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16 Aug 2018 at 01:33
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Bibliography on: CRISPR-Cas


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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: 2018-08-15

Gleditzsch D, Pausch P, Müller-Esparza H, et al (2018)

PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures.

RNA biology [Epub ahead of print].

Adaptive immunity of prokaryotes is mediated by CRISPR-Cas systems that employ a large variety of Cas protein effectors to identify and destroy foreign genetic material. The different targeting mechanisms of Cas proteins rely on the proper protection of the host genome sequence while allowing for efficient detection of target sequences, termed protospacers. A short DNA sequence, the protospacer-adjacent motif (PAM), is frequently used to mark proper target sites. Cas proteins have evolved a multitude of PAM-interacting domains, which enables them to cope with viral anti-CRISPR measures that alter the sequence or accessibility of PAM elements. In this review, we summarize known PAM recognition strategies for all CRISPR-Cas types. Available structures of target bound Cas protein effector complexes highlight the diversity of mechanisms and domain architectures that are employed to guarantee target specificity.

RevDate: 2018-08-15

Maikova A, Severinov K, O Soutourina (2018)

New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System.

Frontiers in microbiology, 9:1740.

Over the last decades the enteric bacterium Clostridium difficile (novel name Clostridioides difficile) - has emerged as an important human nosocomial pathogen. It is a leading cause of hospital-acquired diarrhea and represents a major challenge for healthcare providers. Many aspects of C. difficile pathogenesis and its evolution remain poorly understood. Efficient defense systems against phages and other genetic elements could have contributed to the success of this enteropathogen in the phage-rich gut communities. Recent studies demonstrated the presence of an active CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) subtype I-B system in C. difficile. In this mini-review, we will discuss the recent advances in characterization of original features of the C. difficile CRISPR-Cas system in laboratory and clinical strains, as well as interesting perspectives for our understanding of this defense system function and regulation in this important enteropathogen. This knowledge will pave the way for the development of promising biotechnological and therapeutic tools in the future. Possible applications for the C. difficile strain monitoring and genotyping, as well as for CRISPR-based genome editing and antimicrobials are also discussed.

RevDate: 2018-08-15
CmpDate: 2018-08-15

Wang HX, Song Z, Lao YH, et al (2018)

Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide.

Proceedings of the National Academy of Sciences of the United States of America, 115(19):4903-4908.

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

RevDate: 2018-08-15
CmpDate: 2018-08-15

Storz U (2018)

CRISPR Cas9 - Licensing the unlicensable.

Journal of biotechnology, 265:86-92.

A new gene engineering technology has recently made it through the media, not only because of its technical advantages, but also because it is in the focus of an epic patent battle between two academic institutions. The technology bears the cryptic name "CRISPR Cas9", and allows the manipulation of genes (so called "gene editing") with so far unseen simplicity and efficacy. Dana Carroll of the University of Utah said for this reason that CRISPR Cas9 has brought about the "democratization of gene targeting". However, due to legal battles and conflicting patent estates, third parties may find it difficult to decide where to acquire licenses. This article gives an overview.

RevDate: 2018-08-14

Künne T, Zhu Y, da Silva F, et al (2018)

Role of nucleotide identity in effective CRISPR target escape mutations.

Nucleic acids research pii:5070486 [Epub ahead of print].

Prokaryotes use primed CRISPR adaptation to update their memory bank of spacers against invading genetic elements that have escaped CRISPR interference through mutations in their protospacer target site. We previously observed a trend that nucleotide-dependent mismatches between crRNA and the protospacer strongly influence the efficiency of primed CRISPR adaptation. Here we show that guanine-substitutions in the target strand of the protospacer are highly detrimental to CRISPR interference and interference-dependent priming, while cytosine-substitutions are more readily tolerated. Furthermore, we show that this effect is based on strongly decreased binding affinity of the effector complex Cascade for guanine-mismatched targets, while cytosine-mismatched targets only minimally affect target DNA binding. Structural modeling of Cascade-bound targets with mismatches shows that steric clashes of mismatched guanines lead to unfavorable conformations of the RNA-DNA duplex. This effect has strong implications for the natural selection of target site mutations that lead to effective escape from type I CRISPR-Cas systems.

RevDate: 2018-08-14

Molenda O, Tang S, Lomheim L, et al (2018)

Extrachromosomal circular elements targeted by CRISPR-Cas in Dehalococcoides mccartyi are linked to mobilization of reductive dehalogenase genes.

The ISME journal pii:10.1038/s41396-018-0254-2 [Epub ahead of print].

Dehalococcoides mccartyi are obligate organohalide-respiring bacteria that play an important detoxifying role in the environment. They have small genomes (~1.4 Mb) with a core region interrupted by two high plasticity regions (HPRs) containing dozens of genes encoding reductive dehalogenases involved in organohalide respiration. The genomes of eight new strains of D. mccartyi were closed from metagenomic data from a related set of enrichment cultures, bringing the total number of genomes to 24. Two of the newly sequenced strains and three previously sequenced strains contain CRISPR-Cas systems. These D. mccartyi CRISPR-Cas systems were found to primarily target prophages and genomic islands. The genomic islands were identified either as integrated into D. mccartyi genomes or as circular extrachromosomal elements. We observed active circularization of the integrated genomic island containing vcrABC operon encoding the dehalogenase (VcrA) responsible for the transformation of vinyl chloride to non-toxic ethene. We interrogated archived DNA from established enrichment cultures and found that the CRISPR array acquired three new spacers in 11 years. These data provide a glimpse into dynamic processes operating on the genomes distinct to D. mccartyi strains found in enrichment cultures and provide the first insights into possible mechanisms of lateral DNA exchange in D. mccartyi.

RevDate: 2018-08-14

Devkota S (2018)

The Road less Traveled: Strategies to Enhance the Frequency of Homology-Directed Repair (HDR) for Increased Efficiency of CRISPR/Cas-Mediated Transgenesis.

Non-homologous end joining (NHEJ), and to a lesser extent, the error-free pathway known as homology-directed repair (HDR) are cellular mechanisms for recovery from double-strand DNA breaks (DSB) induced by RNA-guided programmable nuclease CRISPR/Cas. Since NHEJ is equivalent to using a duck tape to stick two pieces of metals together, the outcome of this repair mechanism is prone to error. Any out-of-frame mutations or premature stop codons resulting from NHEJ repair mechanism are extremely handy for loss-of-function studies. Substitution of a mutation on the genome with the correct exogenous repair DNA requires coordination via an error-free HDR, for targeted transgenesis. However, several practical limitations exist in harnessing the potential of HDR to replace a faulty mutation for therapeutic purposes in all cell types and more so in somatic cells. In germ cells after the DSB, copying occurs from the homologous chromosome, which increases the chances of incorporation of exogenous DNA with some degree of homology into the genome compared with somatic cells where copying from the identical sister chromatid is always preferred. This review summarizes several strategies that have been implemented to increase the frequency of HDR with a focus on somatic cells. It also highlights the limitations of this technology in gene therapy and suggests specific solutions to circumvent those barriers.

RevDate: 2018-08-14

Faure G, Shmakov SA, Makarova KS, et al (2018)

Comparative genomics and evolution of trans-activating RNAs in Class 2 CRISPR-Cas systems.

RNA biology [Epub ahead of print].

Trans-activating CRISPR (tracr) RNA is a distinct RNA species that interacts with the CRISPR (cr) RNA to form the dual guide (g) RNA in type II and subtype V-B CRISPR-Cas systems. The tracrRNA-crRNA interaction is essential for pre-crRNA processing as well as target recognition and cleavage. The tracrRNA consists of an antirepeat, which forms an imperfect hybrid with the repeat in the crRNA, and a distal region containing a Rho-independent terminator. Exhaustive comparative analysis of the sequences and predicted structures of the Class 2 CRISPR guide RNAs shows that all these guide RNAs share distinct structural features, in particular, the nexus stem-loop that separates the repeat-antirepeat hybrid from the distal portion of the tracrRNA and the conserved GU pair at that end of the hybrid. These structural constraints might ensure full exposure of the spacer for target recognition. Reconstruction of tracrRNA evolution for 4 tight bacterial groups demonstrates random drift of repeat-antirepeat complementarity within a window of hybrid stability that is, apparently, maintained by selection. An evolutionary scenario is proposed whereby tracrRNAs evolved on multiple occasions, via rearrangement of a CRISPR array to form the antirepeat in different locations with respect to the array. A functional tracrRNA would form if, in the new location, the antirepeat is flanked by sequences that meet the minimal requirements for a promoter and a Rho-independent terminator. Alternatively, or additionally, the antirepeat sequence could be occasionally 'reset' by recombination with a repeat, restoring the functionality of tracrRNAs that drift beyond the required minimal hybrid stability.

RevDate: 2018-08-14
CmpDate: 2018-08-14

Jia Y, Xu RG, Ren X, et al (2018)

Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM.

Proceedings of the National Academy of Sciences of the United States of America, 115(18):4719-4724.

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.

RevDate: 2018-08-14
CmpDate: 2018-08-14

Wu Q, Tian Y, Zhang J, et al (2018)

In vivo CRISPR screening unveils histone demethylase UTX as an important epigenetic regulator in lung tumorigenesis.

Proceedings of the National Academy of Sciences of the United States of America, 115(17):E3978-E3986.

Lung cancer is the leading cause of cancer-related death worldwide. Inactivation of tumor suppressor genes (TSGs) promotes lung cancer malignant progression. Here, we take advantage of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated somatic gene knockout in a KrasG12D/+ mouse model to identify bona fide TSGs. From individual knockout of 55 potential TSGs, we identify five genes, including Utx, Ptip, Acp5, Acacb, and Clu, whose knockout significantly promotes lung tumorigenesis. These candidate genes are frequently down-regulated in human lung cancer specimens and significantly associated with survival in patients with lung cancer. Through crossing the conditional Utx knockout allele to the KrasG12D/+ mouse model, we further find that Utx deletion dramatically promotes lung cancer progression. The tumor-promotive effect of Utx knockout in vivo is mainly mediated through an increase of the EZH2 level, which up-regulates the H3K27me3 level. Moreover, the Utx-knockout lung tumors are preferentially sensitive to EZH2 inhibitor treatment. Collectively, our study provides a systematic screening of TSGs in vivo and identifies UTX as an important epigenetic regulator in lung tumorigenesis.

RevDate: 2018-08-14
CmpDate: 2018-08-14

Shang W, Jiang Y, Boettcher M, et al (2018)

Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation.

Proceedings of the National Academy of Sciences of the United States of America, 115(17):E4051-E4060.

Despite decades of research, mechanisms controlling T cell activation remain only partially understood, which hampers T cell-based immune cancer therapies. Here, we performed a genome-wide CRISPR screen to search for genes that regulate T cell activation. Our screen confirmed many of the known regulators in proximal T cell receptor signaling and, importantly, also uncovered a previously uncharacterized regulator, FAM49B (family with sequence similarity 49 member B). FAM49B deficiency led to hyperactivation of Jurkat T cells following T cell receptor stimulation, as indicated by enhancement of CD69 induction, PAK phosphorylation, and actin assembly. FAM49B directly interacted with the active form of the small GTPase Rac, and genetic disruption of the FAM49B-Rac interaction compromised FAM49B function. Thus, FAM49B inhibits T cell activation by repressing Rac activity and modulating cytoskeleton reorganization.

RevDate: 2018-08-14
CmpDate: 2018-08-14

Duan D (2017)

A New Kid on the Playground of CRISPR DMD Therapy.

Human gene therapy. Clinical development, 28(2):62-64.

RevDate: 2018-08-14
CmpDate: 2018-08-14

Wilson JM (2017)

Jurassic Park, Gene Therapy, and Neuroscience: An Interview with Feng Zhang, PhD.

Human gene therapy. Clinical development, 28(1):4-6.

RevDate: 2018-08-14
CmpDate: 2018-08-14

Hoyos-Flight M, Brady E, Sang H, et al (2017)

Genome Editing and the Future of Farming meeting report.

Transgenic research, 26(2):319-321.

RevDate: 2018-08-14
CmpDate: 2018-08-14

Makino S, Fukumura R, Y Gondo (2016)

Illegitimate translation causes unexpected gene expression from on-target out-of-frame alleles created by CRISPR-Cas9.

Scientific reports, 6:39608 pii:srep39608.

