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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 20 Jul 2019 at 01:36 Created: 

CRISPR-Cas

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

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

Citations The Papers (from PubMed®)

RevDate: 2019-07-19

Nekrasov V (2019)

Sequence-specific nucleases as tools for enhancing disease resistance in crops.

Transgenic research, 28(Suppl 2):75-80.

Genome editing technologies, such as CRISPR/Cas, have recently become valuable tools for plant reverse genetics as well as crop improvement, including enhancement of disease resistance. Targeting susceptibility (S) genes by genome editing has proven to be a viable strategy for generating resistance to both bacterial and fungal pathogens in various crops. Examples include generating loss-of-function mutations in promoter elements of the SWEET S genes, which are targeted by transcription activator-like effectors secreted by many phytopathogenic Xanthomonas bacteria, as well as in the conserved MLO locus that confers susceptibility to powdery mildew fungal pathogens in many monocots and dicots. In addition to genome editing applications, CRISPR/Cas systems can be used as means of defending plants against viruses via targeting viral genomic DNA or RNA. Genome editing is therefore a highly promising approach that enables engineering disease resistance to various plant pathogens directly in elite cultivar background in a highly precise manner. Unlike conventional crop breeding, genome editing approaches are not relying on lengthy and laborious crosses/back-crosses involving parental and progeny lines and can significantly shorten the breeding timeline. Taking into account the high potential of genome editing technologies for both basic and applied plant science, the recent decision of the European Court of Justice to define transgene-free genetically edited crops as GMOs is, clearly, a backward step for the EU.

RevDate: 2019-07-19
CmpDate: 2019-07-19

Zhan H, Zhou Q, Gao Q, et al (2019)

Multiplexed promoterless gene expression with CRISPReader.

Genome biology, 20(1):113 pii:10.1186/s13059-019-1712-5.

BACKGROUND: Genes are comprised of DNA codes and contain promoters and other control elements for reading these codes. The rapid development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has made possible the construction of a novel code-reading system with low dependency on the native control elements.

RESULTS: We develop CRISPReader, a technology for controlling promoterless gene expression in a robust fashion. We demonstrate that this tool is highly efficient in controlling transcription and translation initiation of a targeted transgene. A notable feature of CRISPReader is the ability to "read" the open reading frames of a cluster of gene without traditional regulatory elements or other cofactors. In particular, we use this strategy to construct an all-in-one AAV-CRISPR-Cas9 system by removing promoter-like elements from the expression cassette to resolve the existing AAV packaging size problem. The compact AAV-CRISPR-Cas9 is also more efficient in transactivation, DNA cleavage, and gene editing than the dual-AAV vector encoding two separate Cas9 elements, shown by targeting both reporter and endogenous genes in vitro and in vivo.

CONCLUSIONS: CRISPReader represents a novel approach for gene regulation that enables minimal gene constructs to be expressed and can be used in potential biomedical applications.

RevDate: 2019-07-19
CmpDate: 2019-07-19

Humbert O, Laszlo GS, Sichel S, et al (2019)

Engineering resistance to CD33-targeted immunotherapy in normal hematopoiesis by CRISPR/Cas9-deletion of CD33 exon 2.

Leukemia, 33(3):762-808.

RevDate: 2019-07-19
CmpDate: 2019-07-19

Lyu Q, Dhagia V, Han Y, et al (2018)

CRISPR-Cas9-Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin-Brief Report.

Arteriosclerosis, thrombosis, and vascular biology, 38(9):2184-2190.

Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research.

RevDate: 2019-07-18

Ahmad N, Rahman MU, Mukhtar Z, et al (2019)

A critical look on CRISPR-based genome editing in plants.

Journal of cellular physiology [Epub ahead of print].

