a major challenge in leukemia is identifying differential and druggable hypoxic responses between leukemic and normal cells. Previously, we found that arginase 2 (ARG2), an enzyme of the urea cycle, is overexpressed in CML but not normal progenitors. ARG2 is a target of the hypoxia inducible factors (HIF1-α and HIF2-α), and is required for the generation of polyamines which are required for cell growth. We therefore explored if the clinically-tested arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) would be effective against leukemic cells under hypoxic conditions. Remarkably, nor-NOHA effectively induced apoptosis in ARG2-expressing cells under hypoxia but not normoxia. Co-treatment with nor-NOHA overcame hypoxia-mediated resistance towards BCR-ABL1 kinase inhibitors. While nor-NOHA itself is promising in targeting the leukemia hypoxic response, we unexpectedly found that its anti-leukemic activity was independent of ARG2 inhibition. Genetic ablation of ARG2 using CRISPR/Cas9 had no effect on the viability of leukemic cells and their sensitivity towards nor-NOHA. This discrepancy was further evidenced by the distinct effects of ARG2 knockouts and nor-NOHA on cellular respiration. In conclusion, we show that nor-NOHA has significant but off-target anti-leukemic activity among ARG2-expressing hypoxic cells. Since nor-NOHA has been employed in clinical trials, and is widely used in studies on endothelial dysfunction, immunosuppression and metabolism, the diverse biological effects of nor-NOHA must be cautiously evaluated before attributing its activity to ARG inhibition.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Xu X, Zhang Z, Yang Y, et al (2018)

Genome editing reveals the function of Yorkie during the embryonic and early larval development in silkworm, Bombyx mori.

Insect molecular biology, 27(6):675-685.

As a transcriptional coactivator, Yorkie (Yki) is a major downstream target of the Hippo signalling pathway to regulate the organ size during animal development and regeneration. Previous microarray analysis in the silkworm, Bombyx mori, has shown that genes associated with the Hippo pathway were primarily expressed in gonads and imaginal discs. The RNA-interference-mediated silencing of Yki at the early wandering stage delayed B. mori development and ovary maturation, whereas baculovirus-mediated overexpression at the late larval instar facilitated organ growth and accelerated metamorphosis. Here, we employed CRISPR/Cas9-mediated mutagenesis to investigate the function of Yki in B. mori (BmYki) at the embryonic and early larval stages. Knocking out of BmYki led to reduced body size, moulting defects and, eventually, larval lethality. Sequence analysis of CRISPR/Cas9 mutants exhibited an array of deletions in BmYki. As a critical downstream effector of the Hippo kinase cassette, silencing of BmYki at the embryonic stage is indispensable and the consequence is lethal. Given that the Hippo signalling pathway is evolutionarily conserved, Yki has the potential to be a novel molecular target for genetic-based pest management practices.

RevDate: 2019-03-20
CmpDate: 2019-03-20

Wang Q, Cobine PA, JJ Coleman (2018)

Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein complexes.

Fungal genetics and biology : FG & B, 117:21-29.

The Fusarium oxysporum species complex (FOSC) is an economically important group of pathogenic filamentous fungi that are able to infect both animals and plants. Reverse genetic techniques, including gene disruption/deletion methods, to study these fungi are available although limitations exist resulting in decreased efficiency. Herein we describe a gene editing system developed using a F. oxysporum-optimized Cas9 ribonucleoprotein (RNP) and protoplast transformation method. The Cas9 protein and sgRNA were assembled to form a stable RNP in vitro and this complex was transferred into fungal protoplasts for gene editing with PEG-mediated transformation. In order to determine if the Cas9 RNP system is functional in the FOSC protoplasts and assess the efficacy of the system, two genes, URA5 and URA3, were selected for targeted disruption generating uracil auxotroph mutants that are resistant to 5-fluoroorotic acid, 5-FOA. In addition, a gene in a secondary metabolite biosynthetic cluster, the ortholog of BIK1, was mutated using this system and the maximum efficiency of this gene disruption was about 50%. Further analysis of the bik1 mutant confirmed that this polyketide synthase was involved in the synthesis of the red pigment, bikaverin. The mutants generated in this study displayed the strong expected phenotypes, demonstrating this F. oxysporum-optimized CRISPR/Cas9 system is stable and can efficiently disrupt the genes of interest.

RevDate: 2019-03-20
CmpDate: 2019-02-20

Yan S, Tu Z, Liu Z, et al (2018)

A Huntingtin Knockin Pig Model Recapitulates Features of Selective Neurodegeneration in Huntington's Disease.

Cell, 173(4):989-1002.e13.

Huntington's disease (HD) is characterized by preferential loss of the medium spiny neurons in the striatum. Using CRISPR/Cas9 and somatic nuclear transfer technology, we established a knockin (KI) pig model of HD that endogenously expresses full-length mutant huntingtin (HTT). By breeding this HD pig model, we have successfully obtained F1 and F2 generation KI pigs. Characterization of founder and F1 KI pigs shows consistent movement, behavioral abnormalities, and early death, which are germline transmittable. More importantly, brains of HD KI pig display striking and selective degeneration of striatal medium spiny neurons. Thus, using a large animal model of HD, we demonstrate for the first time that overt and selective neurodegeneration seen in HD patients can be recapitulated by endogenously expressed mutant proteins in large mammals, a finding that also underscores the importance of using large mammals to investigate the pathogenesis of neurodegenerative diseases and their therapeutics.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Caprara G, Prosperini E, Piccolo V, et al (2018)

PARP14 Controls the Nuclear Accumulation of a Subset of Type I IFN-Inducible Proteins.

Journal of immunology (Baltimore, Md. : 1950), 200(7):2439-2454.