CRISPR-Cas9 is efficient enough to knock out both alleles directly by introducing out-of-frame mutations. We succeeded in making biallelic on-target frameshift mutations of the endogenous Gli3 gene; however, the GLI3 protein was expressed in all six of the established cell lines carrying homozygous out-of-frame mutations. We developed a dual-tagged expression vector and proved that illegitimate translation (ITL) was the cause of the unexpected Gli3 expression. Thus, gene expression must be examined even if designed on-target out-of-frame sequences are introduced by genome editing. In addition, it is highly recommended to pre-examine the occurrence of ITL in vitro prior to the design and construction of any genome-editing vectors. In vitro assay systems such as the dual-tagged ITL assay system developed in this study should aid the identification and elucidation of ITL-based human diseases and gene expression.

RevDate: 2018-08-14
CmpDate: 2018-08-14

Malkki H (2016)

Huntington disease: Selective deactivation of Huntington disease mutant allele by CRISPR-Cas9 gene editing.

Nature reviews. Neurology, 12(11):614-615.

RevDate: 2018-08-13

Ka D, Jang DM, Han BW, et al (2018)

Molecular organization of the type II-A CRISPR adaptation module and its interaction with Cas9 via Csn2.

Nucleic acids research pii:5068242 [Epub ahead of print].

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide microbial adaptive immunity against invading foreign nucleic acids. In type II-A CRISPR-Cas systems, the Cas1-Cas2 integrase complex and the subtype-specific Csn2 comprise the CRISPR adaptation module, which cooperates with the Cas9 nuclease effector for spacer selection. Here, we report the molecular organization of the Streptococcus pyogenes type II-A CRISPR adaptation module and its interaction with Cas9 via Csn2. We determined the crystal structure of S. pyogenes type II-A Cas2. Chromatographic and calorimetric analyses revealed the stoichiometry and topology of the type II-A adaptation module composed of Cas1, Cas2 and Csn2. We also demonstrated that Cas9 interacts with Csn2 in a direct and stoichiometric manner. Our results reveal a network of molecular interactions among type II-A Cas proteins and highlight the role of Csn2 in coordinating Cas components involved in the adaptation and interference stages of CRISPR-mediated immunity.

RevDate: 2018-08-11

Killelea T, Hawkins M, Howard JL, et al (2018)

DNA replication roadblocks caused by Cascade interference complexes are alleviated by RecG DNA repair helicase.

RNA biology [Epub ahead of print].

Cascade complexes underpin E. coli CRISPR-Cas immunity systems by stimulating 'adaptation' reactions that update immunity and by initiating 'interference' reactions that destroy invader DNA. Recognition of invader DNA in Cascade catalysed R-loops provokes DNA capture and its subsequent integration into CRISPR loci by Cas1 and Cas2. DNA capture processes are unclear but may involve RecG helicase, which stimulates adaptation during its role responding to genome instability. We show that Cascade is a potential source of genome instability because it blocks DNA replication and that RecG helicase alleviates this by dissociating Cascade. This highlights how integrating in vitro CRISPR-Cas interference and adaptation reactions with DNA replication and repair reactions will help to determine precise mechanisms underpinning prokaryotic adaptive immunity.

RevDate: 2018-08-13
CmpDate: 2018-08-13

Guo X, Gao M, Wang Y, et al (2018)

LDL Receptor Gene-ablated Hamsters: A Rodent Model of Familial Hypercholesterolemia With Dominant Inheritance and Diet-induced Coronary Atherosclerosis.

EBioMedicine, 27:214-224.

Familial hypercholesterolemia (FH) is an autosomal dominant genetic disease caused mainly by LDL receptor (Ldlr) gene mutations. Unlike FH patients, heterozygous Ldlr knockout (KO) mice do not show a dominant FH trait. Hamsters, like humans, have the cholesteryl ester transfer protein, intestine-only ApoB editing and low hepatic cholesterol synthesis. Here, we generated Ldlr-ablated hamsters using CRISPR/Cas9 technology. Homozygous Ldlr KO hamsters on a chow diet developed hypercholesterolemia with LDL as the dominant lipoprotein and spontaneous atherosclerosis. On a high-cholesterol/high-fat (HCHF) diet, these animals exhibited severe hyperlipidemia and atherosclerotic lesions in the aorta and coronary arteries. Moreover, the heterozygous Ldlr KO hamsters on a short-term HCHF diet also had overt hypercholesterolemia, which could be effectively ameliorated with several lipid-lowering drugs. Importantly, heterozygotes on 3-month HCHF diets developed accelerated lesions in the aortas and coronary arteries. Our findings demonstrate that the Ldlr KO hamster is an animal model of choice for human FH and has great potential in translational research of hyperlipidemia and coronary heart disease.

RevDate: 2018-08-13
CmpDate: 2018-08-13

Higo A, Isu A, Fukaya Y, et al (2018)

Application of CRISPR Interference for Metabolic Engineering of the Heterocyst-Forming Multicellular Cyanobacterium Anabaena sp. PCC 7120.

Plant & cell physiology, 59(1):119-127.

Anabaena sp. PCC 7120 (A. 7120) is a heterocyst-forming multicellular cyanobacterium that performs nitrogen fixation. This cyanobacterium has been extensively studied as a model for multicellularity in prokaryotic cells. We have been interested in photosynthetic production of nitrogenous compounds using A. 7120. However, the lack of efficient gene repression tools has limited its usefulness. We originally developed an artificial endogenous gene repression method in this cyanobacterium using small antisense RNA. However, the narrow dynamic range of repression of this method needs to be improved. Recently, clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) technology was developed and was successfully applied in some unicellular cyanobacteria. The technology requires expression of nuclease-deficient CRISPR-associated protein 9 (dCas9) and a single guide RNA (sgRNA) that is complementary to a target sequence, to repress expression of the target gene. In this study, we employed CRISPRi technology for photosynthetic production of ammonium through repression of glnA, the only gene encoding glutamine synthetase that is essential for nitrogen assimilation in A. 7120. By strictly regulating dCas9 expression using the TetR gene induction system, we succeeded in fine-tuning the GlnA protein in addition to the level of glnA transcripts. Expression of sgRNA by the heterocyst-specific nifB promoter led to efficient repression of GlnA in heterocysts, as well as in vegetative cells. Finally, we showed that ammonium is excreted into the medium only when inducers of expression of dCas9 were added. In conclusion, CRISPRi enables temporal control of desired products and will be a useful tool for basic science.

RevDate: 2018-08-13
CmpDate: 2018-08-13

Kc R, Srivastava A, Wilkowski JM, et al (2016)

Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection.

Scientific reports, 6:32048 pii:srep32048.

CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies.

RevDate: 2018-08-13
CmpDate: 2018-08-13

Vasiliou SK, Diamandis EP, Church GM, et al (2016)

CRISPR-Cas9 System: Opportunities and Concerns.

Clinical chemistry, 62(10):1304-1311.

RevDate: 2018-08-10

Goodman DA, KM Stedman (2018)

Comparative genetic and genomic analysis of the novel fusellovirus Sulfolobus spindle-shaped virus 10.

Virus evolution, 4(2):vey022 pii:vey022.

Viruses that infect thermophilic Archaea are unique in both their structure and genetic makeup. The lemon-shaped fuselloviruses-which infect members of the order Sulfolobales, growing optimally at 80 °C and pH 3-are some of the most ubiquitous and best studied viruses of the thermoacidophilic Archaea. Nonetheless, much remains to be learned about these viruses. In order to investigate fusellovirus evolution, we have isolated and characterized a novel fusellovirus, Sulfolobus spindle-shaped virus 10 (formerly SSV-L1). Comparative genomic analyses highlight significant similarity with both SSV8 and SSV9, as well as conservation of promoter elements within the Fuselloviridae. SSV10 encodes five ORFs with no homology within or outside of the Fuselloviridae, as well as a putatively functional Cas4-like ORF, which may play a role in evading CRISPR-mediated host defenses. Moreover, we demonstrate the ability of SSV10 to withstand mutation in a fashion consistent with mutagenesis in SSV1.

RevDate: 2018-08-10
CmpDate: 2018-08-10

Chavez A, Pruitt BW, Tuttle M, et al (2018)

Precise Cas9 targeting enables genomic mutation prevention.

Proceedings of the National Academy of Sciences of the United States of America, 115(14):3669-3673.

Here, we present a generalized method of guide RNA "tuning" that enables Cas9 to discriminate between two target sites that differ by a single-nucleotide polymorphism. We employ our methodology to generate an in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.

RevDate: 2018-08-10
CmpDate: 2018-08-10

Kunkel GH, Chaturvedi P, Thelian N, et al (2018)

Mechanisms of TFAM-mediated cardiomyocyte protection.

Canadian journal of physiology and pharmacology, 96(2):173-181.

Although mitochondrial transcription factor A (TFAM) is a protective component of mitochondrial DNA and a regulator of calcium and reactive oxygen species (ROS) production, the mechanism remains unclear. In heart failure, TFAM is significantly decreased and cardiomyocyte instability ensues. TFAM inhibits nuclear factor of activated T cells (NFAT), which reduces ROS production; additionally, TFAM transcriptionally activates SERCA2a to decrease free calcium. Therefore, decreasing TFAM vastly increases protease expression and hypertrophic factors, leading to cardiomyocyte functional decline. To examine this hypothesis, treatments of 1.0 μg of a TFAM vector and 1.0 μg of a CRISPR-Cas9 TFAM plasmid were administered to HL-1 cardiomyocytes via lipofectamine transfection. Western blotting and confocal microscopy analysis show that CRISPR-Cas9 knockdown of TFAM significantly increased proteases Calpain1, MMP9, and regulators Serca2a, and NFAT4 protein expression. CRISPR knockdown of TFAM in HL-1 cardiomyocytes upregulates degradation factors, leading to cardiomyocyte instability. Hydrogen peroxide oxidative stress decreased TFAM expression and increased Calpain1, MMP9, and NFAT4 protein expression. TFAM overexpression normalizes pathological hypertrophic factor NFAT4 in the presence of oxidative stress.

RevDate: 2018-08-09

Immethun CM, TS Moon (2018)

Synthetic Gene Regulation in Cyanobacteria.

Advances in experimental medicine and biology, 1080:317-355.

Cyanobacteria are appealing hosts for green chemical synthesis due to their use of light and carbon dioxide. To optimize product yields and titers, specific and tunable regulation of the metabolic pathways is needed. Synthetic biology has increased and diversified the genetic tools available for biological process control. While early tool development focused on commonly used heterotrophs, there has been a recent expansion of tools for cyanobacteria. CRISPR-Cas9 has been used to edit the genome of cyanobacterial strains, while transcriptional regulation has been accomplished with CRISPR interference and RNA riboswitches. Promoter development has produced a significant number of transcriptional regulators, including those that respond to chemicals, environmental signals, and metabolic states. Trans-acting RNAs have been utilized for posttranscriptional and translational control. The regulation of translation initiation is beginning to be explored with ribosome binding sites and riboswitches, while protein degradation tags have been used to control expression levels. Devices built from multiple parts have also been developed to create more complex behaviors. These advances in development of synthetic cyanobacterial regulatory parts provide the groundwork for creation of new, even more sophisticated bioprocess control devices, bolstering the viability of cyanobacteria as sustainable biotechnology platforms.

RevDate: 2018-08-09

Robbins JP, Perfect L, Ribe EM, et al (2018)

Clusterin Is Required for β-Amyloid Toxicity in Human iPSC-Derived Neurons.

Frontiers in neuroscience, 12:504.

Our understanding of the molecular processes underlying Alzheimer's disease (AD) is still limited, hindering the development of effective treatments, and highlighting the need for human-specific models. Advances in identifying components of the amyloid cascade are progressing, including the role of the protein clusterin in mediating β-amyloid (Aβ) toxicity. Mutations in the clusterin gene (CLU), a major genetic AD risk factor, are known to have important roles in Aβ processing. Here we investigate how CLU mediates Aβ-driven neurodegeneration in human induced pluripotent stem cell (iPSC)-derived neurons. We generated a novel CLU-knockout iPSC line by CRISPR/Cas9-mediated gene editing to investigate Aβ-mediated neurodegeneration in cortical neurons differentiated from wild type and CLU knockout iPSCs. We measured response to Aβ using an imaging assay and measured changes in gene expression using qPCR and RNA sequencing. In wild type neurons imaging indicated that neuronal processes degenerate following treatment with Aβ25-35 peptides and Aβ1-42 oligomers, in a dose dependent manner, and that intracellular levels of clusterin are increased following Aβ treatment. However, in CLU knockout neurons Aβ exposure did not affect neurite length, suggesting that clusterin is an important component of the amyloid cascade. Transcriptomic data were analyzed to elucidate the pathways responsible for the altered response to Aβ in neurons with the CLU deletion. Four of the five genes previously identified as downstream to Aβ and Dickkopf-1 (DKK1) proteins in an Aβ-driven neurotoxic pathway in rodent cells were also dysregulated in human neurons with the CLU deletion. AD and lysosome pathways were the most significantly dysregulated pathways in the CLU knockout neurons, and pathways relating to cytoskeletal processes were most dysregulated in Aβ treated neurons. The absence of neurodegeneration in the CLU knockout neurons in response to Aβ compared to the wild type neurons supports the role of clusterin in Aβ-mediated AD pathogenesis.