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing, derived from prokaryotic immunity system, is rapidly emerging as an alternative platform for introducing targeted alterations in genomes. The CRISPR-based tools have been deployed for several other applications including gene expression studies, detection of mutation patterns in genomes, epigenetic regulation, chromatin imaging, etc. Unlike the traditional genetic engineering approaches, it is simple, cost-effective, and highly specific in inducing genetic variations. Despite its popularity, the technology has limitations such as off-targets, low mutagenesis efficiency, and its dependency on in-vitro regeneration protocols for the recovery of stable plant lines. Several other issues such as persisted CRISPR activity in subsequent generations, the potential for transferring to its wild type population, the risk of reversion of edited version to its original phenotype particularly in cross-pollinated plant species when released into the environment and the scarcity of validated targets have been overlooked. This article briefly highlights these undermined aspects, which may challenge the wider applications of this platform for improving crop genetics.

RevDate: 2019-07-18
CmpDate: 2019-07-18

Pinto DO, Scott TA, DeMarino C, et al (2019)

Effect of transcription inhibition and generation of suppressive viral non-coding RNAs.

Retrovirology, 16(1):13 pii:10.1186/s12977-019-0475-0.

BACKGROUND: HIV-1 patients receiving combination antiretroviral therapy (cART) survive infection but require life-long adherence at high expense. In chronic cART-treated patients with undetectable viral titers, cell-associated viral RNA is still detectable, pointing to low-level viral transcriptional leakiness. To date, there are no FDA-approved drugs against HIV-1 transcription. We have previously shown that F07#13, a third generation Tat peptide mimetic with competitive activity against Cdk9/T1-Tat binding sites, inhibits HIV-1 transcription in vitro and in vivo.

RESULTS: Here, we demonstrate that increasing concentrations of F07#13 (0.01, 0.1, 1 µM) cause a decrease in Tat levels in a dose-dependent manner by inhibiting the Cdk9/T1-Tat complex formation and subsequent ubiquitin-mediated Tat sequestration and degradation. Our data indicate that complexes I and IV contain distinct patterns of ubiquitinated Tat and that transcriptional inhibition induced by F07#13 causes an overall reduction in Tat levels. This reduction may be triggered by F07#13 but ultimately is mediated by TAR-gag viral RNAs that bind suppressive transcription factors (similar to 7SK, NRON, HOTAIR, and Xist lncRNAs) to enhance transcriptional gene silencing and latency. These RNAs complex with PRC2, Sin3A, and Cul4B, resulting in epigenetic modifications. Finally, we observed an F07#13-mediated decrease of viral burden by targeting the R region of the long terminal repeat (HIV-1 promoter region, LTR), promoting both paused polymerases and increased efficiency of CRISPR/Cas9 editing in infected cells. This implies that gene editing may be best performed under a repressed transcriptional state.

CONCLUSIONS: Collectively, our results indicate that F07#13, which can terminate RNA Polymerase II at distinct sites, can generate scaffold RNAs, which may assemble into specific sets of "RNA Machines" that contribute to gene regulation. It remains to be seen whether these effects can also be seen in various clades that have varying promoter strength, mutant LTRs, and in patient samples.

RevDate: 2019-07-18
CmpDate: 2019-07-18

Wu Z, Chen Z, Gao X, et al (2019)

Combination of ssDNA recombineering and CRISPR-Cas9 for Pseudomonas putida KT2440 genome editing.

Applied microbiology and biotechnology, 103(6):2783-2795.

Pseudomonas putida KT2440 is a Gram-negative, biosafety strain that plays important roles in environmental and biotechnological applications. Highly efficient genome editing strategy is essential to the elucidation of gene function and construction of metabolic engineered strains. Building on our previously established recombineering-mediated markerless and scarless P. putida KT2440 chromosomal gene deletion methods, herein we combined single-stranded DNA (ssDNA) recombineering and CRISPR-Cas9 technologies for P. putida KT2440 genome editing. Firstly, an inactive kanamycin resistance gene was knocked into the P. putida KT2440 chromosome. Then, based on kanamycin selection, recombinase gene selection, ssDNA recombineering condition optimization, and gRNA expression promoter selection were performed. A two-plasmid genome editing system was established; the first is a broad host range, RK2 replicon-based plasmid cloned with the tightly regulated redβ and cas9 genes; the second is a broad host range, pBBR1 replicon-based, sgRNA expression plasmid. Gene point mutations and gene deletions were carried out; the genome editing efficiency is as high as 100%. The method will expedite the P. putida KT2440 metabolic engineering and synthetic biology studies.