The enzymes of the poly-ADP-ribose polymerase (PARP) superfamily control many relevant cellular processes, but a precise understanding of their activities in different physiological or disease contexts is largely incomplete. We found that transcription of several Parp genes was dynamically regulated upon murine macrophage activation by endotoxin. PARP14 was strongly induced by several inflammatory stimuli and translocated into the nucleus of stimulated cells. Quantitative mass spectrometry analysis showed that PARP14 bound to a group of IFN-stimulated gene (ISG)-encoded proteins, most with an unknown function, and it was required for their nuclear accumulation. Moreover, PARP14 depletion attenuated transcription of primary antiviral response genes regulated by the IFN regulatory transcription factor 3, including Ifnb1, thus reducing IFN-β production and activation of ISGs involved in the secondary antiviral response. In agreement with the above-mentioned data, PARP14 hindered Salmonella typhimurium proliferation in murine macrophages. Overall, these data hint at a role of PARP14 in the control of antimicrobial responses and specifically in nuclear activities of a subgroup of ISG-encoded proteins.

RevDate: 2019-03-20
CmpDate: 2019-03-20

Di Cristina M, VB Carruthers (2018)

New and emerging uses of CRISPR/Cas9 to genetically manipulate apicomplexan parasites.

Parasitology, 145(9):1119-1126.

Although the application of CRISPR/Cas9 genome engineering approaches was first reported in apicomplexan parasites only 3 years ago, this technology has rapidly become an essential component of research on apicomplexan parasites. This review briefly describes the history of CRISPR/Cas9 and the principles behind its use along with documenting its implementation in apicomplexan parasites, especially Plasmodium spp. and Toxoplasma gondii. We also discuss the recent use of CRISPR/Cas9 for whole genome screening of gene knockout mutants in T. gondii and highlight its use for seminal genetic manipulations of Cryptosporidium spp. Finally, we consider new variations of CRISPR/Cas9 that have yet to be implemented in apicomplexans. Whereas CRISPR/Cas9 has already accelerated rapid interrogation of gene function in apicomplexans, the full potential of this technology is yet to be realized as new variations and innovations are integrated into the field.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Kornete M, Marone R, LT Jeker (2018)

Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells.

Journal of immunology (Baltimore, Md. : 1950), 200(7):2489-2501.

Adoptive cell transfer is an important approach for basic research and emerges as an effective treatment for various diseases, including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. In this article, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology-directed repair, systematically optimize experimental parameters, and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells, with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Repairing the cause of the scurfy syndrome, a 2-bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Che P, Anand A, Wu E, et al (2018)

Developing a flexible, high-efficiency Agrobacterium-mediated sorghum transformation system with broad application.

Plant biotechnology journal, 16(7):1388-1395.

Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi-arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here, we report a ternary vector (also known as cohabitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium-mediated transformation of sorghum. We report regeneration frequencies ranging from 6% to 29% in Tx430 using different selectable markers and single copy, backbone free 'quality events' ranging from 45% to 66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84-7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9-mediated gene knockouts in Tx430.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Lin CS, Hsu CT, Yang LH, et al (2018)

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration.

Plant biotechnology journal, 16(7):1295-1310.

Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Tirado-Gonzalez I, Czlonka E, Nevmerzhitskaya A, et al (2018)

CRISPR/Cas9-edited NSG mice as PDX models of human leukemia to address the role of niche-derived SPARC.

Leukemia, 32(4):1049-1052.

RevDate: 2019-03-20
CmpDate: 2019-02-19

DiEuliis D, J Giordano (2018)

Gene editing using CRISPR/Cas9: implications for dual-use and biosecurity.

Protein & cell, 9(3):239-240.

RevDate: 2019-03-20
CmpDate: 2019-02-20

Du J, Shang J, Chen F, et al (2018)

A CRISPR/Cas9-Based Screening for Non-Homologous End Joining Inhibitors Reveals Ouabain and Penfluridol as Radiosensitizers.

Molecular cancer therapeutics, 17(2):419-431.

Non-homologous end joining (NHEJ) is the major pathway responsible for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSB), and correspondingly regulates the cellular response to IR. Identification of NHEJ inhibitors could substantially enhance the tumor radiosensitivity and improve the therapeutic efficiency of radiotherapy. In this study, we demonstrated a screening for NHEJ inhibitors using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and high-resolution melting (HRM) analysis. Because NHEJ is regarded as an error-prone mechanism, the NHEJ-mediated ligation of the site-specific DSB induced by Cas9 nuclease would eventually cause the mutation of the targeted sequence. Then, HRM analysis, a reliable and rapid assay for detecting sequence variation, was performed to evaluate the mutation efficiency of the targeted site. Validating analysis confirmed the NHEJ activities were positively correlated with the mutation frequencies. Next, an approved drug library containing 1,540 compounds was interrogated by using this screening strategy. Our results identified ouabain, a cardiotonic agent, and penfluridol, an antipsychotic agent, have the capacity to restrain NHEJ activity. Further experiments in vitro revealed the radiosensitizing effects of these compounds. Overall, we presented a cell-based screening for NHEJ inhibitors, which could promote the discovery of novel radiosensitizers. Mol Cancer Ther; 17(2); 419-31. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

RevDate: 2019-03-14
CmpDate: 2019-03-14

Ong SH, Li Y, Koike-Yusa H, et al (2017)

Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries.

Scientific reports, 7(1):7384.

Genome-wide CRISPR-based knockout (CRISPR-KO) screening is an emerging technique which enables systematic genetic analysis of a cellular or molecular phenotype in question. Continuous improvements, such as modifications to the guide RNA (gRNA) scaffold and the development of gRNA on-target prediction algorithms, have since been made to increase their screening performance. We compared the performance of three available second-generation human genome-wide CRISPR-KO libraries that included at least one of the improvements, and examined the effect of gRNA scaffold, number of gRNAs per gene and number of replicates on screen performance. We identified duplicated screens using a library with 6 gRNAs per gene as providing the best trade-off. Despite the improvements, we found that each improved library still has library-specific false negatives and, for the first time, estimated the false negative rates of CRISPR-KO screens, which are between 10% and 20%. Our newly-defined optimal screening parameters would be helpful in designing screens and constructing bespoke gRNA libraries.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Feehan JM, Chiu CN, Stanar P, et al (2017)

Modeling Dominant and Recessive Forms of Retinitis Pigmentosa by Editing Three Rhodopsin-Encoding Genes in Xenopus Laevis Using Crispr/Cas9.