RevDate: 2018-08-09
CmpDate: 2018-08-09

Tobin SC (2016)

Recent advances in CRISPR/Cas9 technology for targeting latent HIV-1 reservoirs.

AIDS (London, England), 30(11):N17.

RevDate: 2018-08-07

Buckner MMC, Ciusa ML, LJV Piddock (2018)

Strategies to combat antimicrobial resistance: anti-plasmid and plasmid curing.

FEMS microbiology reviews pii:5061628 [Epub ahead of print].

Antimicrobial resistance (AMR) is a global problem hindering treatment of bacterial infections, rendering many aspects of modern medicine less effective. AMR genes (ARGs), are frequently located on plasmids, which are self-replicating elements of DNA. They are often transmissible between bacteria, and some have spread globally. Novel strategies to combat AMR are needed, and plasmid curing and anti-plasmid approaches could reduce ARG prevalence, and sensitize bacteria to antibiotics. We discuss the use of curing agents as laboratory tools including chemicals (e.g. detergents and intercalating agents), drugs used in medicine including ascorbic acid, psychotropic drugs (e.g. chlorpromazine), antibiotics (e.g. aminocoumarins, quinolones, and rifampicin), and plant-derived compounds. Novel strategies are examined; these include conjugation inhibitors (e.g. linoleic, oleic, 2-hexadecynoic and tanzawaic acids, and TraE inhibitors), systems designed around plasmid incompatibility, phages, and CRISPR/Cas based approaches. Currently there is a general lack of in vivo curing options. This review highlights this important shortfall, which if filled, could provide a promising mechanism to reduce ARG prevalence in humans and animals. Plasmid curing mechanisms which are not suitable for in vivo use could still prove important for reducing the global burden of AMR, as high levels of ARGs exist in the environment.

RevDate: 2018-08-07

Millen AM, Samson JE, Tremblay D, et al (2018)

Lactococcus lactis type III-A CRISPR-Cas system cleaves bacteriophage RNA.

RNA biology [Epub ahead of print].

CRISPR-Cas defends microbial cells against invading nucleic acids including viral genomes. Recent studies have shown that type III-A CRISPR-Cas systems target both RNA and DNA in a transcription-dependent manner. We previously found a type III-A system on a conjugative plasmid in Lactococcus lactis which provided resistance against virulent phages of the Siphoviridae family. Its naturally occurring spacers are oriented to generate crRNAs complementary to target phage mRNA, suggesting transcription-dependent targeting. Here, we show that only constructs whose spacers produce crRNAs complementary to the phage mRNA confer phage resistance in L. lactis. In vivo nucleic acid cleavage assays showed that cleavage of phage dsDNA genome was not detected within phage-infected L. lactis cells. On the other hand, Northern blots indicated that the lactococcal CRISPR-Cas cleaves phage mRNA in vivo. These results cannot exclude that single-stranded phage DNA is not being targeted, but phage DNA replication has been shown to be impaired.

RevDate: 2018-08-08
CmpDate: 2018-08-08

Wu RS, Lam II, Clay H, et al (2018)

A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish.

Developmental cell, 46(1):112-125.e4.

Zebrafish is a powerful model for forward genetics. Reverse genetic approaches are limited by the time required to generate stable mutant lines. We describe a system for gene knockout that consistently produces null phenotypes in G0 zebrafish. Yolk injection of sets of four CRISPR/Cas9 ribonucleoprotein complexes redundantly targeting a single gene recapitulated germline-transmitted knockout phenotypes in >90% of G0 embryos for each of 8 test genes. Early embryonic (6 hpf) and stable adult phenotypes were produced. Simultaneous multi-gene knockout was feasible but associated with toxicity in some cases. To facilitate use, we generated a lookup table of four-guide sets for 21,386 zebrafish genes and validated several. Using this resource, we targeted 50 cardiomyocyte transcriptional regulators and uncovered a role of zbtb16a in cardiac development. This system provides a platform for rapid screening of genes of interest in development, physiology, and disease models in zebrafish.

RevDate: 2018-08-07
CmpDate: 2018-08-07

Carleton JB, Berrett KC, J Gertz (2018)

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines.

Journal of visualized experiments : JoVE.

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these enhancers combine to produce a transcriptional response. As millions of enhancers have been identified, high-throughput tools are needed to determine enhancer function on a genome-wide scale. Current methods for studying enhancer function include making genetic deletions using nuclease-proficient Cas9, but it is difficult to study the combinatorial effects of multiple enhancers using this technique, as multiple successive clonal cell lines must be generated. Here, we present Enhancer-i, a CRISPR interference-based method that allows for functional interrogation of multiple enhancers simultaneously at their endogenous loci. Enhancer-i makes use of two repressive domains fused to nuclease-deficient Cas9, SID and KRAB, to achieve enhancer deactivation via histone deacetylation at targeted loci. This protocol utilizes transient transfection of guide RNAs to enable transient inactivation of targeted regions and is particularly effective at blocking inducible transcriptional responses to stimuli in tissue culture settings. Enhancer-i is highly specific both in its genomic targeting and its effects on global gene expression. Results obtained from this protocol help to understand whether an enhancer is contributing to gene expression, the magnitude of the contribution, and how the contribution is affected by other nearby enhancers.

RevDate: 2018-08-08
CmpDate: 2018-08-08

Thomas SP, Hoang TT, Ressler VT, et al (2018)

Human angiogenin is a potent cytotoxin in the absence of ribonuclease inhibitor.

RNA (New York, N.Y.), 24(8):1018-1027.

Angiogenin (ANG) is a secretory ribonuclease that promotes the proliferation of endothelial cells, leading to angiogenesis. This function relies on its ribonucleolytic activity, which is low for simple RNA substrates. Upon entry into the cytosol, ANG is sequestered by the ribonuclease inhibitor protein (RNH1). We find that ANG is a potent cytotoxin for RNH1-knockout HeLa cells, belying its inefficiency as a nonspecific catalyst. The toxicity does, however, rely on the ribonucleolytic activity of ANG and a cytosolic localization, which lead to the accumulation of particular tRNA fragments (tRFs), such as tRF-5 Gly-GCC. These up-regulated tRFs are highly cytotoxic at physiological concentrations. Although ANG is well-known for its promotion of cell growth, our results reveal that ANG can also cause cell death.

RevDate: 2018-08-07
CmpDate: 2018-08-07

LeBlanc C, Zhang F, Mendez J, et al (2018)

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress.

The Plant journal : for cell and molecular biology, 93(2):377-386.

The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9.

RevDate: 2018-08-08
CmpDate: 2018-08-08

Zhou X, Bai X, Y Xing (2018)

A Rice Genetic Improvement Boom by Next Generation Sequencing.

Current issues in molecular biology, 27:109-126.

Rice (Oryza sativa L.) is a staple food crop for people worldwide, and a key goal has been to increase its grain yield. An increasing population that relies on a decreasing level of farmland has rendered the traditional method for the isolation and use of genetic loci in rice breeding unsatisfactory. Recently, the rapid development in next generation sequencing (NGS) has boosted the number of genome sequences for hundreds to thousands of rice varieties. A MutMap strategy and bulk segregation analysis (BSA) has been developed to directly identify candidate genes based on NGS. The genome-wide association analysis (GWAS) has become a commonly used approach toward identifying the genetic loci and candidate genes for several traits that are closely associated with grain yield. The Multi-parent Advanced Generation Inter-Cross population (MAGIC) is introduced here to discuss potential applications for mapping QTLs for rice varietal development. These strategies broaden the capacity of functional gene identification and its application as a complementary method to insert mutants that comprise T-DNA and transposons. High-throughput SNP analysis platforms, such as the SNP array, provide novel strategies for genomic-assisted selections (GAS) for rice genetic improvements. Moreover, accurate genome sequence information enables genome editing for the utilization of key recessive genes that control important agronomic traits. This review summarizes how NGS accelerates rice genetic improvements through the identification and utilization of key functional genes that regulate agronomic traits.

RevDate: 2018-08-08
CmpDate: 2018-08-08

Shinwari ZK, Tanveer F, AT Khalil (2018)

Ethical Issues Regarding CRISPR Mediated Genome Editing.

Current issues in molecular biology, 26:103-110.

CRISPR-Cas9 has emerged as a simple, precise and most rapid genome editing technology. With a number of promising applications ranging from agriculture and environment to clinical therapeutics, it is greatly transforming the field of molecular biology. However, there are certain ethical, moral and safety concerns related to the attractive applications of this technique. The most contentious issues concerning human germline modifications are the challenges to human safety and morality such as risk of unforeseen, undesirable effects in clinical applications particularly to correct or prevent genetic diseases, matter of informed consent and the risk of exploitation for eugenics. Stringent regulations and guidelines as well as worldwide debate and awareness are required to ensure responsible and wise use of CRISPR mediated genome editing technology. There is a need for an extensive dialogue among scientists, ethicists, industrialists and policy makers on its societal implications. The opinion of different elements of the society including the general public as well as religious scholars is also critical. In countries with existing legislative framework, it might be appropriate to allow CRISPR based research to proceed with proper justification. However, much anticipated future clinical applications must be strictly regulated with newly established regulations.

RevDate: 2018-08-08
CmpDate: 2018-08-08

Khan FA, Pandupuspitasari NS, ChunJie H, et al (2018)

Applications of CRISPR/Cas9 in Reproductive Biology.

Current issues in molecular biology, 26:93-102.

Genome editing is unraveling its benefits in wide areas of scientific development and understanding. The advances of genome editing from ZFNs and TALLENs to CRISPRs defines it wide applicability. Reproduction is the fundamental process by which all organisms maintain their generations. CRISPR/Cas9, a new versatile genome editing tool is recently tamed to correct several disease causing genetic mutations spreading its arms to improve reproductive health. It not only edit harmful genetic mutations but is also applied to control the spread of parasitic diseases like malaria by introducing selfish genetic elements, propagated through generations and population via reproduction. These applications made us to review the recent developments of CRISPRs use in reproductive biology.

RevDate: 2018-08-08
CmpDate: 2018-08-08

Kaboli S, H Babazada (2018)

CRISPR Mediated Genome Engineering and its Application in Industry.

Current issues in molecular biology, 26:81-92.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) method has been dramatically changing the field of genome engineering. It is a rapid, highly efficient and versatile tool for precise modification of genome that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This novel RNA-guided genome-editing technique has become a revolutionary tool in biomedical science and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing tool, summarize the recent advances in CRISPR/Cas9 technology to engineer the genomes of a wide variety of organisms, and discuss their applications to treatment of fungal and viral disease. We also discuss advantageous of CRISPR/Cas9 technology to drug design, creation of animal model, and to food, agricultural and energy sciences. Adoption of the CRISPR/Cas9 technology in biomedical and biotechnological researches would create innovative applications of it not only for breeding of strains exhibiting desired traits for specific industrial and medical applications, but also for investigation of genome function.

RevDate: 2018-08-08
CmpDate: 2018-08-08

Jamal M, Ullah A, Ahsan M, et al (2018)

Improving CRISPR-Cas9 On-Target Specificity.

Current issues in molecular biology, 26:65-80.

The CRISPR-Cas9 has revolutionized the field of molecular biology, medical genetics and medicine. The technology is robust, facile and simple to achieve genome targeting in cells and organisms. However, to propagate these nucleases for therapeutic application, the on-target specificity is of paramount importance. Although the binding and cleavage of off-target sites by Cas9 is issue of concern, however the specificity of CRISPR technology is greatly improved in current research employing the use of engineer nucleases, improved gRNA selection, novel Cas9 orhtologs and the advancement in methods to detect and screen off-target sites and its effects. Here we summarize the advances in this state-of-the-art technology that will equip the genome editing tools to be applied in clinical research. The researcher should optimize these methods with emphasize to achieve perfection in the specificity.

RevDate: 2018-08-08
CmpDate: 2018-08-08

Sameeullah M, Khan FA, Özer G, et al (2018)

CRISPR/Cas9-Mediated Immunity in Plants Against Pathogens.