RevDate: 2019-07-18
CmpDate: 2019-07-18

Dolgin E (2019)

Genomic focus brings tea research to the boil.

Nature, 566(7742):S12-S13.

RevDate: 2019-07-18
CmpDate: 2019-07-18

Liang C, Zhao J, Lu J, et al (2019)

Development and Characterization of MDR1 (Mdr1a/b) CRISPR/Cas9 Knockout Rat Model.

Drug metabolism and disposition: the biological fate of chemicals, 47(2):71-79.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology is widely used as a tool for gene editing in rat genome site-specific engineering. Multidrug resistance 1 [MDR1 (also known as P-glycoprotein)] is a key efflux transporter that plays an important role not only in the transport of endogenous and exogenous substances, but also in tumor MDR. In this report, a novel MDR1 (Mdr1a/b) double-knockout (KO) rat model was generated by the CRISPR/Cas9 system without any off-target effect detected. Western blot results showed that MDR1 was completely absent in the liver, small intestine, brain, and kidney of KO rats. Further pharmacokinetic studies of digoxin, a typical substrate of MDR1, confirmed the deficiency of MDR1 in vivo. To determine the possible compensatory mechanism of Mdr1a/b (-/-) rats, the mRNA levels of the CYP3A subfamily and transporter-related genes were compared in the brain, liver, kidney, and small intestine of KO and wild-type rats. In general, a new Mdr1a/b (-/-) rat model has been successfully generated and characterized. This rat model is a useful tool for studying the function of MDR1 in drug absorption, tumor MDR, and drug target validation.

RevDate: 2019-07-18
CmpDate: 2019-07-18

He Z, Zhang T, Jiang L, et al (2018)

Use of CRISPR/Cas9 technology efficiently targetted goat myostatin through zygotes microinjection resulting in double-muscled phenotype in goats.

Bioscience reports, 38(6):.

Myostatin gene (MSTN) can inhibit the proliferation of myoblast, which in turn promotes muscle growth and inhibits adipocyte differentiation in livestock. MSTN mutation may lead to muscle hypertrophy or double-muscled (DM) phenotype. MSTN mutation animal, such as sheep, dog, and rabbit have been generated through CRISPR/Cas9 technology. However, goats with promising MSTN mutation have not been generated. We designed two sgRNAs loci targetting exon3 of MSTN gene to destroy the MSTN cysteines knots. We got seven goats from seven recipients, in which six were MSTN knocked-out (KO) goats, with a mutation rate of 85.7%. Destroyed cysteine knots caused MSTN structure inactivation. The average body weight gain (BWG) per day of MSTN KO goats was significantly higher than that of wild-type (WT) goats. MSTN KO goats showed abnormal sugar, fat, and protein metabolism compared with wild-type controls (MSTN+/+). Inheritance of mutations was observed in offspring of MSTN KO goats by PCR analysis.

RevDate: 2019-07-18
CmpDate: 2019-07-18

Tang D, Subramanian J, Haley B, et al (2019)

Pyruvate Kinase Muscle-1 Expression Appears to Drive Lactogenic Behavior in CHO Cell Lines, Triggering Lower Viability and Productivity: A Case Study.

Biotechnology journal, 14(4):e1800332.

Chinese hamster ovary (CHO) cell lines are used to express a variety of therapeutic proteins. However, lactogenic behavior displayed by some CHO cell lines during manufacturing processes may result in a decline in viability, productivity, and possible alterations in product quality. In cultured cells, lactate is produced during glycolysis through irreversible conversion of phosphoenolpyruvate to pyruvate and then lactate via sequential function of pyruvate kinase and lactate dehydrogenase (LDH) enzymes. In the process of cell line development (CLD), two lactogenic cell lines expressing different antibody molecules are identified. The lactogenic behaviors of these cell lines can be differentially mitigated through optimization of either nutrient feeds or culture pH, depending on the cell line. Analysis of various proteins involved in the glycolysis pathway reveal a direct correlation between the pyruvate kinase muscle-1 (PKM-1) isoform levels and lactogenic behavior. CRISPR mediated knockout of the PKM-1 isoform abolishes lactate accumulation even under lactogenic conditions. Furthermore, a cell line lacking expression of both PKM-1 and PKM-2 enzymes capable of maintaining productivity, viability, and growth without displaying lactogenic behavior is identified. Targeted deletion of PKM in CHO cells may be tolerated due to expression of PKL (liver) and PKR (red blood cell) isoforms of pyruvate kinase. All together, these findings suggest that PKM-1 up-regulation during antibody production could trigger lactogenic behavior and that this enzyme is dispensable for CHO cell survival.