Scientific reports, 7(1):6920.

The utility of Xenopus laevis, a common research subject for developmental biology, retinal physiology, cell biology, and other investigations, has been limited by lack of a robust gene knockout or knock-down technology. Here we describe manipulation of the X. laevis genome using CRISPR/Cas9 to model the human disorder retinitis pigmentosa, and to introduce point mutations or exogenous DNA sequences. We introduced and characterized in-frame and out-of-frame insertions and deletions in three genes encoding rhodopsin by co-injection of Cas9 mRNA, eGFP mRNA, and single guide RNAs into fertilized eggs. Deletions were characterized by direct sequencing and cloning; phenotypes were assessed by assays of rod opsin in retinal extracts, and confocal microscopy of cryosectioned and immunolabeled contralateral eyes. We obtained germline transmission of editing to F1 offspring. In-frame deletions frequently caused dominant retinal degeneration associated with rhodopsin biosynthesis defects, while frameshift phenotypes were consistent with knockout. We inserted eGFP or point mutations into rhodopsin genes by co-injection of repair fragments with homology to the Cas9 target sites. Our techniques can produce high frequency gene editing in X. laevis, permitting analysis in the F0 generation, and advancing the utility of X. laevis as a subject for biological research and disease modeling.

RevDate: 2019-03-20
CmpDate: 2019-03-20

Keeler AM, ElMallah MK, TR Flotte (2017)

Gene Therapy 2017: Progress and Future Directions.

Clinical and translational science, 10(4):242-248.

RevDate: 2019-03-20
CmpDate: 2017-06-12

Zheng X, Li SY, Zhao GP, et al (2017)

An efficient system for deletion of large DNA fragments in Escherichia coli via introduction of both Cas9 and the non-homologous end joining system from Mycobacterium smegmatis.

Biochemical and biophysical research communications, 485(4):768-774.

Accompanied with the internal non-homologous end joining (NHEJ) system, Cas9 can be used to easily inactivate a gene or delete a fragment through introduction of DNA double-stranded breaks (DSBs) in eukaryotic cells. While in most prokaryotes (e.g. Escherichia coli), due to the lack of NHEJ, homologous recombination (HR) is required for repair of DSBs, which is less convenient. Here, a markerless system was developed for rapid gene inactivation or fragment deletion in E. coli via introduction of both Cas9 and a bacterial NHEJ system. Three bacterial NHEJ systems, i.e. Mycobacterium smegmatis (Msm), Mycobacterium tuberculosis (Mtb) and Bacillus subtilis (Bs), were tested in E. coli, and the MsmNHEJ system showed the best efficiency. With the employment of Cas9 and MsmNHEJ, we efficiently mutated lacZ gene, deleted glnALG operon and two large DNA fragments (67 kb and 123 kb) in E. coli, respectively. Moreover, the system was further designed to allow for continuous inactivation of genes or deletion of DNA fragments in E. coli. We envision this system can be extended to other bacteria, especially those with low HR efficiency.

RevDate: 2019-03-20
CmpDate: 2019-02-19

Hohmann S (2017)

Editor's comment on "CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein".

Molecular genetics and genomics : MGG, 292(3):535-536.

RevDate: 2019-03-20
CmpDate: 2017-06-12

Zhu QM, Ko KA, Ture S, et al (2017)

Novel Thrombotic Function of a Human SNP in STXBP5 Revealed by CRISPR/Cas9 Gene Editing in Mice.

Arteriosclerosis, thrombosis, and vascular biology, 37(2):264-270.

OBJECTIVE: To identify and characterize the effect of a SNP (single-nucleotide polymorphism) in the STXBP5 locus that is associated with altered thrombosis in humans. GWAS (genome-wide association studies) have identified numerous SNPs associated with human thrombotic phenotypes, but determining the functional significance of an individual candidate SNP can be challenging, particularly when in vivo modeling is required. Recent GWAS led to the discovery of STXBP5 as a regulator of platelet secretion in humans. Further clinical studies have identified genetic variants of STXBP5 that are linked to altered plasma von Willebrand factor levels and thrombosis in humans, but the functional significance of these variants in STXBP5 is not understood.

APPROACH AND RESULTS: We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) techniques to produce a precise mouse model carrying a human coding SNP rs1039084 (encoding human p. N436S) in the STXBP5 locus associated with decreased thrombosis. Mice carrying the orthologous human mutation (encoding p. N437S in mouse STXBP5) have lower plasma von Willebrand factor levels, decreased thrombosis, and decreased platelet secretion compared with wild-type mice. This thrombosis phenotype recapitulates the phenotype of humans carrying the minor allele of rs1039084. Decreased plasma von Willebrand factor and platelet activation may partially explain the decreased thrombotic phenotype in mutant mice.

CONCLUSIONS: Using precise mammalian genome editing, we have identified a human nonsynonymous SNP rs1039084 in the STXBP5 locus as a causal variant for a decreased thrombotic phenotype. CRISPR/Cas9 genetic editing facilitates the rapid and efficient generation of animals to study the function of human genetic variation in vascular diseases.

RevDate: 2019-03-19

Varble A, Meaden S, Barrangou R, et al (2019)

Recombination between phages and CRISPR-cas loci facilitates horizontal gene transfer in staphylococci.

Nature microbiology pii:10.1038/s41564-019-0400-2 [Epub ahead of print].