Current issues in molecular biology, 26:55-64.

Global crop production is highly threatened due to pathogen invasion. The huge quantity of pesticides application, although harmful to the environment and human health, is carried out to prevent the crop losses worldwide, every year. Therefore, understanding the molecular mechanisms of pathogenicity and plant resistance against pathogen is important. The resistance against pathogens is regulated by three important phytohormones viz. salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). Here we review possible role of CRISPR technology to understand the plant pathogenicity by mutating genes responsible for pathogen invasion or up-regulating the phytohormones genes or resistant genes. Thus hormone biosynthesis genes, receptor and feeding genes of pathogens could be important targets for modifications using CRISPR/Cas9 following multiplexing tool box strategy in order to edit multiple genes simultaneously to produce super plants. Here we put forward our idea thatthe genes would be either mutated in case of plant receptor protein targets of pathogens or up-regulation of resistant genes or hormone biosynthesis genes will be better choice for resistance against pathogens.

RevDate: 2018-08-08
CmpDate: 2018-08-08

Khurshid H, Jan SA, Shinwari ZK, et al (2018)

An Era of CRISPR/ Cas9 Mediated Plant Genome Editing.

Current issues in molecular biology, 26:47-54.

Recently the engineered nucleases have revolutionized genome editing to perturb gene expression at specific sites in complex eukaryotic genomes. Three important classes of these genome editing tools are Moreover, the more recent type II Clustered Regularly Inter-spaced Short Palindromic Repeats/Crispr associated protein (CRISPR/Cas9) system has become the most favorite plant genome editing tool for its precision and RNA based specificity unlike its counterparts which rely on protein based specificity. Plasmid-mediated co-delivery of multiple sgRNAs and Cas9 to the Plant cell can simultaneously alter more than one target loci which enable multiplex genome editing. In this review, we discuss recent advancements in the CRISPR/ Cas9 technology mechanism, theory and its applications in plants and agriculture. We also suggest that the CRISPR/ Cas9 as an effective genome editing tool, has vast potential for crop improvement and studying gene regulation mechanism and chromatin remodeling.

RevDate: 2018-08-08
CmpDate: 2018-08-08

Jamal M, Ullah A, Ahsan M, et al (2018)

Treating Genetic Disorders Using State-Of-The-Art Technology.

Current issues in molecular biology, 26:33-46.

CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated Protein 9), basically a bacterial immune system is now widely applicable to engineer genomes of a number of cells and organisms because of its simplicity and robustness. In research avenue the system has been optimized to regulate gene expression, modify epigenome and edit target locus. These applications make CRISPR/Cas9, a technology of choice to edit disease causing mutations as well as the epigenome more efficiently than ever before. Meanwhile its application in in vivo and ex vivo cells is encouraging the scientific community for more vigorous gene therapy and in clinical setups for therapeutic genome editing. Here we review the recent advances that CRISPR-Cas9 mediated genome editing has achieved and is reported in previous studies and address the challenges associated with it.

RevDate: 2018-08-06

Strohkendl I, Saifuddin FA, Rybarski JR, et al (2018)

Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a.

Molecular cell pii:S1097-2765(18)30546-X [Epub ahead of print].

Class 2 CRISPR-Cas nucleases are programmable genome editing tools with promising applications in human health and disease. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence. This result implies substantial reversibility during R-loop formation-a late transition state-and defies common descriptions of a "seed" region. Our results provide a quantitative basis for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity for Cas12a than for the Cas9 nuclease.

RevDate: 2018-08-06
CmpDate: 2018-08-06

Farboud B, Jarvis E, Roth TL, et al (2018)

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms.

Journal of visualized experiments : JoVE.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break. The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

RevDate: 2018-08-06
CmpDate: 2018-08-06

Tröder SE, Ebert LK, Butt L, et al (2018)

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes.

PloS one, 13(5):e0196891 pii:PONE-D-18-07297.

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

RevDate: 2018-08-06
CmpDate: 2018-08-06

Minegishi Y, Nakaya N, SI Tomarev (2018)

Mutation in the Zebrafish cct2 Gene Leads to Abnormalities of Cell Cycle and Cell Death in the Retina: A Model of CCT2-Related Leber Congenital Amaurosis.

Investigative ophthalmology & visual science, 59(2):995-1004.

Purpose: The compound heterozygous mutations in the β subunit of chaperonin containing TCP-1 (CCT), encoded by CCT2, lead to the Leber congenital amaurosis (LCA). In this study, a cct2 mutant line of zebrafish was established to investigate the role of CCT2 mutations in LCA in vertebrates.

Methods: A cct2 mutant zebrafish line was produced using the CRISPR-Cas9 system. Changes in the eyes of developing wild-type and mutant larvae were monitored using microscopy, immunostaining, TUNEL, and EdU assays. Phenotypic rescue of mutant phenotype was investigated by injection of CCT2 RNA into zebrafish embryos.

Results: The cct2 mutation (L394H-7del) led to the synthesis of a mutated cctβ protein with the L394H replacement and deletion of 7 amino acid residues (positions 395-401). The homozygous cct2-L394H-7del mutant exhibited a small eye phenotype at 2 days post fertilization (dpf) and was embryonically lethal after 5 dpf. In homozygous cct2-L394H-7del mutants, the retinal ganglion cell differentiation was attenuated, retinal cell cycle was affected, and the neural retinal cell death was significantly increased at 2 dpf compared with wild-type. Injection of RNA encoding wild-type human CCTβ rescued the small eye phenotype, reduced retinal cell death, and restored the levels of CCTβ protein and the major client protein Gβ1 that were significantly reduced in the homozygous cct2-L394H-7del mutant compared with wild-type. These results indicate that cct2 plays an essential role in retinal development by regulating the cell cycle.

Conclusions: The retinal pathology observed in the homozygous cct2-L394H-7del mutants resembles the retinal pathology of human LCA patients.

RevDate: 2018-08-06
CmpDate: 2018-08-06

Lemos BR, Kaplan AC, Bae JE, et al (2018)

CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

Proceedings of the National Academy of Sciences of the United States of America, 115(9):E2040-E2047.

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

RevDate: 2018-08-06
CmpDate: 2018-08-06

He J, Ford HC, Carroll J, et al (2018)

Assembly of the membrane domain of ATP synthase in human mitochondria.

Proceedings of the National Academy of Sciences of the United States of America, 115(12):2988-2993.

The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F1-c8 complex inhibited by the ATPase inhibitor protein IF1 and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast Fo membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae.

RevDate: 2018-08-06
CmpDate: 2018-08-06

Tian W, Shi J, Qin J, et al (2018)

Brain lipid binding protein mediates the proliferation of human glioblastoma cells by regulating ERK1/2 signaling pathway in vitro.

In vitro cellular & developmental biology. Animal, 54(2):156-162.

Brain lipid binding protein (BLBP) is highly expressed in the radial glial cells (RGCs) of the central nervous system (CNS), in glioblastomas, and, in vitro, in U251 cells. In this report, we have demonstrated that increased BLBP expression in glioblastoma is associated with poor survival and used a double-vector CRISPR/Cas9 lentiviral system to deplete endogenous BLBP from U251 cells, we found that loss of BLBP induced cell growth inhibition and S-phase arrest. Moreover, an increase in P53 and a decrease in p-ERK1/2 were observed after BLBP depletion, suggesting a potential mechanism by which loss of BLBP results in growth inhibition.

RevDate: 2018-08-06
CmpDate: 2018-08-06

Yang Y, Zhu G, Li R, et al (2017)

The RNA Editing Factor SlORRM4 Is Required for Normal Fruit Ripening in Tomato.

Plant physiology, 175(4):1690-1702.

RNA editing plays a key posttranscriptional role in gene expression. Existing studies on cytidine-to-uridine RNA editing in plants have focused on maize (Zea mays), rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana). However, the importance and regulation of RNA editing in several critical agronomic processes are not well understood, a notable example of which is fruit ripening. Here, we analyzed the expression profile of 33 RNA editing factors and identified 11 putative tomato (Solanum lycopersicum) fruit ripening-related factors. A rapid virus-induced gene silencing assay indicated that the organelle RNA recognition motif-containing protein SlORRM4 affected tomato fruit ripening. Knocking out SlORRM4 expression using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome editing strategy delayed tomato fruit ripening by lowering respiratory rate and ethylene production. Additionally, the expression of numerous genes associated with fruit ripening and mitochondrial functions changed significantly when SlORRM4 was knocked out. Moreover, the loss of SlORRM4 function significantly reduced RNA editing of many mitochondrial transcripts, leading to low-level expression of some core subunits that are critical for mitochondrial complex assembly (i.e. Nad3, Cytc1, and COX II). Taken together, these results indicate that SlORRM4 is involved in RNA editing of transcripts in ripening fruit that influence mitochondrial function and key aspects of fruit ripening.

RevDate: 2018-08-06
CmpDate: 2018-08-06

Ueda M, Matsui A, Tanaka M, et al (2017)

The Distinct Roles of Class I and II RPD3-Like Histone Deacetylases in Salinity Stress Response.

Plant physiology, 175(4):1760-1773.

Histone acetylation is an essential process in the epigenetic regulation of diverse biological processes, including environmental stress responses in plants. Previously, our research group identified a histone deacetylase (HDAC) inhibitor (HDI) that confers salt tolerance in Arabidopsis (Arabidopsis thaliana). In this study, we demonstrate that class I HDAC (HDA19) and class II HDACs (HDA5/14/15/18) control responses to salt stress through different pathways. The screening of 12 different selective HDIs indicated that seven newly reported HDIs enhance salt tolerance. Genetic analysis, based on a pharmacological study, identified which HDACs function in salinity stress tolerance. In the wild-type Columbia-0 background, hda19 plants exhibit tolerance to high-salinity stress, while hda5/14/15/18 plants exhibit hypersensitivity to salt stress. Transcriptome analysis revealed that the effect of HDA19 deficiency on the response to salinity stress is distinct from that of HDA5/14/15/18 deficiencies. In hda19 plants, the expression levels of stress tolerance-related genes, late embryogenesis abundant proteins that prevent protein aggregation and positive regulators such as ABI5 and NAC019 in abscisic acid signaling, were induced strongly relative to the wild type. Neither of these elements was up-regulated in the hda5/14/15/18 plants. The mutagenesis of HDA19 by genome editing in the hda5/14/15/18 plants enhanced salt tolerance, suggesting that suppression of HDA19 masks the phenotype caused by the suppression of class II HDACs in the salinity stress response. Collectively, our results demonstrate that HDIs that inhibit class I HDACs allow the rescue of plants from salinity stress regardless of their selectivity, and they provide insight into the hierarchal regulation of environmental stress responses through HDAC isoforms.

RevDate: 2018-08-06
CmpDate: 2018-08-06

Wang Y, Chen L, Tian Z, et al (2018)

CRISPR-Cas9 mediated gene knockout in human coronary artery endothelial cells reveals a pro-inflammatory role of TLR2.

Cell biology international, 42(2):187-193.

Endothelial inflammatory responses promote the development and progression of atherosclerosis. It was reported that Toll-like receptors 2 (TLR2) is associated with endothelial inflammation. However, the effect of TLR2 on inflammatory responses in human coronary artery endothelial cells (HCAECs) remains largely unknown. Here, we tested the hypothesis that TLR2 can enhance inflammatory reactions in HCAECs after stimulated by TLR2 agonist. First, we used CRISPR-Cas9 technology to knockout TLR2 gene in HCAECs. Then, TLR2-KO and wild type HCAECs were treated with TLR2 agonist peptidoglycan (PGN). The expression levels of intercellular cell adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), and interleukin-8 (IL-8) were analyzed by real-time PCR, Western blot, and ELISA. The expression status of myeloid differentiation primary response gene 88 (MyD88), phosphorylated IRAK-1 (pIRAK-1) and phosphorylated NF-κB (pNF-κB) were detected by Western blot. Our results show that after treated with TLR2 agonist, the expression levels of ICAM-1, IL-6, and IL-8 were downregulated in TLR2-KO cells compared to those of wild type cells. Further, Western blots of MyD88, pIRAK-1, and pNF-κB show that the expression levels of these pro-inflammatory molecules were much lower in TLR2-KO cells compared to that of wild type cells by stimulating with TLR2 agonist. We suggest that TLR2 may affect inflammatory reaction in HCAECs by introducing pro-inflammatory molecules like MyD88, pIRAK-1, and pNF-κB.

RevDate: 2018-08-06
CmpDate: 2018-08-06

Ikeda F, Yoshida K, Toki T, et al (2017)

Exome sequencing identified RPS15A as a novel causative gene for Diamond-Blackfan anemia.