RevDate: 2019-07-18
CmpDate: 2019-07-18

Luo YL, Xu CF, Li HJ, et al (2018)

Macrophage-Specific in Vivo Gene Editing Using Cationic Lipid-Assisted Polymeric Nanoparticles.

ACS nano, 12(2):994-1005.

The CRISPR/Cas9 gene editing technology holds promise for the treatment of multiple diseases. However, the inability to perform specific gene editing in targeted tissues and cells, which may cause off-target effects, is one of the critical bottlenecks for therapeutic application of CRISPR/Cas9. Herein, macrophage-specific promoter-driven Cas9 expression plasmids (pM458 and pM330) were constructed and encapsulated in cationic lipid-assisted PEG-b-PLGA nanoparticles (CLAN). The obtained nanoparticles encapsulating the CRISPR/Cas9 plasmids were able to specifically express Cas9 in macrophages as well as their precursor monocytes both in vitro and in vivo. More importantly, after further encoding a guide RNA targeting Ntn1 (sgNtn1) into the plasmid, the resultant CLANpM330/sgNtn1 successfully disrupted the Ntn1 gene in macrophages and their precursor monocytes in vivo, which reduced expression of netrin-1 (encoded by Ntn1) and subsequently improved type 2 diabetes (T2D) symptoms. Meanwhile, the Ntn1 gene was not disrupted in other cells due to specific expression of Cas9 by the CD68 promoter. This strategy provides alternative avenues for specific in vivo gene editing with the CRISPR/Cas9 system.

RevDate: 2019-07-18
CmpDate: 2019-07-18

Park J, S Bae (2018)

Cpf1-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cpf1.

Bioinformatics (Oxford, England), 34(6):1077-1079.

Summary: Following the type II CRISPR-Cas9 system, type V CRISPR-Cpf1 endonucleases have been found to be applicable for genome editing in various organisms in vivo. However, there are as yet no web-based tools capable of optimally selecting guide RNAs (gRNAs) among all possible genome-wide target sites. Here, we present Cpf1-Database, a genome-wide gRNA library design tool for LbCpf1 and AsCpf1, which have DNA recognition sequences of 5'-TTTN-3' at the 5' ends of target sites. Cpf1-Database provides a sophisticated but simple way to design gRNAs for AsCpf1 nucleases on the genome scale. One can easily access the data using a straightforward web interface, and using the powerful collections feature one can easily design gRNAs for thousands of genes in short time.

Free access at http://www.rgenome.net/cpf1-database/.

Contact: sangsubae@hanyang.ac.kr.

RevDate: 2019-07-17
CmpDate: 2019-07-17

Zhang Y, Feng J, Wang P, et al (2019)

CRISPR/Cas9-mediated efficient genome editing via protoplast-based transformation in yeast-like fungus Aureobasidium pullulans.

Gene, 709:8-16.