CRISPR (clustered regularly interspaced short palindromic repeats) loci and their associated (cas) genes encode an adaptive immune system that protects prokaryotes from viral1 and plasmid2 invaders. Following viral (phage) infection, a small fraction of the prokaryotic cells are able to integrate a small sequence of the invader's genome into the CRISPR array1. These sequences, known as spacers, are transcribed and processed into small CRISPR RNA guides3-5 that associate with Cas nucleases to specify a viral target for destruction6-9. Although CRISPR-cas loci are widely distributed throughout microbial genomes and often display hallmarks of horizontal gene transfer10-12, the drivers of CRISPR dissemination remain unclear. Here, we show that spacers can recombine with phage target sequences to mediate a form of specialized transduction of CRISPR elements. Phage targets in phage 85, ΦNM1, ΦNM4 and Φ12 can recombine with spacers in either chromosomal or plasmid-borne CRISPR loci in Staphylococcus, leading to either the transfer of CRISPR-adjacent genes or the propagation of acquired immunity to other bacteria in the population, respectively. Our data demonstrate that spacer sequences not only specify the targets of Cas nucleases but also can promote horizontal gene transfer.

RevDate: 2019-03-19

Perumal E, So Youn K, Sun S, et al (2019)

PTEN inactivation induces epithelial-mesenchymal transition and metastasis by intranuclear translocation of β-catenin and snail/slug in non-small cell lung carcinoma cells.

Lung cancer (Amsterdam, Netherlands), 130:25-34.

OBJECTIVE: Epithelial-mesenchymal transition (EMT) is the key event in distant metastasis of diverse tumors including lung cancer. Recent evidence suggests the involvement of phosphatase and tensin homolog (PTEN) in EMT phenotypes. However, the molecular mechanism of EMT induced by PTEN inactivation is not clear in lung cancer. We aimed to investigate the role of PTEN inactivation in acquisition of EMT in lung cancer cells.

METHODS: We knocked out the PTEN in PTEN proficient lung cancer cells lines (A549 and NCI-H460) using CRISPR/Cas-9 system and observed the growth, EMT phenotypes, and EMT related molecules. We also explored the in vivo effect of PTEN inactivation on tumor cell growth and distant metastasis using nude mouse injection.

RESULTS: PTEN knockout (KO) cells showed faster growth, migration and invasion than PTEN wild-type (WT) cells. When we injected the cells into nude mice, PTEN-KO cells showed faster growth and higher metastatic potential. In PTEN-KO cells, the levels of phosphorylated AKT (Ser-473 and Thr-308) were profoundly elevated and the expressions of phosphorylated GSK-3β (Ser9, inactive form) increased, while that of β-catenin decreased. Regarding the EMT markers, the expression of E-cadherin decreased but those of N-cadherin, vimentin and MMP-2 increased in the PTEN-KO cells. Especially, PTEN-KO cells showed the almost complete intra-nuclear shift of β-catenin and no β-catenin signal was observed in the cell membrane. Accordingly, PTEN-KO cells exhibited morphological changes such as loss of cell-to-cell contact, pseudopodia and the round shape, which are the typical phenotypes of EMT. Snail and Slug were also dominantly accumulated in the nucleus after PTEN inactivation.

CONCLUSION: All these data consistently support that PTEN inactivation contributes to EMT by nuclear translocation of β-catenin and Snail/Slug in lung cancer cells.

RevDate: 2019-03-19
CmpDate: 2019-03-19

Benoit CR, Stanton AE, Tartanian AC, et al (2018)

Functional and phylogenetic characterization of noncanonical vitamin B12-binding proteins in zebrafish suggests involvement in cobalamin transport.

The Journal of biological chemistry, 293(45):17606-17621.

In humans, transport of food-derived cobalamin (vitamin B12) from the digestive system into the bloodstream involves three paralogous proteins: transcobalamin (TC), haptocorrin (HC), and intrinsic factor (IF). Each of these proteins contains two domains, an α-domain and a β-domain, which together form a cleft in which cobalamin binds. Zebrafish (Danio rerio) are thought to possess only a single cobalamin transport protein, referred to as Tcn2, which is a transcobalamin homolog. Here, we used CRISPR/Cas9 mutagenesis to create null alleles of tcn2 in zebrafish. Fish homozygous for tcn2-null alleles were viable and exhibited no obvious developmentally or behaviorally abnormal phenotypes. For this reason, we hypothesized that previously unidentified cobalamin-carrier proteins encoded in the zebrafish genome may provide an additional pathway for cobalamin transport. We identified genes predicted to code for two such proteins, Tcn-beta-a (Tcnba) and Tcn-beta-b (Tcnbb), which differ from all previously characterized cobalamin transport proteins as they lack the α-domain. These β-domain-only proteins are representative of an undescribed class of cobalamin-carrier proteins that are highly conserved throughout the ray-finned fishes. We observed that the genes encoding the three cobalamin transport homologs, tcn2, tcnba, and tcnbb, are expressed in unique spatial and temporal patterns in the developing zebrafish. Moreover, exogenously expressed recombinant Tcnba and Tcnbb bound cobalamin with high affinity, comparable with binding by full-length Tcn2. Taken together, our results suggest that this noncanonical protein structure has evolved to fully function as a cobalamin-carrier protein, thereby allowing for a compensatory cobalamin transport mechanism in the tcn2-/- zebrafish.

RevDate: 2019-03-19
CmpDate: 2019-03-19

Tan Y, Jin C, Ma W, et al (2018)

Dismissal of RNA Polymerase II Underlies a Large Ligand-Induced Enhancer Decommissioning Program.

Molecular cell, 71(4):526-539.e8.

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.

RevDate: 2019-03-19
CmpDate: 2019-03-19

Blokhin I, Khorkova O, Hsiao J, et al (2018)

Developments in lncRNA drug discovery: where are we heading?.

Expert opinion on drug discovery, 13(9):837-849.