Haematologica, 102(3):e93-e96.

RevDate: 2018-08-05

Javed MR, Sadaf M, Ahmed T, et al (2018)

CRISPR-Cas System: History and Prospects as a Genome Editing Tool in Microorganisms.

Current microbiology pii:10.1007/s00284-018-1547-4 [Epub ahead of print].

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR or more precisely CRISPR-Cas) system has proven to be a highly efficient and simple tool for achieving site-specific genome modifications in comparison to Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). The discovery of bacterial defense system that uses RNA-guided DNA cleaving enzymes for producing double-strand breaks along CRISPR has provided an exciting alternative to ZFNs and TALENs for gene editing & regulation, as the CRISPR-associated (Cas) proteins remain the same for different gene targets and only the short sequence of the guide RNA needs to be changed to redirect the site-specific cleavage. Therefore, in recent years the CRISPR-Cas system has emerged as a revolutionary engineering tool for carrying out precise and controlled genetic modifications in many microbes such as Escherichia coli, Staphylococcus aureus, Lactobacillus reuteri, Clostridium beijerinckii, Streptococcus pneumonia, and Saccharomyces cerevisiae. Though, concerns about CRISPR-Cas effectiveness in interlinked gene modifications and off-target effects need to be addressed. Nevertheless, it holds a great potential to speed up the pace of gene function discovery by interacting with previously intractable organisms and by raising the extent of genetic screens. Therefore, the potential applications of this system in microbial adaptive immune system, genome editing, gene regulations, functional genomics & biosynthesis along ethical issues, and possible harmful effects have been reviewed.

RevDate: 2018-08-04

Nishiyama J (2018)

Genome editing in the mammalian brain using the CRISPR-Cas system.

Neuroscience research pii:S0168-0102(18)30152-4 [Epub ahead of print].

Recent advances in genome editing technologies such as the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 have enabled the rapid and efficient modification of endogenous genomes in a variety of cell types, accelerating biomedical research. In particular, precise genome editing in somatic cells in vivo allows for the rapid generation of genetically modified cells in living animals and holds great promise for the possibility of directly correcting genetic defects associated with human diseases. However, because of the limited suitability and efficiency of these technologies in the brain, especially in postmitotic neurons, the practical application of genome editing technologies has been largely limited in the field of neuroscience. Recent technological advances overcome significant challenges facing genome editing in the brain and have enabled us to precisely edit the genome in both mitotic cells and mature postmitotic neurons in vitro and in vivo, providing powerful means for studying gene function and dysfunction in the brain. This review highlights the development of genome editing technologies for the brain and discusses their applications, limitations, and future challenges.

RevDate: 2018-08-04

Mendoza BJ, CT Trinh (2018)

In Silico Processing of the Complete CRISPR-Cas Spacer Space for Identification of PAM Sequences.

Biotechnology journal [Epub ahead of print].

Despite extensive exploration of the diversity of CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR associated) systems, biological applications have been mostly confined to Class 2 systems, specifically the Cas9 and Cas12 (formerly Cpf1) single effector proteins. A key limitation of exploring and utilizing other CRISPR-Cas systems with unique functionalities, particularly Class I types and their multi-protein effector complex, is the knowledge of the system's protospacer adjacent motif (PAM) sequence identity. In this work, we developed a systematic pipeline, named CASPERpam, that enables us to comprehensively assess the PAM sequences of all the available CRISPR-Cas systems in the NCBI database of bacterial genomes. The CASPERpam analysis revealed that within the 30,389 assemblies previously screened for CRISPR arrays, there exists 26,364 spacers that match somewhere in the viral, bacterial, and plasmid databases of NCBI, using the constraints of 95% sequence identity and 95% sequence coverage for blast hits. When grouping these results by species, we were able to identify putative PAM sequences for 1,049 among 1,493 unique species. The remaining species either have insufficient data or an undetermined result from the analysis. Finally, we were able to assign a confidence score to each species' PAM prediction and generate categories that largely cover the revealed diversity of PAM motifs, providing a baseline for further experimental studies including PAM assays. We envision CASPERpam is a useful bioinformatic tool for understanding and harnessing the diversity of CRISPR-Cas systems.

RevDate: 2018-08-03

Plaza Reyes A, F Lanner (2018)

Time Matters: Gene Editing at the Mouse 2-Cell Embryo Stage Boosts Knockin Efficiency.

Cell stem cell, 23(2):155-157.

Although endonuclease-mediated genome editing techniques offer significant improvement over traditional methods, they are still ineffective for introduction of large DNA sequences. Recently in Nature Biotechnology, Gu et al. (2018) developed a CRISPR-Cas strategy termed 2C-HR-CRISPR that generates fluorescent reporter tagging of genes with up to 95% knockin efficiency in mouse embryos.

RevDate: 2018-08-03

Simhadri VL, McGill J, McMahon S, et al (2018)

Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population.

Molecular therapy. Methods & clinical development, 10:105-112 pii:S2329-0501(18)30060-3.

The repurposing of the CRISPR/Cas microbial adaptive immune system for gene editing has resulted in an exponential rise in new technologies and promising approaches for treating numerous human diseases. While some of the approaches being currently developed involve ex vivo editing by CRISPR/Cas9, many more potential applications will require in vivo editing. The in vivo use of this technology comes with challenges, one of which is the immune response to Cas9, a protein of microbial origin. Thus, the prevalence of pre-existing antibodies to Cas9 could also be a relevant parameter. There are many avenues for how CRISPR/Cas9 technologies will be applied in vivo, including the mode of delivery. These may be expected to invoke different immunological pathways. Nonetheless, as with all protein therapeutics, it may be desirable to monitor for anti-Cas9 antibodies during clinical development. This will require the development of robust and reliable assays. Here, we describe ELISA-based assays that are capable of detecting antibodies to Cas9 from Staphylococcus aureus (SaCas9) and Streptococcs pyogenes (SpCas9) in human sera. Furthermore, using these assays to screen for pre-existing antibodies in 200 human serum samples, we found the prevalence of anti-SaCas9 and anti-SpCas9 antibodies to be 10% and 2.5%, respectively.

RevDate: 2018-08-03

Aglawe SB, Barbadikar KM, Mangrauthia SK, et al (2018)

New breeding technique "genome editing" for crop improvement: applications, potentials and challenges.

3 Biotech, 8(8):336.

Crop improvement is a continuous process in agriculture which ensures ample supply of food, fodder and fiber to burgeoning world population. Despite tremendous success in plant breeding and transgenesis to improve the yield-related traits, there have been several limitations primarily with the specificity in genetic modifications and incompatibility of host species. Because of this, new breeding techniques (NBTs) are gaining worldwide attention for crop improvement programs. Among the NBTs, genome editing (GE) using site-directed nucleases (SDNs) is an important and potential technique that overcomes limitations associated with classical breeding and transgenesis. These SDNs specifically target a compatible region in the gene/genome. The meganucleases (MgN), zinc finger nucleases (ZFN), transcription activator-like effectors nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated endonuclease (Cas) are being successfully employed for GE. These can be used for desired or targeted modifications of the native endogenous gene(s) or targeted insertion of cis/trans elements in the genomes of recipient organisms. Applications of these techniques appear to be endless ever since their discovery and several modifications in original technologies have further brought precision and accuracy in these methods. In this review, we present an overview of GE using SDNs with an emphasis on CRISPR/Cas system, their advantages, limitations and also practical considerations while designing experiments have been discussed. The review also emphasizes on the possible applications of CRISPR for improving economic traits in crop plants.

RevDate: 2018-08-03

González-Torres P, T Gabaldón (2018)

Genome Variation in the Model Halophilic Bacterium Salinibacter ruber.

Frontiers in microbiology, 9:1499.

The halophilic bacterium Salinibacter ruber is an abundant and ecologically important member of halophilic communities worldwide. Given its broad distribution and high intraspecific genetic diversity, S. ruber is considered one of the main models for ecological and evolutionary studies of bacterial adaptation to hypersaline environments. However, current insights on the genomic diversity of this species is limited to the comparison of the genomes of two co-isolated strains. Here, we present a comparative genomic analysis of eight S. ruber strains isolated at two different time points in each of two different Mediterranean solar salterns. Our results show an open pangenome with contrasting evolutionary patterns in the core and accessory genomes. We found that the core genome is shaped by extensive homologous recombination (HR), which results in limited sequence variation within population clusters. In contrast, the accessory genome is modulated by horizontal gene transfer (HGT), with genomic islands and plasmids acting as gateways to the rest of the genome. In addition, both types of genetic exchange are modulated by restriction and modification (RM) or CRISPR-Cas systems. Finally, genes differentially impacted by such processes reveal functional processes potentially relevant for environmental interactions and adaptation to extremophilic conditions. Altogether, our results support scenarios that conciliate "Neutral" and "Constant Diversity" models of bacterial evolution.

RevDate: 2018-08-03
CmpDate: 2018-08-03

Lee J, Yun HH, Kim S, et al (2018)

Implication of BIS in the Migration and Invasion of A549 Non-small Cell Lung Cancer Cells.

Anticancer research, 38(8):4525-4533.

BACKGROUND/AIM: High expression of the Bcl-2-interacting cell death suppressor (BIS), an anti-apoptotic protein, in various human cancers is linked to a poor outcome. The purpose of this study was to clarify whether BIS is associated with the migration and invasive characteristics of A549 cells.

MATERIALS AND METHODS: BIS-knockout (KO) cells were prepared by the CRISPR/Cas9 method. The aggressive behaviors of A549 cells were analyzed by wound healing and a transwell invasion assay as well as 3D spheroid culture.

RESULTS: BIS depletion resulted in significant inhibition of the migration and invasive potential of A549 cells which was accompanied by an increased ratio of E-cadherin/N-cadherin and a decrease in the mRNA levels of Zeb1, Snail, Slug and MMP-2. NF-κB activity was suppressed in BIS-KO A549 cells due to the decrease in p65 protein levels, but not in mRNA levels.

CONCLUSION: BIS regulates cell invasion and the induction of the epithelial-mesenchymal transition (EMT) phenotype in A549 cells probably via the NF-κB signaling pathway.

RevDate: 2018-08-03
CmpDate: 2018-08-03

Martel D, Beneke T, Gluenz E, et al (2017)

Characterisation of Casein Kinase 1.1 in Leishmania donovani Using the CRISPR Cas9 Toolkit.

BioMed research international, 2017:4635605.

The recent adaptation of CRISPR Cas9 genome editing to Leishmania spp. has opened a new era in deciphering Leishmania biology. The method was recently improved using a PCR-based CRISPR Cas9 approach, which eliminated the need for cloning. This new approach, which allows high-throughput gene deletion, was successfully validated in L. mexicana and L. major. In this study, we validated the toolkit in Leishmania donovani targeting the flagellar protein PF16, confirming that the tagged protein localizes to the flagellum and that null mutants lose their motility. We then used the technique to characterise CK1.1, a member of the casein kinase 1 family, which is involved in the regulation of many cellular processes. We showed that CK1.1 is a low-abundance protein present in promastigotes and in amastigotes. We demonstrated that CK1.1 is not essential for promastigote and axenic amastigote survival or for axenic amastigote differentiation, although it may have a role during stationary phase. Altogether, our data validate the use of PCR-based CRISPR Cas9 toolkit in L. donovani, which will be crucial for genetic modification of hamster-derived, disease-relevant parasites.

RevDate: 2018-08-02

Crawley AB, Henriksen ED, Stout E, et al (2018)

Characterizing the activity of abundant, diverse and active CRISPR-Cas systems in lactobacilli.

Scientific reports, 8(1):11544 pii:10.1038/s41598-018-29746-3.

CRISPR-Cas systems provide immunity against phages and plasmids in bacteria and archaea. Despite the popularity of CRISPR-Cas9 based genome editing, few endogenous systems have been characterized to date. Here, we sampled 1,262 publically available lactobacilli genomes found them to be enriched with CRISPR-Cas adaptive immunity. While CRISPR-Cas is ubiquitous in some Lactobacillus species, CRISPR-Cas content varies at the strain level in most Lactobacillus species. We identified that Type II is the most abundant type across the genus, with II-A being the most dominant sub-type. We found that many Type II-A systems are actively transcribed, and encode spacers that efficiently provide resistance against plasmid uptake. Analysis of various CRISPR transcripts revealed that guide sequences are highly diverse in terms of crRNA and tracrRNA length and structure. Interference assays revealed highly diverse target PAM sequences. Lastly, we show that these systems can be readily repurposed for self-targeting by expressing an engineered single guide RNA. Our results reveal that Type II-A systems in lactobacilli are naturally active in their native host in terms of expression and efficiently targeting invasive and genomic DNA. Together, these systems increase the possible Cas9 targeting space and provide multiplexing potential in native hosts and heterologous genome editing purpose.