Aureobasidium pullulans, a yeast-like fungus with strong environmental adaptability, remains a potential host for bio-production of different valuable metabolites. However, its potential application is limited by low-efficient genetic manipulation. In this study, CRISPR/Cas9-mediated genome editing via protoplast-based transformation system was developed. To test CRISPR/Cas9 mediated genomic mutagenesis, the orotidine 5-phosphate decarboxylase (umps) gene was used as a counter-selectable selection marker. By co-transforming of two plasmids harboring cas9 gene and a guide RNA targeting umps, respectively, the CRISPR/Cas9 system could significantly increase frequency of mutation in the targeting site of guide RNA. To further validate that CRISPR/Cas9 stimulated homologous recombination with donor DNA, a color reporter system of beta-glucuronidase (gus) gene was developed for calculating positive mutation rate. The results showed that positive mutation rate with CRISPR/Cas9 system was ~40% significantly higher than only with the donor DNA (~4%). Furthermore, the different posttranscriptional RNA processing schemes were analyzed by compared the effects of flanking gRNA with self-cleaving ribozymes or tRNA. The result demonstrated that gRNA processed by self-cleaving ribozymes achieves higher positive mutant rate. This study provided foundation for a simple and powerful genome editing tool for A. pullulans. Moreover, a counter-selectable selection marker (umps) and a color reporter system (gus) were being developed as genetic parts for strain engineering.

RevDate: 2019-07-17
CmpDate: 2019-07-17

Klimke A, Güttler S, Kuballa P, et al (2019)

Use of CRISPR/Cas9 for the Modification of the Mouse Genome.

Methods in molecular biology (Clifton, N.J.), 1953:213-230.

The use of CRISPR/Cas9 to modify the mouse genome has gained immense interest in the past few years since it allows the direct modification of embryos, bypassing the need of labor-intensive procedures for the manipulation of embryonic stem cells. By shortening the overall timelines and reducing the costs for the generation of new genetically modified mouse lines (Li et al., Nat Biotechnol 31: 681-683, 2013), this technology has rapidly become a major tool for in vivo drug discovery applications.

RevDate: 2019-07-17
CmpDate: 2019-07-17

Horn M, Metge F, MS Denzel (2019)

Unbiased Forward Genetic Screening with Chemical Mutagenesis to Uncover Drug-Target Interactions.

Methods in molecular biology (Clifton, N.J.), 1953:23-31.

The steadily increasing throughput in next-generation sequencing technologies is revolutionizing a number of fields in biology. One application requiring massive parallel sequencing is forward genetic screening based on chemical mutagenesis. Such screens interrogate the entire genome in an entirely unbiased fashion and can be applied to a number of research questions. CRISPR/Cas9-based screens, which are largely limited to a gene's loss of function, have already been very successful in identifying drug targets and pathways related to the drug's mode of action. By inducing single nucleotide changes using an alkylating reagent, it is possible to generate amino acid changes that perturb the interaction between a drug and its direct target, resulting in drug resistance. This chemogenomic approach combined with latest sequencing technologies allows deconvolution of drug targets and characterization of drug-target binding interfaces at amino acid resolution, therefore nicely complementing existing biochemical approaches. Here we describe a general protocol for a chemical mutagenesis-based forward genetic screen applicable for drug-target deconvolution.

RevDate: 2019-07-17
CmpDate: 2019-07-17

Zhao S, Xing Z, Liu Z, et al (2019)

Efficient somatic and germline genome engineering of Bactrocera dorsalis by the CRISPR/Cas9 system.

Pest management science, 75(7):1921-1932.

BACKGROUND: Bactrocera dorsalis (Hendel), a very destructive insect pest of many fruits and vegetables, is widespread in many Asian countries. To facilitate control of this pest, it is essential to investigate its genetics and gene function using targeted gene disruption.

RESULTS: Here, we describe successful targeted mutagenesis of the white and transformer genes in B. dorsalis through use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. Co-injection of the white sgRNA and Cas9 mRNA into B. dorsalis embryos caused eye color change, and the white mutations in the germline were heritable. CRISPR-mediated knockout of the sex determination gene transformer (tra) in B. dorsalis resulted in a male-biased sex ratio and adult flies with abnormal outer and interior reproductive organs. Small indels and substitutions were induced by CRIRPR for both genes.

CONCLUSION: Our data demonstrate that somatic and germline genome engineering of the pest B. dorsalis can be performed efficiently using the CRISPR/Cas9 system, opening the door to the use of the CRISPR-mediated method for functional annotations of genes in B. dorsalis and for its population control using, for example, such as gene drive. © 2018 Society of Chemical Industry.

RevDate: 2019-07-17
CmpDate: 2019-07-17

Billker O (2018)

CRISPRing the Elephant in the Room.