INTRODUCTION: The central dogma of molecular biology, which states that the only role of long RNA transcripts is to convey information from gene to protein, was brought into question in recent years due to discovery of the extensive presence and complex roles of long noncoding RNAs (lncRNAs). Furthermore, lncRNAs were found to be involved in pathogenesis of multiple diseases and thus represent a new class of therapeutic targets. Translational efforts in the lncRNA field have been augmented by progress in optimizing the chemistry and delivery platforms of lncRNA-targeting modalities, including oligonucleotide-based drugs and CRISPR-Cas9. Areas covered: This review covers the current advances in characterizing diversity and biological functions of lncRNA focusing on their therapeutic potential in selected therapeutic areas. Expert opinion: Due to accelerating parallel progress in lncRNA biology and lncRNA-compatible therapeutic modalities, it is likely that lncRNA-dependent mechanisms of pathogenesis will soon be targeted in various disorders, including neurological, psychiatric, cardiovascular, infectious diseases, and cancer. Significant efforts, however, are still required to better understand the biology of both lncRNAs and lncRNA-targeting drugs. Further work is needed in the areas of lncRNA nomenclature, database representation, intra/interfield communication, and education of the community at large.

RevDate: 2019-03-19
CmpDate: 2019-03-19

Shou J, Li J, Liu Y, et al (2018)

Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion.

Molecular cell, 71(4):498-509.e4.

Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site in vivo and in vitro, generating double-strand break ends with 5' overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.

RevDate: 2019-03-19
CmpDate: 2019-03-19

Contreras LM (2018)

Methods and advances in RNA characterization and design.

Methods (San Diego, Calif.), 143:1-3.

RevDate: 2019-03-19
CmpDate: 2019-03-19

Paul MW, Zelensky AN, Wyman C, et al (2018)

Single-Molecule Dynamics and Localization of DNA Repair Proteins in Cells.

Methods in enzymology, 600:375-406.

Direct observation of individual protein molecules in their native environment, at nanometer resolution, in a living cell, in motion is not only fascinating but also uniquely informative. Several recent major technological advances in genomic engineering, protein and synthetic fluorophore development, and light microscopy have dramatically increased the accessibility of this approach. This chapter describes the procedures for modifying endogenous genomic loci to producing fluorescently tagged proteins, their high-resolution visualization, and analysis of their dynamics in mammalian cells, using DNA repair proteins BRCA2 and RAD51 as an example.

RevDate: 2019-03-19
CmpDate: 2019-03-19

Lee YJ, TS Moon (2018)

Design rules of synthetic non-coding RNAs in bacteria.

Methods (San Diego, Calif.), 143:58-69.

One of the long-term goals of synthetic biology is to develop designable genetic parts with predictable behaviors that can be utilized to implement diverse cellular functions. The discovery of non-coding RNAs and their importance in cellular processing have rapidly attracted researchers' attention towards designing functional non-coding RNA molecules. These synthetic non-coding RNAs have simple design principles governed by Watson-Crick base pairing, but exhibit increasingly complex functions. Importantly, due to their specific and modular behaviors, synthetic non-coding RNAs have been widely adopted to modulate transcription and translation of target genes. In this review, we summarize various design rules and strategies employed to engineer synthetic non-coding RNAs. Specifically, we discuss how RNA molecules can be transformed into powerful regulators and utilized to control target gene expression. With the establishment of generalizable non-coding RNA design rules, the research community will shift its focus to RNA regulators from protein regulators.

RevDate: 2019-03-18

Moses C, P Kaur (2019)

Applications of CRISPR systems in respiratory health: Entering a new 'red pen' era in genome editing.

Respirology (Carlton, Vic.) [Epub ahead of print].

Respiratory diseases, such as influenza infection, acute tracheal bronchitis, pneumonia, tuberculosis, chronic obstructive pulmonary disease, asthma, lung cancer and nasopharyngeal carcinoma, continue to significantly impact human health. Diseases of the lung and respiratory tract are influenced by environmental conditions and socio-economic factors; however, many of these serious respiratory disorders are also rooted in genetic or epigenetic causes. Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, isolated from the immune system of prokaryotes, provide a tool to manipulate gene sequences and gene expression with significant implications for respiratory research. CRISPR/Cas systems allow preclinical modelling of causal factors involved in many respiratory diseases, providing new insights into their underlying mechanisms. CRISPR can also be used to screen for genes involved in respiratory processes, development and pathology, identifying novel disease drivers or drug targets. Finally, CRISPR/Cas systems can potentially correct genetic mutations and edit epigenetic marks that contribute to respiratory disorders, providing a form of personalized medicine that could be used in conjunction with other technologies such as stem cell reprogramming and transplantation. CRISPR gene editing is a young field of research, and concerns regarding its specificity, as well as the need for efficient and safe delivery methods, need to be addressed further. However, CRISPR/Cas systems represent a significant step forward for research and therapy in respiratory health, and it is likely we will see the breakthroughs generated from this technology continue.

RevDate: 2019-03-18

Lu ZJ, Yu Q, Zhou SH, et al (2019)

Construction of a GLUT-1 and HIF-1α gene knockout cell model in HEp-2 cells using the CRISPR/Cas9 technique.

Cancer management and research, 11:2087-2096 pii:cmar-11-2087.

Background: Glucose transporter (GLUT)-mediated glucose uptake is an important process in the development of laryngeal carcinoma, one of the most common malignancies of the head and neck. GLUT-1, together with HIF-1α, is also an indicator of hypoxia. Both proteins play a critical role in glucose uptake and glycolysis in laryngeal carcinoma cells under hypoxic stress. A double gene knockout model in which HIF-1α and GLUT-1 are no longer expressed can provide important information about carcinogenesis in laryngeal carcinoma.

Purpose: In this study we used the CRISPR/Cas 9 system to induce HIF-1α and GLUT-1 double gene knockout in HEp-2 cells and then used the knocked-out cells to study the role of these markers in laryngeal carcinoma, including in chemoradioresistance.

Methods: High-grade small-guide RNAs (sgRNAs) of HIF-1α and GLUT-1 were designed using an online tool and inserted into the pUC57-T7-gRNA vector. The recombinant plasmids were transfected into HEp-2 cells and positive cells were screened using the dilution method. Gene mutation and expression were determined by sequence analysis and immunoblotting.