RevDate: 2018-08-02
CmpDate: 2018-08-02

Obinata H, Sugimoto A, S Niwa (2018)

Streptothricin acetyl transferase 2 (Sat2): A dominant selection marker for Caenorhabditis elegans genome editing.

PloS one, 13(5):e0197128 pii:PONE-D-18-05527.

Studies on Caenorhabditis elegans would benefit from the introduction of new selectable markers to allow more complex types of experiments to be conducted with this model animal. We established a new antibiotic selection marker for C. elegans transformation based on nourseothricin (NTC) and its resistance-encoding gene, streptothricin-acetyl transferase 2 (Sat2). NTC was able to efficiently prevent worm development at very low concentrations, and the worms expressing Sat2 were able to survive on the selection plates without any developmental defects. Using CRISPR/Cas9 and NTC selection, we were able to easily insert a 13-kb expression cassette into a defined locus in C. elegans. The structure and spectrum of NTC differs from other antibiotics like hygromycin B and geneticin, making it possible to use NTC alongside them. Indeed, we confirmed NTC-sat2 selection could work with the hygromycin B selection system simultaneously. Thus, the new NTC-Sat2 system can act as a useful dominant marker for gene transfer and genome editing in C. elegans.

RevDate: 2018-08-02
CmpDate: 2018-08-02

Schuh RS, de Carvalho TG, Giugliani R, et al (2018)

Gene editing of MPS I human fibroblasts by co-delivery of a CRISPR/Cas9 plasmid and a donor oligonucleotide using nanoemulsions as nonviral carriers.

European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V, 122:158-166.

Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by the deficiency of alpha-L-iduronidase (IDUA). This study shows the use of nanoemulsions co-complexed with the plasmid of CRISPR/Cas9 system and a donor oligonucleotide aiming at MPS I gene editing in vitro. Nanoemulsions composed of MCT, DOPE, DOTAP, DSPE-PEG, and water were prepared by high-pressure homogenization. The DNA was complexed by adsorption (NA) or encapsulation (NE) of preformed DNA/DOTAP complexes with nanoemulsions at +4/-1 charge ratio. The incubation in pure DMEM or supplemented with serum showed that the complexation with DNA was stable after 1 h of incubation, but the complexes tended to release the adsorbed DNA after 24 h of incubation, while the encapsulated DNA remained complexed in the oil core of the nanoemulsions even 48 h after incubation with DMEM. The treatment of MPS I patient's fibroblasts homozygous for the p.Trp402∗ mutation led to a significant increase in IDUA activity at 2, 15, and 30 days when compared to MPS I untreated fibroblasts. Flow cytometry and confocal microscopy demonstrated that there was a reduction in the area of lysosomes to values similar to normal, an indicator of correction of the cellular phenotype. These results show that the nanoemulsions co-complexed with the CRISPR/Cas9 system and a donor oligonucleotide could effectively transfect MPS I p.Trp402∗ patient's fibroblasts, as well as enable the production of IDUA, and represent a potential new treatment option for MPS I.

RevDate: 2018-08-02
CmpDate: 2018-08-02

Moutal A, Yang X, Li W, et al (2017)

CRISPR/Cas9 editing of Nf1 gene identifies CRMP2 as a therapeutic target in neurofibromatosis type 1-related pain that is reversed by (S)-Lacosamide.

Pain, 158(12):2301-2319.

Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease linked to mutations of the Nf1 gene. Patients with NF1 commonly experience severe pain. Studies on mice with Nf1 haploinsufficiency have been instructive in identifying sensitization of ion channels as a possible cause underlying the heightened pain suffered by patients with NF1. However, behavioral assessments of Nf1 mice have led to uncertain conclusions about the potential causal role of Nf1 in pain. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (CRISPR/Cas9) genome editing system to create and mechanistically characterize a novel rat model of NF1-related pain. Targeted intrathecal delivery of guide RNA/Cas9 nuclease plasmid in combination with a cationic polymer was used to generate allele-specific C-terminal truncation of neurofibromin, the protein encoded by the Nf1 gene. Rats with truncation of neurofibromin, showed increases in voltage-gated calcium (specifically N-type or CaV2.2) and voltage-gated sodium (particularly tetrodotoxin-sensitive) currents in dorsal root ganglion neurons. These gains-of-function resulted in increased nociceptor excitability and behavioral hyperalgesia. The cytosolic regulatory protein collapsin response mediator protein 2 (CRMP2) regulates activity of these channels, and also binds to the targeted C-terminus of neurofibromin in a tripartite complex, suggesting a possible mechanism underlying NF1 pain. Prevention of CRMP2 phosphorylation with (S)-lacosamide resulted in normalization of channel current densities, excitability, as well as of hyperalgesia following CRISPR/Cas9 truncation of neurofibromin. These studies reveal the protein partners that drive NF1 pain and suggest that CRMP2 is a key target for therapeutic intervention.

RevDate: 2018-08-02
CmpDate: 2018-08-02

Katayama S, Moriguchi T, Ohtsu N, et al (2016)

A Powerful CRISPR/Cas9-Based Method for Targeted Transcriptional Activation.

Angewandte Chemie (International ed. in English), 55(22):6452-6456.

Targeted transcriptional activation of endogenous genes is important for understanding physiological transcriptional networks, synthesizing genetic circuits, and inducing cellular phenotype changes. The CRISPR/Cas9 system has great potential to achieve this purpose, however, it has not yet been successfully used to efficiently activate endogenous genes and induce changes in cellular phenotype. A powerful method for transcriptional activation by using CRISPR/Cas9 was developed. Replacement of a methylated promoter with an unmethylated one by CRISPR/Cas9 was sufficient to activate the expression of the neural cell gene OLIG2 and the embryonic stem cell gene NANOG in HEK293T cells. Moreover, CRISPR/Cas9-based OLIG2 activation induced the embryonic carcinoma cell line NTERA-2 to express the neuronal marker βIII-tubulin.

RevDate: 2018-07-31

Sicard A, Boardman DA, MK Levings (2018)

Taking regulatory T-cell therapy one step further.

Current opinion in organ transplantation [Epub ahead of print].

PURPOSE OF REVIEW: Adoptive cell therapy using CD4FOXP3 regulatory T cells (Treg) has emerged as a promising therapeutic strategy to treat autoimmunity and alloimmunity. Preclinical studies suggest that the efficacy of Treg therapy can be improved by modifying the antigen specificity, stability and function of therapeutic Tregs. We review recent innovations that considerably enhance the possibilities of controlling these parameters.

RECENT FINDINGS: Antigen-specific Tregs can be generated by genetically modifying polyclonal Tregs to express designated T-cell receptors or single-chain chimeric antigen receptors. The benefits of this approach can be further extended by using novel strategies to fine-tune the antigen-specificity and affinity of Treg in vivo. CRISPR/Cas 9 technology now enables the modification of therapeutic Tregs so they are safer, more stable and long lived. The differentiation and homing properties of Tregs can also be modulated by gene editing or modifying ex-vivo stimulation conditions.

SUMMARY: A new wave of innovation has considerably increased the number of strategies that could be used to increase the therapeutic potential of Treg therapy. However, the increased complexity of these approaches may limit their wide accessibility. Third-party therapy with off-the-shelf Treg products could be a solution.

RevDate: 2018-07-31

Li HY, Kao CY, Lin WH, et al (2018)

Characterization of CRISPR-Cas Systems in Clinical Klebsiella pneumoniae Isolates Uncovers Its Potential Association With Antibiotic Susceptibility.

Frontiers in microbiology, 9:1595.

Prokaryotic CRISPR-Cas systems limit the acquisition of genetic elements and provide immunity against invasive bacteriophage. The characteristics of CRISPR-Cas systems in clinical Klebsiella pneumoniae isolates are still unknown. Here, 97 K. pneumoniae genomes retrieved from the Integrated Microbial Genomes & Microbiomes genome database and 176 clinical isolates obtained from patients with bloodstream (BSI, n = 87) or urinary tract infections (UTI, n = 89) in Taiwan, were used for analysis. Forty out of ninety-seven genomes (41.2%) had CRISPR-Cas systems identified by the combination of CRISPRFinder and cas1 gene sequence alignment. The phylogenetic trees revealed that CRISPR-Cas systems in K. pneumoniae were divided into two types (type I-E, 23; subtype I-E∗, 17) based on the sequences of Cas1 and Cas3 proteins and their location in the chromosome. The distribution of type I-E and I-E∗ CRISPR-Cas systems was associated with the multilocus sequence typing and the pulsed-field gel electrophoresis results. Importantly, no CRISPR-Cas system was identified in published genomes of clonal complex 258 isolates (ST11 and ST258), which comprise the largest multi-drug resistant K. pneumoniae clonal group worldwide. PCR with cas-specific primers showed that 30.7% (54/176) of the clinical isolates had a CRISPR-Cas system. Among clinical isolates, more type I-E CRISPR-Cas systems were found in UTI isolates (BSI, 5.7%; UTI, 11.2%), and subtype I-E∗ CRISPR-Cas systems were dominant in BSI isolates (BSI, 28.7%; UTI, 15.7%) (p = 0.042). Isolates which had subtype I-E∗ CRISPR-Cas system were more susceptible to ampicillin-sulbactam (p = 0.009), cefazolin (p = 0.016), cefuroxime (p = 0.039), and gentamicin (p = 0.012), compared to the CRISPR-negative isolates. The strains containing subtype I-E∗ CRISPR-Cas systems had decreased numbers of plasmids, prophage regions, and acquired antibiotic resistance genes in their published genomes. Here, we first revealed subtype I-E∗ CRISPR-Cas system in K. pneumoniae potentially interfering with the acquisition of phages and plasmids harboring antibiotic resistance determinants, and thus maintained these isolates susceptible to antibiotics.

RevDate: 2018-07-31

Seal BS, Drider D, Oakley BB, et al (2018)

Microbial-derived products as potential new antimicrobials.

Veterinary research, 49(1):66 pii:10.1186/s13567-018-0563-5.

Due to the continuing global concerns involving antibiotic resistance, there is a need for scientific forums to assess advancements in the development of antimicrobials and their alternatives that might reduce development and spread of antibiotic resistance among bacterial pathogens. The objectives of the 2nd International Symposium on Alternatives to Antibiotics were to highlight promising research results and novel technologies that can provide alternatives to antibiotics for use in animal health and production, assess challenges associated with their authorization and commercialization for use, and provide actionable strategies to support their development. The session on microbial-derived products was directed at presenting novel technologies that included exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials, probiotics development via fecal microbiome transplants among monogastric production animals such as chickens and mining microbial sources such as bacteria or yeast to identify new antimicrobial compounds. Other research has included continuing development of antimicrobial peptides such as newly discovered bacteriocins as alternatives to antibiotics, use of bacteriophages accompanied by development of unique lytic proteins with specific cell-wall binding domains and novel approaches such as microbial-ecology guided discovery of anti-biofilm compounds discovered in marine environments. The symposium was held at the Headquarters of the World Organisation for Animal Health (OIE) in Paris, France during 12-15 December 2016.

RevDate: 2018-07-30

Heidrich N, Hagmann A, Bauriedl S, et al (2018)

The CRISPR/Cas system in Neisseria meningitidis affects bacterial adhesion to human nasopharyngeal epithelial cells.

RNA biology [Epub ahead of print].

Neisseria meningitidis, a commensal β-proteobacterium of the human nasopharynx, constitutes a worldwide leading cause of sepsis and epidemic meningitis. A recent genome-wide association study suggested an association of its type II-C CRISPR/Cas system with carriage and thus less invasive lineages. Here, we show that knock-out strains lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells which constitutes a central step in the pathogenesis of invasive meningococcal disease. Transcriptome sequencing data further suggest that meningococcal Cas9 does not affect the expression of surface adhesins but rather exerts its effect on cell adhesion in an indirect manner. Consequently, we speculate that the meningococcal CRISPR/Cas system exerts novel functions beyond its established role in defence against foreign DNA.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Prior H, MacConnachie L, Martinez JL, et al (2018)

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins.

Journal of visualized experiments : JoVE.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Chu FC, Wu PS, Pinzi S, et al (2018)

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing.

Journal of visualized experiments : JoVE.