Cell host & microbe, 24(6):754-755.

The importance of guanylyl-cyclases (GCs) in apicomplexa has remained elusive due to the large size of the genes. Two recent studies, including Brown and Sibley, 2018 in this issue of Cell Host & Microbe, make elegant use of genome editing with CRISPR-Cas9 to characterize roles of GCs in Toxoplasma and Plasmodium.

RevDate: 2019-07-17
CmpDate: 2019-07-17

Bressan RB, SM Pollard (2018)

Genome Editing in Human Neural Stem and Progenitor Cells.

Results and problems in cell differentiation, 66:163-182.

Experimental tools for precise manipulation of mammalian genomes enable reverse genetic approaches to explore biology and disease. Powerful genome editing technologies built upon designer nucleases, such as CRISPR/Cas9, have recently emerged. Parallel progress has been made in methodologies for the expansion and differentiation of human pluripotent and tissue stem cells. Together these innovations provide a remarkable new toolbox for human cellular genetics and are opening up vast opportunities for discoveries and applications across the breadth of life sciences research. In this chapter, we review the emergence of genome editing technologies and how these are being deployed in studies of human neurobiology, neurological disease, and neuro-oncology. We focus our discussion on CRISPR/Cas9 and its application in studies of human neural stem and progenitor cells.

RevDate: 2019-07-17
CmpDate: 2019-07-17

Wang P, Liu Z, Zhang X, et al (2018)

CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells.

Protein & cell, 9(11):945-965.

Vascular cell functionality is critical to blood vessel homeostasis. Constitutive NF-κB activation in vascular cells results in chronic vascular inflammation, leading to various cardiovascular diseases. However, how NF-κB regulates human blood vessel homeostasis remains largely elusive. Here, using CRISPR/Cas9-mediated gene editing, we generated RelA knockout human embryonic stem cells (hESCs) and differentiated them into various vascular cell derivatives to study how NF-κB modulates human vascular cells under basal and inflammatory conditions. Multi-dimensional phenotypic assessments and transcriptomic analyses revealed that RelA deficiency affected vascular cells via modulating inflammation, survival, vasculogenesis, cell differentiation and extracellular matrix organization in a cell type-specific manner under basal condition, and that RelA protected vascular cells against apoptosis and modulated vascular inflammatory response upon tumor necrosis factor α (TNFα) stimulation. Lastly, further evaluation of gene expression patterns in IκBα knockout vascular cells demonstrated that IκBα acted largely independent of RelA signaling. Taken together, our data reveal a protective role of NF-κB/RelA in modulating human blood vessel homeostasis and map the human vascular transcriptomic landscapes for the discovery of novel therapeutic targets.

RevDate: 2019-07-17
CmpDate: 2019-07-17

Thielen AJF, van Baarsen IM, Jongsma ML, et al (2018)

CRISPR/Cas9 generated human CD46, CD55 and CD59 knockout cell lines as a tool for complement research.

Journal of immunological methods, 456:15-22.

BACKGROUND: To prevent unwanted complement activation and subsequent damage, complement activation must be tightly regulated on healthy host cells. Dysregulation of the complement system contributes to the pathology of diseases like Paroxysmal Nocturnal Hemoglobinuria and atypical Hemolytic Uremic Syndrome. To investigate complement regulator deficiencies, primary patient cells may be used, but access to patient cells may be limited and cells are heterogeneous between different patients. To inhibit regulator function on healthy host cells, blocking antibodies can be used, though it may be difficult to exclude antibody-mediated effects. To circumvent these issues, we created single and combined complement regulator human knockout cells to be able to in vitro investigate complement activation and regulation on human cells.

METHODS: CRISPR/Cas9 was used to knockout (KO) complement regulatory proteins CD46, CD55 and/or CD59 in human HAP1 cells. Single cell derived cell lines were profiled by Sanger sequencing and flow cytometry. To confirm the lack of complement regulatory function, the cells were exposed to complement in normal human serum and subsequently C3 and C4 deposition on the cell surface were detected by using flow cytometry.