Results: In HIF-1α and GLUT-1 double gene knockout HEp-2 cells, a 171-bp deletion in the HIF-1α genomic sequence was detected, whereas multiple base insertions resulted in frameshift mutations in the GLUT-1 gene. Neither HIF-1α nor GLUT-1 protein was expressed in positive cells. The proliferation, migration, and invasion of HEp-2 cells were significantly decreased afterward. The possible mechanism may be that the inhibition PI3K/AKT/mTOR pathway by HIF-1α and GLUT-1 double gene knockout using CRISPR/Cas9 technique lead to reduction of glucose uptake and lactic acid generation.

Conclusion: Our HIF-1α and GLUT-1 double gene knockout HEp-2 cell model, obtained using a CRISPR/Cas9-based system, may facilitate studies of the pathogenesis of laryngeal carcinoma.

RevDate: 2019-03-18
CmpDate: 2019-03-18

Xiao B, Yin S, Hu Y, et al (2019)

Epigenetic editing by CRISPR/dCas9 in Plasmodium falciparum.

Proceedings of the National Academy of Sciences of the United States of America, 116(1):255-260.

Genetic manipulation remains a major obstacle for understanding the functional genomics of the deadliest malaria parasite Plasmodium falciparum Although the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system has been successfully applied to introduce permanent changes in the parasite genome, its use is still limited. Here we show that fusing different epigenetic effector domains to a Cas9 null mutant efficiently and specifically reprograms the expression of target genes in P. falciparum By precisely writing and erasing histone acetylation at the transcription start site regions of the invasion-related genes reticulocyte binding protein homolog 4 (rh4) and erythrocyte binding protein 175 (eba-175), respectively, we achieved significant activation of rh4 and repression of eba-175, leading to the switch of the parasite invasion pathways into human erythrocytes. By using the epigenetic knockdown system, we have also characterized the effects of PfSET1, previously identified as an essential gene, on expression of mainly trophozoite- and schizont-specific genes, and therefore regulation of the growth of the mature forms of P. falciparum This epigenetic CRISPR/dCas9 system provides a powerful approach for regulating gene expression at the transcriptional level in P. falciparum.

RevDate: 2019-03-18
CmpDate: 2019-03-18

Shi X, He W, Guo S, et al (2019)

RNA-seq Analysis of the SCN1A-KO Model based on CRISPR/Cas9 Genome Editing Technology.

Neuroscience, 398:1-11.

Dravet syndrome (DS) is a disease that is primarily caused by the inactivation of the SCN1A-encoded voltage-gated sodium channel alpha subunit (Nav1.1). In this study, we constructed an SCN1A gene knockout model using CRISPR/Cas9 genome editing technology to deprive the Nav1.1 function in vitro. With mRNA-seq analysis we found abundant gene changes after SCN1A knockout, which associated with various signaling pathways, such as cancer pathways, the PI3K-AKT signaling pathway, the MAPK signaling pathway, and pathways involved in HTLV-I infection. We also noticed changes in the spliceosome, decreased glycolytic capacity, disturbances in calcium signaling pathways, and changes in the potassium, sodium, chloride, and calcium plasma channels after SCN1A knockout. In this study, we have been the first time to discover these changes and summarize them here and hope it would provide some clue for the study of Nav1.1 in the nervous system.

RevDate: 2019-03-18
CmpDate: 2019-03-18

Fujii M, Clevers H, T Sato (2019)

Modeling Human Digestive Diseases With CRISPR-Cas9-Modified Organoids.

Gastroenterology, 156(3):562-576.

Insights into the stem cell niche have allowed researchers to cultivate adult tissue stem cells as organoids that display structural and phenotypic features of healthy and diseased epithelial tissues. Organoids derived from patients' tissues are used as models of disease and to test drugs. CRISPR-Cas9 technology can be used to genetically engineer organoids for studies of monogenic diseases and cancer. We review the derivation of organoids from human gastrointestinal tissues and how CRISPR-Cas9 technology can be used to study these organoids. We discuss burgeoning technologies that are broadening our understanding of diseases of the digestive system.

RevDate: 2019-03-18
CmpDate: 2019-03-18

DeWeerdt S (2018)

The genomics of brain cancer.

Nature, 561(7724):S54-S55.

RevDate: 2019-03-18
CmpDate: 2019-03-18

Wang H, Park H, Liu J, et al (2018)

An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9.

G3 (Bethesda, Md.), 8(11):3607-3616.

Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ∼35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2 We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.

RevDate: 2019-03-18
CmpDate: 2019-03-18

Huynh N, Zeng J, Liu W, et al (2018)

A Drosophila CRISPR/Cas9 Toolkit for Conditionally Manipulating Gene Expression in the Prothoracic Gland as a Test Case for Polytene Tissues.

G3 (Bethesda, Md.), 8(11):3593-3605.

Targeting gene function with spatial or temporal specificity is a key goal in molecular genetics. CRISPR-Cas9 has greatly facilitated this strategy, but some standard approaches are problematic. For instance, simple tissue-specific or global overexpression of Cas9 can cause significant lethality or developmental delays even in the absence of gRNAs. In particular, we found that Gal4-mediated expression of UAS-Cas9 in the Drosophila prothoracic gland (PG) was not a suitable strategy to disrupt gene expression, since Cas9 alone caused widespread lethality. The PG is widely used for studying endocrine gland function during animal development, but tools validating PG-specific RNAi phenotypes are lacking. Here, we present a collection of modular gateway-compatible CRISPR-Cas9 tools that allow precise modulation of target gene activity with temporal and spatial specificity. We also demonstrate that Cas9 fused to the progesterone ligand-binding domain can be used to activate gene expression via RU486. Using these approaches, we were able to avoid the lethality associated with simple GAL4-mediated overexpression of Cas9 in the PG. Given that the PG is a polytene tissue, we conclude that these tools work effectively in endoreplicating cells where Cas9 has to target multiple copies of the same locus. Our toolkit can be easily adapted for other tissues and can be used both for gain- and loss-of-function studies.