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR's genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Del'Guidice T, Lepetit-Stoffaes JP, Bordeleau LJ, et al (2018)

Membrane permeabilizing amphiphilic peptide delivers recombinant transcription factor and CRISPR-Cas9/Cpf1 ribonucleoproteins in hard-to-modify cells.

PloS one, 13(4):e0195558 pii:PONE-D-17-43853.

Delivery of recombinant proteins to therapeutic cells is limited by a lack of efficient methods. This hinders the use of transcription factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoproteins to develop cell therapies. Here, we report a soluble peptide designed for the direct delivery of proteins to mammalian cells including human stem cells, hard-to-modify primary natural killer (NK) cells, and cancer cell models. This peptide is composed of a 6x histidine-rich domain fused to the endosomolytic peptide CM18 and the cell penetrating peptide PTD4. A less than two-minute co-incubation of 6His-CM18-PTD4 peptide with spCas9 and/or asCpf1 CRISPR ribonucleoproteins achieves robust gene editing. The same procedure, co-incubating with the transcription factor HoxB4, achieves transcriptional regulation. The broad applicability and flexibility of this DNA- and chemical-free method across different cell types, particularly hard-to-transfect cells, opens the way for a direct use of proteins for biomedical research and cell therapy manufacturing.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Mertz L (2018)

A CRISPR Approach for a Common Inherited Disease: Researchers at Duke University Hope Gene Editing Can Eliminate Mutations That Lead to Duchenne Muscular Dystrophy.

IEEE pulse, 9(2):12-14.

Gene editing and CRISPR (a group of repeated DNA sequences in bacteria) typically target disease-causing mutated genes by eliminating the bad gene altogether, by correcting the problem DNA to restore proper gene functioning, or by modifying a different gene to compensate for the faulty gene's lost function. One research group at Duke University in Durham, North Carolina, however, is using a different strategy to fight one of the most common inherited genetic diseases: Duchenne muscular dystrophy (DMD).

RevDate: 2018-07-31
CmpDate: 2018-07-31

Nguyen DD, S Chang (2017)

Development of Novel Therapeutic Agents by Inhibition of Oncogenic MicroRNAs.

International journal of molecular sciences, 19(1): pii:ijms19010065.

MicroRNAs (miRs, miRNAs) are regulatory small noncoding RNAs, with their roles already confirmed to be important for post-transcriptional regulation of gene expression affecting cell physiology and disease development. Upregulation of a cancer-causing miRNA, known as oncogenic miRNA, has been found in many types of cancers and, therefore, represents a potential new class of targets for therapeutic inhibition. Several strategies have been developed in recent years to inhibit oncogenic miRNAs. Among them is a direct approach that targets mature oncogenic miRNA with an antisense sequence known as antimiR, which could be an oligonucleotide or miRNA sponge. In contrast, an indirect approach is to block the biogenesis of miRNA by genome editing using the CRISPR/Cas9 system or a small molecule inhibitor. The development of these inhibitors is straightforward but involves significant scientific and therapeutic challenges that need to be resolved. In this review, we summarize recent relevant studies on the development of miRNA inhibitors against cancer.

RevDate: 2018-07-31
CmpDate: 2018-07-31

Baliou S, Adamaki M, Kyriakopoulos AM, et al (2018)

Role of the CRISPR system in controlling gene transcription and monitoring cell fate (Review).

Molecular medicine reports, 17(1):1421-1427.

Even though the accrual of transcripts is implicated in distinct disease states, our knowledge regarding their functional role remains obscure. The CRISPR system has surged at the forefront of genome engineering tools in the field of RNA modulation. In the present review, we discuss some exciting applications of the CRISPR system, including the manipulation of RNA sequences, the visualization of chromosomal loci in living cells and the modulation of transcription. The CRISPR system has been documented to be very reliable and specific in altering gene expression, via leveraging inactive catalytically dead CRISPR-associated protein 9 (Cas9). In the present review, the CRISPR system is presented as an eminent tool for the meticulous analysis of gene regulation, loci mapping and complex pathways.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Zhao C, Zheng X, Qu W, et al (2017)

CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif.

International journal of biological sciences, 13(12):1470-1478 pii:ijbsv13p1470.

Designing efficient and specific CRISPR single-guide RNAs (sgRNAs) is vital for the successful application of CRISPR technology. Currently, a growing number of new RNA-guided endonucleases with a different protospacer adjacent motif (PAM) have been discovered, suggesting the necessity to develop a versatile tool for designing sgRNA to meet the requirement of different RNA-guided DNA endonucleases. Here, we report the development of a flexible sgRNA design program named "CRISPR-offinder". Support for user-defined PAM and sgRNA length was provided to increase the targeting range and specificity. Additionally, evaluation of on- and off-target scoring algorithms was integrated into the CRISPR-offinder. The CRISPR-offinder has provided the bench biologist a rapid and efficient tool for identification of high quality target sites, and it is freely available at https://sourceforge.net/projects/crispr-offinder-v1-2/ or http://www.biootools.com.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Yau EH, TM Rana (2018)

Next-Generation Sequencing of Genome-Wide CRISPR Screens.

Methods in molecular biology (Clifton, N.J.), 1712:203-216.

Genome-wide functional genomic screens utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system have proven to be a powerful tool for systematic genomic perturbation in mammalian cells and provide an alternative to previous screens utilizing RNA interference technology. The wide availability of these libraries through public plasmid repositories as well as the decreasing cost and speed in quantifying these screens using high-throughput next-generation sequencing (NGS) allows for the adoption of the technology in a variety of laboratories interested in diverse biologic questions. Here, we describe the protocol to generate next-generation sequencing libraries from genome-wide CRISPR genomic screens.

RevDate: 2018-07-31
CmpDate: 2018-07-31

Gorter de Vries AR, de Groot PA, van den Broek M, et al (2017)

CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus.

Microbial cell factories, 16(1):222 pii:10.1186/s12934-017-0835-1.

BACKGROUND: The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae.

RESULTS: This study reports a new CRISPR-Cas9 method for accurate gene deletion in S. pastorianus. This method combined the Streptococcus pyogenes cas9 gene expressed from either a chromosomal locus or from a mobile genetic element in combination with a plasmid-borne gRNA expression cassette. While the well-established gRNA expression system using the RNA polymerase III dependent SNR52 promoter failed, expression of a gRNA flanked with Hammerhead and Hepatitis Delta Virus ribozymes using the RNA polymerase II dependent TDH3 promoter successfully led to accurate deletion of all four alleles of the SeILV6 gene in strain CBS1483. Furthermore the expression of two ribozyme-flanked gRNAs separated by a 10-bp linker in a polycistronic array successfully led to the simultaneous deletion of SeATF1 and SeATF2, genes located on two separate chromosomes. The expression of this array resulted in the precise deletion of all five and four alleles mediated by homologous recombination in the strains CBS1483 and Weihenstephan 34/70 respectively, demonstrating the multiplexing abilities of this gRNA expression design.

CONCLUSIONS: These results firmly established that CRISPR-Cas9 significantly facilitates and accelerates genome editing in S. pastorianus.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Garcia PL, Hossain MI, Andrabi SA, et al (2018)

Generation and Characterization of SULT4A1 Mutant Mouse Models.

Drug metabolism and disposition: the biological fate of chemicals, 46(1):41-45.

Sulfotransferase 4A1 (SULT4A1) belongs to the cytosolic sulfotransferase (SULT) superfamily of enzymes that catalyze sulfonation reactions with a variety of endogenous and exogenous substrates. Of the SULTs, SULT4A1 was shown to have the highest sequence homology between vertebrate species, yet no known function or enzymatic activity has been identified for this orphan SULT. To better understand SULT4A1 function in mammalian brain, two mutant SULT4A1 mouse strains were generated utilizing clustered regulatory interspaced short palindromic repeats (CRISPR)-content-addressable storage (Cas) 9 technology. The first strain possessed a 28-base pair (bp) deletion (Δ28) in exon 1 that resulted in a frameshift mutation with premature termination. The second strain possessed a 12-bp in-frame deletion (Δ12) immediately preceding an active site histidine111 common to the SULT family. Homozygous pups of both strains present with severe and progressive neurologic symptoms, including tremor, absence seizures, abnormal gait, ataxia, decreased weight gain compared with littermates, and death around postnatal days 21-25. SULT4A1 immunostaining was decreased in brains of heterozygous pups and not detectable in homozygous pups of both SULT4A1 mutants. SULT4A1 localization in subcellular fractions of mouse brain showed SULT4A1 associated with mitochondrial, cytosolic, and microsomal fractions, a novel localization pattern for SULTs. Finally, primary cortical neurons derived from embryonic (E15) CD-1 mice expressed high levels of SULT4A1 throughout the cell except in nuclei. Taken together, SULT4A1 appears to be an essential neuronal protein required for normal brain function, at least in mammals. Mouse models will be valuable in future studies to investigate the regulation and functions of SULT4A1 in the mammalian brain.

RevDate: 2018-07-31
CmpDate: 2018-07-31

Chew WL (2018)

Immunity to CRISPR Cas9 and Cas12a therapeutics.

Wiley interdisciplinary reviews. Systems biology and medicine, 10(1):.

Genome-editing therapeutics are poised to treat human diseases. As we enter clinical trials with the most promising CRISPR-Cas9 and CRISPR-Cas12a (Cpf1) modalities, the risks associated with administering these foreign biomolecules into human patients become increasingly salient. Preclinical discovery with CRISPR-Cas9 and CRISPR-Cas12a systems and foundational gene therapy studies indicate that the host immune system can mount undesired responses against the administered proteins and nucleic acids, the gene-edited cells, and the host itself. These host defenses include inflammation via activation of innate immunity, antibody induction in humoral immunity, and cell death by T-cell-mediated cytotoxicity. If left unchecked, these immunological reactions can curtail therapeutic benefits and potentially lead to mortality. Ways to assay and reduce the immunogenicity of Cas9 and Cas12a proteins are therefore critical for ensuring patient safety and treatment efficacy, and for bringing us closer to realizing the vision of permanent genetic cures. WIREs Syst Biol Med 2018, 10:e1408. doi: 10.1002/wsbm.1408 This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Translational, Genomic, and Systems Medicine > Translational Medicine Translational, Genomic, and Systems Medicine > Therapeutic Methods.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Arias-Fuenzalida J, Jarazo J, Qing X, et al (2017)

FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's Disease Modeling.

Stem cell reports, 9(5):1423-1431.

Genome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ)-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE). FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson's-disease-associated mutations in α-synuclein and present their comparative phenotypes.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Liu T, Zhang Z, Yu C, et al (2017)

Tetrandrine antagonizes acute megakaryoblastic leukaemia growth by forcing autophagy-mediated differentiation.

British journal of pharmacology, 174(23):4308-4328.

BACKGROUND AND PURPOSE: The poor prognosis of acute megakaryoblastic leukaemia (AMKL) means there is a need to develop novel therapeutic methods to treat this condition. It was recently shown that inducing megakaryoblasts to undergo terminal differentiation is effective as a treatment for AMKL. This encouraged us to identify a compound that induces megakaryocyte differentiation, which could then act as a potent anti-leukaemia agent.

EXPERIMENTAL APPROACH: The effects of tetrandrine on the expression of CD41 and cell morphology were investigated in AMKL cells. We used CRISPR/Cas9 knockout system to knock out ATG7 and verify the role of autophagy in tetrandrine-induced megakaryocyte differentiation. shNotch1 and CA-Akt were transfected into K562 cells to examine the downstream pathways of ROS signalling and the mechanistic basis of the tetrandrine-induced megakaryocyte differentiation. The anti-leukaemia effects of tetrandrine were analysed both in vitro and in vivo.

KEY RESULTS: A low dose of tetrandrine induced cell cycle arrest and megakaryocyte differentiation in AMKL cells via activation of autophagy. Molecularly, we demonstrated that this effect is mediated by activation of Notch1 and Akt and subsequent accumulation of ROS. In contrast, in normal mouse fetal liver cells, although tetrandrine induced autophagy, it did not affect cell proliferation or promote megakaryocyte differentiation, suggesting a specific effect of tetrandrine in malignant megakaryoblasts. Finally, tetrandrine also showed in vivo efficacy in an AMKL xenograft mouse model.

CONCLUSIONS AND IMPLICATIONS: Modulating autophagy-mediated differentiation may be a novel strategy for treating AMKL, and tetrandrine has the potential to be developed as a differentiation-inducing agent for AMKL chemotherapy.

RevDate: 2018-07-31
CmpDate: 2018-07-31

Barkin SL (2017)

At the intersection of systematic and seismic: examining the way forward for pediatric research.

Pediatric research, 82(6):897-898.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Ehrke-Schulz E, Zhang W, Gao J, et al (2017)

Recent Advances in Preclinical Developments Using Adenovirus Hybrid Vectors.

Human gene therapy, 28(10):833-841.

Adenovirus (Ad)-based vectors are efficient gene-transfer vehicles to deliver foreign DNA into living organisms, offering large cargo capacity and low immunogenicity and genotoxicity. As Ad shows low integration rates of their genomes into host chromosomes, vector-derived gene expression decreases due to continuous cell cycling in regenerating tissues and dividing cell populations. To overcome this hurdle, adenoviral delivery can be combined with mechanisms leading to maintenance of therapeutic DNA and long-term effects of the desired treatment. Several hybrid Ad vectors (AdV) exploiting various strategies for long-term treatment have been developed and characterized. This review summarizes recent developments of preclinical approaches using hybrid AdVs utilizing either the Sleeping Beauty transposase system for somatic integration into host chromosomes or designer nucleases, including transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease for permanent gene editing. Further options on how to optimize these vectors further are discussed, which may lead to future clinical applications of these versatile gene-therapy tools.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Graham DM (2017)

Cas9 cuts to the quick in the cortex.

Lab animal, 46(5):191.

RevDate: 2018-07-30
CmpDate: 2018-07-30

Gregorowius D, Biller-Andorno N, A Deplazes-Zemp (2017)

The role of scientific self-regulation for the control of genome editing in the human germline: The lessons from the Asilomar and the Napa meetings show how self-regulation and public deliberation can lead to regulation of new biotechnologies.

EMBO reports, 18(3):355-358.

RevDate: 2018-07-28

Richardson CD, Kazane KR, Feng SJ, et al (2018)

CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway.

Nature genetics pii:10.1038/s41588-018-0174-0 [Epub ahead of print].

CRISPR-Cas genome editing creates targeted DNA double-strand breaks (DSBs) that are processed by cellular repair pathways, including the incorporation of exogenous DNA via single-strand template repair (SSTR). To determine the genetic basis of SSTR in human cells, we developed a coupled inhibition-cutting system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. We found that human Cas9-induced SSTR requires the Fanconi anemia (FA) pathway, which is normally implicated in interstrand cross-link repair. The FA pathway does not directly impact error-prone, non-homologous end joining, but instead diverts repair toward SSTR. Furthermore, FANCD2 protein localizes to Cas9-induced DSBs, indicating a direct role in regulating genome editing. Since FA is itself a genetic disease, these data imply that patient genotype and/or transcriptome may impact the effectiveness of gene editing treatments and that treatments biased toward FA repair pathways could have therapeutic value.

RevDate: 2018-07-27

Hupfeld M, Trasanidou D, Ramazzini L, et al (2018)

A functional type II-A CRISPR-Cas system from Listeria enables efficient genome editing of large non-integrating bacteriophage.

Nucleic acids research, 46(13):6920-6933.

CRISPR-Cas systems provide bacteria with adaptive immunity against invading DNA elements including bacteriophages and plasmids. While CRISPR technology has revolutionized eukaryotic genome engineering, its application to prokaryotes and their viruses remains less well established. Here we report the first functional CRISPR-Cas system from the genus Listeria and demonstrate its native role in phage defense. LivCRISPR-1 is a type II-A system from the genome of L. ivanovii subspecies londoniensis that uses a small, 1078 amino acid Cas9 variant and a unique NNACAC protospacer adjacent motif. We transferred LivCRISPR-1 cas9 and trans-activating crRNA into Listeria monocytogenes. Along with crRNA encoding plasmids, this programmable interference system enables efficient cleavage of bacterial DNA and incoming phage genomes. We used LivCRISPR-1 to develop an effective engineering platform for large, non-integrating Listeria phages based on allelic replacement and CRISPR-Cas-mediated counterselection. The broad host-range Listeria phage A511 was engineered to encode and express lysostaphin, a cell wall hydrolase that specifically targets Staphylococcus peptidoglycan. In bacterial co-culture, the armed phages not only killed Listeria hosts but also lysed Staphylococcus cells by enzymatic collateral damage. Simultaneous killing of unrelated bacteria by a single phage demonstrates the potential of CRISPR-Cas-assisted phage engineering, beyond single pathogen control.

RevDate: 2018-07-27

Viswanatha R, Li Z, Hu Y, et al (2018)

Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in Drosophila cells.

eLife, 7: pii:36333.

Genome-wide screens in Drosophila cells have offered numerous insights into gene function, yet a major limitation has been the inability to stably deliver large multiplexed DNA libraries to cultured cells allowing barcoded pooled screens. Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in Drosophila S2R+ cells. Under basal growth conditions, 1235 genes were essential for cell fitness at a false-discovery rate of 5%, representing the highest-resolution fitness gene set yet assembled for Drosophila, including 407 genes which likely duplicated along the vertebrate lineage and whose orthologs were underrepresented in human CRISPR screens. We additionally performed context-specific fitness screens for resistance to or synergy with trametinib, a Ras/ERK/ETS inhibitor, or rapamycin, an mTOR inhibitor, and identified key regulators of each pathway. The results present a novel, scalable, and versatile platform for functional genomic screens in invertebrate cells.

RevDate: 2018-07-27
CmpDate: 2018-07-27

Cohen J (2018)

A 'gene drive' makes its debut in mammals.

Science (New York, N.Y.), 361(6398):118.

RevDate: 2018-07-27
CmpDate: 2018-07-27

Rylee JC, Siniard DJ, Doucette K, et al (2018)

Expanding the genetic toolkit of Tribolium castaneum.

PloS one, 13(4):e0195977 pii:PONE-D-18-05739.

The red flour beetle, Tribolium castaneum, is an important model insect and agricultural pest. However, many standard genetic tools are lacking or underdeveloped in this system. Here, we present a set of new reagents to augment existing Tribolium genetic tools. We demonstrate a new GAL4 driver line that employs the promoter of a ribosomal protein gene to drive expression of a UAS responder in the fat body. We also present a novel dual fluorescent reporter that labels cell membranes and nuclei with different fluorophores for the analysis of cellular morphology. This approach also demonstrates the functionality of the viral T2A peptide for bicistronic gene expression in Tribolium. To facilitate classical genetic analysis, we created lines with visible genetic markers by CRISPR-mediated disruption of the yellow and ebony body color loci with a cassette carrying an attP site, enabling future φC31-mediated integration. Together, the reagents presented here will facilitate more robust genetic analysis in Tribolium and serve as a blueprint for the further development of this powerful model's genetic toolkit.

RevDate: 2018-07-27
CmpDate: 2018-07-27

Hara S, S Takada (2018)

Genome editing for the reproduction and remedy of human diseases in mice.

Journal of human genetics, 63(2):107-113.

With the recent progress in genome-editing technologies, such as the CRISPR/Cas9 system, genetically modified animals carrying nucleotide substitutions or large chromosomal rearrangements can be produced rapidly and at low cost. Such genome-editing techniques have been applied in the generation of animal models, especially mice, for reproducing human disease mutations, such as single-nucleotide polymorphisms (SNPs) or large chromosomal rearrangements identified by genome-wide screening analyses. While application methods are under development for various complex mutations involving genome editing for mimicking human disease-causing mutations in mice, functional studies of mouse models carrying replicated human mutations are gradually being published. In this review, we discuss the recent progress in application methods of the CRISPR/Cas9 system, focusing on the production of mouse models of diseases.

RevDate: 2018-07-27
CmpDate: 2018-07-27

Kurata M, Yamamoto K, Moriarity BS, et al (2018)

CRISPR/Cas9 library screening for drug target discovery.

Journal of human genetics, 63(2):179-186.

CRISPR/Cas9-based tools have rapidly developed in recent years. These include CRISPR-based gene activation (CRISPRa) or inhibition (CRISPRi), for which there are libraries. CRISPR libraries for loss of function have been widely used to identify new biological mechanisms, such as drug resistance and cell survival signals. CRISPRa is highly useful in screening for gain of functions, and CRISPRi is a more powerful tool than RNA interference (RNAi) libraries in screening for loss of functions. Positive selection using a CRISPR library can detect survival cells with specific conditions, such as drug treatment, and it can easily clarify drug resistance mechanisms. Negative selection is capable of detecting dead or slow-growing cells efficiently, and it can identify survival-essential genes, which can be promising candidates for molecularly targeted drugs. In addition, negative selection can be applied for synthetic lethality interactions, where the perturbation of both genes simultaneously results in the loss of viability, but that of either gene alone does not affect viability. This mechanism is highly important to identifying the optimal combination of molecularly targeted drugs. Survival-co-essential genes in cancer cells can be identified using new methods, such as the paired guide RNA system and in combination with single-cell RNA sequencing techniques. These efficient methods can clarify interesting biological mechanisms and suggest candidates for molecularly targeted drugs. This review identifies what types of screenings were performed and suggests ideas for the next CRISPR screenings to develop new drugs.

RevDate: 2018-07-27
CmpDate: 2018-07-27

Kim SM, Yang Y, Oh SJ, et al (2017)

Cancer-derived exosomes as a delivery platform of CRISPR/Cas9 confer cancer cell tropism-dependent targeting.

Journal of controlled release : official journal of the Controlled Release Society, 266:8-16.

An intracellular delivery system for CRISPR/Cas9 is crucial for its application as a therapeutic genome editing technology in a broad range of diseases. Current vehicles carrying CRISPR/Cas9 limit in vivo delivery because of low tolerance and immunogenicity; thus, the in vivo delivery of genome editing remains challenging. Here, we report that cancer-derived exosomes function as natural carriers that can efficiently deliver CRISPR/Cas9 plasmids to cancer. Compared to epithelial cell-derived exosomes, cancer-derived exosomes provide potential vehicles for effective in vivo delivery via selective accumulation in ovarian cancer tumors of SKOV3 xenograft mice, most likely because of their cell tropism. CRISPR/Cas9-loaded exosomes can suppress expression of poly (ADP-ribose) polymerase-1 (PARP-1), resulting in the induction of apoptosis in ovarian cancer. Furthermore, the inhibition of PARP-1 by CRISPR/Cas9-mediated genome editing enhances the chemosensitivity to cisplatin, showing synergistic cytotoxicity. Based on these results, tumor-derived exosomes may be very promising for cancer therapeutics in the future.

RevDate: 2018-07-27
CmpDate: 2018-07-27

Liu C, Zhang L, Liu H, et al (2017)

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications.

Journal of controlled release : official journal of the Controlled Release Society, 266:17-26.

The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes.

RevDate: 2018-07-27
CmpDate: 2018-07-27

Gao M, D Liu (2017)

CRISPR/Cas9-based Pten knock-out and Sleeping Beauty Transposon-mediated Nras knock-in induces hepatocellular carcinoma and hepatic lipid accumulation in mice.

Cancer biology & therapy, 18(7):505-512.

Both Pten and Nras are downstream mediators of receptor tyrosine kinase activation that plays important roles in controlling cell survival and proliferation. Here, we investigated whether and how Pten loss cross-talks with Nras activation in driving liver cancer development in mice. Somatic disruption of hepatic Pten and overexpression of Nras were achieved in out-bred immunocompetent CD-1 mice through a hydrodynamic delivery of plasmids carrying Sleeping Beauty transposon-based integration of Nras and the CRISPR/Cas9-mediated Pten knockout system. Concurrent Pten knockout and Nras knock-in induced hepatocellular carcinoma, while individual gene manipulation failed. Tumor development was associated with liver fibrosis, hyperlipidemia, hepatic deposition of lipid droplets and glycogen, and hepatomegaly. At the molecular level, lipid droplet formation was primarily contributed by upregulated expression of genes responsible for lipogenesis and fatty acid sequestration, such as Srebpf1, Acc, Pparg and its downstream targets. Our findings demonstrated that Pten disruption was synergized by Nras overexpression in driving hepatocyte malignant transformation, which correlated with extensive formation of lipid droplets.

RevDate: 2018-07-27
CmpDate: 2018-07-27

Oulhen N, GM Wessel (2016)

Albinism as a visual, in vivo guide for CRISPR/Cas9 functionality in the sea urchin embryo.

Molecular reproduction and development, 83(12):1046-1047.

RevDate: 2018-07-27
CmpDate: 2018-07-27

Gutschner T, L Chin (2016)

Genome engineering - Matching supply with demand.

Cell cycle (Georgetown, Tex.), 15(11):1395-1396.


RJR Experience and Expertise


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


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


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


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


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


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


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


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

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

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Collection of publications by R J Robbins

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