RevDate: 2019-03-18
CmpDate: 2019-03-18

Luther DC, Lee YW, Nagaraj H, et al (2018)

Delivery approaches for CRISPR/Cas9 therapeutics in vivo: advances and challenges.

Expert opinion on drug delivery, 15(9):905-913.

INTRODUCTION: Therapeutic gene editing is becoming a viable biomedical tool with the emergence of the CRISPR/Cas9 system. CRISPR-based technologies have promise as a therapeutic platform for many human genetic diseases previously considered untreatable, providing a flexible approach to high-fidelity gene editing. For many diseases, such as sickle-cell disease and beta thalassemia, curative therapy may already be on the horizon, with CRISPR-based clinical trials slated for the next few years. Translation of CRISPR-based therapy to in vivo application however, is no small feat, and major hurdles remain for efficacious use of the CRISPR/Cas9 system in clinical contexts. Areas covered: In this topical review, we highlight recent advances to in vivo delivery of the CRISPR/Cas9 system using various packaging formats, including viral, mRNA, plasmid, and protein-based approaches. We also discuss some of the barriers which have yet to be overcome for successful translation of this technology. Expert opinion: This review focuses on the challenges to efficacy for various delivery formats, with specific emphasis on overcoming these challenges through the development of carrier vehicles for transient approaches to CRISPR/Cas9 delivery in vivo.

RevDate: 2019-03-18
CmpDate: 2019-03-18

Callahan SJ, Tepan S, Zhang YM, et al (2018)

Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ).

Disease models & mechanisms, 11(9):.

Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations. Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method referred to as Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest to drive fluorophores, oncogenes or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model via Cas9-mediated inactivation of Rb1 in the context of BRAFV600E in spatially constrained melanocytes. Unlike prior models that take ∼4 months to develop, we found that TEAZ leads to tumor onset in ∼7 weeks, and these tumors develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence, and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types, such as the heart, with the use of suitable transgenic promoters. The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other in vivo systems.

RevDate: 2019-03-18
CmpDate: 2019-03-18

Loyola-Vargas VM, RN Avilez-Montalvo (2018)

Plant Tissue Culture: A Battle Horse in the Genome Editing Using CRISPR/Cas9.

Methods in molecular biology (Clifton, N.J.), 1815:131-148.

Plant tissue culture (PTC) is a set of techniques for culturing cells, tissues, or organs in an aseptic medium with a defined chemical composition, in a controlled environment. Tissue culture, when combined with molecular biology techniques, becomes a powerful tool for the study of metabolic pathways, elucidation of cellular processes, genetic improvement and, through genetic engineering, the generation of cell lines resistant to biotic and abiotic stress, obtaining improved plants of agronomic interest, or studying the complex cellular genome. In this chapter, we analyze in general the use of plant tissue culture, in particular protoplasts and calli, in the implementation of CRISPR/Cas9 technology.

RevDate: 2019-03-18
CmpDate: 2019-03-18

Rouillon C, Athukoralage JS, Graham S, et al (2018)

Control of cyclic oligoadenylate synthesis in a type III CRISPR system.

eLife, 7:.

The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3' end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The 'RNA shredding' activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence.

RevDate: 2019-03-18
CmpDate: 2019-03-18

Rauch F, Geng Y, Lamplugh L, et al (2018)

Crispr-Cas9 engineered osteogenesis imperfecta type V leads to severe skeletal deformities and perinatal lethality in mice.

Bone, 107:131-142.

Osteogenesis imperfecta (OI) type V is caused by an autosomal dominant mutation in the IFITM5 gene, also known as BRIL. The c.-14C>T mutation in the 5'UTR of BRIL creates a novel translational start site adding 5 residues (MALEP) in frame with the natural coding of BRIL. A neomorphic function has been proposed for the MALEP-BRIL but the mechanisms at play are still unknown. In order to further understand the effects of MALEP-BRIL in vivo, we generated a knockin (KI) mouse model having the exact genetic -14C>T replica of patients with OI type V. Live KI descendants were never obtained from 2 male mosaic founders. Skeletal staining with alizarin red/alcian blue and μCT imaging of KI embryos revealed striking skeletal anomalies such as hypomineralized skull, short and bent long bones, and frail and wavy ribs. Histology and histochemical labeling revealed that midshaft of long bones was filled with hypertrophic chondrocytes, lacked a defined primary ossification center with the absence of defined cortices. Gene expression monitoring at E15.5 and E17.5 showed no change in Osx but decreased Bril itself as well as other differentiated osteoblast markers (Ibsp, Bglap, Sost). However, upregulation of Ptgs2 and Nr4a3 suggested that a pro-inflammatory reaction was activated. Primary osteoblasts from KI calvaria showed delayed differentiation and mineralization, with decreased abundance of BRIL. However, the upregulation AdipoQ and Fabp4 in young cultures indicated a possible switch in fate towards adipogenesis. Altogether our data suggest that the low level expression of MALEP-BRIL in Osx+ mesenchymal progenitors blunted their further differentiation into mature osteoblasts, which may have resulted in part from an inflammatory response.

RevDate: 2019-03-17

Makarova SS, Khromov AV, Spechenkova NA, et al (2018)

Application of the CRISPR/Cas System for Generation of Pathogen-Resistant Plants.

Biochemistry. Biokhimiia, 83(12):1552-1562.

The use of the CRISPR/Cas9 prokaryotic adaptive immune system has led to a breakthrough in targeted genome editing in eukaryotes. The CRISPR/Cas technology allows to generate organisms with desirable characteristics by introducing deletions/insertions into selected genome loci resulting in the knockout or modification of target genes. This review focuses on the current state of the CRISPR/Cas use for the generation of plants resistant to viruses, bacteria, and parasitic fungi. Resistance to DNA- and RNA-containing viruses is usually provided by expression in transgenic plants of the Cas endonuclease gene and short guide RNAs (sgRNAs) targeting certain sites in the viral or the host plant genomes to ensure either direct cleavage of the viral genome or modification of the plant host genome in order to decrease the efficiency of virus replication. Editing of plant genes involved in the defense response to pathogens increases plants resistance to bacteria and pathogenic fungi. The review explores strategies and prospects of the development of pathogen-resistant plants with a focus on the generation of non-transgenic (non-genetically modified) organisms, in particular, by using plasmid (DNA)-free systems for delivery of the Cas/sgRNA editing complex into plant cells.

RevDate: 2019-03-16

Zhang YT, Jiang JY, Shi TQ, et al (2019)

Application of the CRISPR/Cas system for genome editing in microalgae.

Applied microbiology and biotechnology pii:10.1007/s00253-019-09726-x [Epub ahead of print].

Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.

RevDate: 2019-03-15

Musharova O, Sitnik V, Vlot M, et al (2019)

Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation.

Molecular microbiology [Epub ahead of print].

CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» - acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference, and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition. This article is protected by copyright. All rights reserved.

RevDate: 2019-03-15

Atmadjaja AN, Holby V, Harding AJ, et al (2019)

CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT).

FEMS microbiology letters pii:5381555 [Epub ahead of print].

The solventogenic clostridia have long been known for their ability to convert sugars from complex feedstocks into commercially important solvents. Although the acetone-butanol-ethanol (ABE) process fell out of favour decades ago, renewed interest in sustainability and 'green' chemistry has re-established our appetite for reviving technologies such as these, albeit with 21st century improvements. As CRISPR-Cas genome editing tools are being developed and applied to the solventogenic clostridia, their industrial potential is growing. Through integration of new pathways, the beneficial traits and historical track record of clostridial fermentation can be exploited to generate a much wider range of industrially relevant products. Here we show the application of genome editing using the endogenous CRISPR-Cas mechanism of Clostridium saccharoperbutylacetonicum N1-4(HMT), to generate a deletion, SNP and to integrate new DNA into the genome. These technological advancements pave the way for application of clostridial species to the production of an array of products.

RevDate: 2019-03-15

Ashley CL, Abendroth A, McSharry BP, et al (2019)

Interferon-Independent Upregulation of Interferon-Stimulated Genes during Human Cytomegalovirus Infection is Dependent on IRF3 Expression.

Viruses, 11(3): pii:v11030246.

The antiviral activity of type I interferons (IFNs) is primarily mediated by interferon-stimulated genes (ISGs). Induction of ISG transcription is achieved when type I IFNs bind to their cognate receptor and activate the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathways. Recently it has become clear that a number of viruses are capable of directly upregulating a subset of ISGs in the absence of type I IFN production. Using cells engineered to block either the response to, or production of type I IFN, the regulation of IFN-independent ISGs was examined in the context of human cytomegalovirus (HCMV) infection. Several ISGs, including IFIT1, IFIT2, IFIT3, Mx1, Mx2, CXCL10 and ISG15 were found to be upregulated transcriptionally following HCMV infection independently of type I IFN-initiated JAK-STAT signaling, but dependent on intact IRF3 signaling. ISG15 protein regulation mirrored that of its transcript with IFNβ neutralization failing to completely inhibit ISG15 expression post HCMV infection. In addition, no detectable ISG15 protein expression was observed following HCMV infection in IRF3 knockdown CRISPR/Cas-9 clones indicating that IFN-independent control of ISG expression during HCMV infection of human fibroblasts is absolutely dependent on IRF3 expression.

RevDate: 2019-03-15
CmpDate: 2019-03-15

Veach RA, MH Wilson (2018)

CRISPR/Cas9 engineering of a KIM-1 reporter human proximal tubule cell line.

PloS one, 13(9):e0204487.

We used the CRISPR/Cas9 system to knock-in reporter transgenes at the kidney injury molecule-1 (KIM-1) locus and isolated human proximal tubule cell (HK-2) clones. PCR verified targeted knock-in of the luciferase and eGFP reporter at the KIM-1 locus. HK-2-KIM-1 reporter cells responded to various stimuli including hypoxia, cisplatin, and high glucose, indicative of upregulation of KIM-1 expression. We attempted using CRISPR/Cas9 to also engineer the KIM-1 reporter in telomerase-immortalized human RPTEC cells. However, these cells demonstrated an inability to undergo homologous recombination at the target locus. KIM-1-reporter human proximal tubular cells could be valuable tools in drug discovery for molecules inhibiting kidney injury. Additionally, our gene targeting strategy could be used in other cell lines to evaluate the biology of KIM-1 in vitro or in vivo.

RevDate: 2019-03-15
CmpDate: 2019-03-15

Curtin SJ (2018)

Editing the Medicago truncatula Genome: Targeted Mutagenesis Using the CRISPR-Cas9 Reagent.

Methods in molecular biology (Clifton, N.J.), 1822:161-174.

Medicago truncatula is an annual plant used for studying legume biology, in particular symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi. Efforts to decipher the genetic basis of these ecologically and economically important traits are a major goal of plant and crop biology. M. truncatula is an excellent model system for this purpose, as it has several publicly available sequenced genomes, has a rapid seed-to-seed generation time, and is highly transformable. Various mutagenesis platforms such as Tnt1 retrotransposons and RNAi knockdown have been used successfully in forward and reverse genetic studies to identify and functionally characterize candidate genes. The CRISPR/Cas9 reagent is the most recent mutagenesis platform and is highly effective at generating site-directed double-stranded breaks (DSB) in M. truncatula. This protocol will demonstrate the construction of reagents using two genome engineering platforms that have successfully generated mutant plants in M. truncatula, M. sativa, and soybean systems. The reagents are easy to assemble, can be quickly retrofitted to test novel regulatory sequences for improved efficiency, and can be used for more advanced genome engineering strategies such as gene insertion or gene replacement.


ESP Quick Facts

ESP Origins

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.

ESP Support

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.

ESP Rationale

Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.

ESP Goal

In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.

ESP Usage

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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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

Electronic Scholarly Publishing
21454 NE 143rd Street
Woodinville, WA 98077

E-mail: RJR8222 @

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).


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


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 )