RESULTS: We created single KO cell lines that completely lacked CD46, CD55 or CD59. We additionally generated double CD46/CD55, CD46/CD59 and CD55/CD59 KOs and triple CD46/CD55/CD59 KOs. Upon classical pathway activation, deletion of CD46 resulted in increased C3 and C4 deposition, while deleting CD55 mainly resulted to increased C3 deposition, confirming their reported function in complement regulation. Upon alternative pathway activation, C3 deposition was only observed on the triple CD46/CD55/CD59 KO cells and not on any of the other cell lines, suggesting that human cells are resistant to spontaneous complement activation and suggesting a role for CD59 in C3 regulation.

CONCLUSIONS: The generation of complement regulator KO cell lines provides a relevant tool for future in vitro investigations of complement activation and regulation on human cells. Furthermore, these cell lines may also be helpful to evaluate therapeutic complement inhibitors and may shed light on novel roles of complement regulatory proteins as we here observed for CD59.

RevDate: 2019-07-17
CmpDate: 2019-07-17

Zhao H, Tu Z, Xu H, et al (2017)

Altered neurogenesis and disrupted expression of synaptic proteins in prefrontal cortex of SHANK3-deficient non-human primate.

Cell research, 27(10):1293-1297.

RevDate: 2019-07-16

Ethiraj P, Sambandam Y, Hathaway-Schrader JD, et al (2019)

RANKL triggers resistance to TRAIL-induced cell death in oral squamous cell carcinoma.

Journal of cellular physiology [Epub ahead of print].

Oral squamous cell carcinoma (OSCC) occurs as a malignancy of the oral cavity. RANK ligand (RANKL) is essential for osteoclast formation/bone resorption. Recently, we showed autoregulation of receptor activator of nuclear factor-κB ligand (RANKL) stimulates OSCC cell proliferation. OSCC cells show resistance to tumor necrosis factor related apoptosis inducing ligand (TRAIL) treatment. Therefore, we hypothesize that RANKL promotes resistance for TRAIL induction of OSCC apoptotic cell death. In this study, SCC14A and SCC74A cells cultured with TRAIL revealed high-level expression of RANKL which increased resistance to TRAIL inhibition of tumor cell proliferation. RANKL stimulation inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling positive staining in TRAIL-treated cells. CRISPR/Cas-9 knockout of RANKL (RANKL-KO) increased caspase-9, caspase-3 activity and cytochrome c release in OSCC cells. RANKL inhibited proapoptotic proteins BAD and BAX expression. TRAIL treatment suppressed the SQSTM1/p62 and RANKL restored the expression. Interestingly, RANKL alone significantly increased proteasome activity. RANKL-KO in OSCC cells inhibited autophagic activity as evidenced by decreased light chain 3B-II and beclin-1 expression. Thus, RANKL stimulation of OSCC tumor cells triggered resistance for TRAIL-induced OSCC cell death. Taken together, blockade of RANKL may inhibit OSCC tumor progression and enhance the potential of TRAIL induced OSCC tumor cell apoptosis.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Jiang M, Hu H, Kai J, et al (2019)

Different knockout genotypes of OsIAA23 in rice using CRISPR/Cas9 generating different phenotypes.

Plant molecular biology, 100(4-5):467-479.

KEY MESSAGE: We have isolated several Osiaa23 rice mutants with different knockout genotypes, resulting in different phenotypes, which suggested that different genetic backgrounds or mutation types influence gene function. The Auxin/Indole-3-Acetic Acid (Aux/IAA) gene family performs critical roles in auxin signal transduction in plants. In rice, the gene OsIAA23 (Os06t0597000) is known to affect development of roots and shoots, but previous knockouts in OsIAA23 have been sterile and difficult for research continuously. Here, we isolate new Osiaa23 mutants using the CRISPR/Cas9 system in japonica (Wuyunjing24) and indica (Kasalath) rice, with extensive genome re-sequencing to confirm the absence of off-target effects. In Kasalath, mutants with a 13-amino acid deletion showed profoundly greater dwarfing, lateral root developmental disorder, and fertility deficiency, relative to mutants with a single amino acid deletion, demonstrating that those 13 amino acids in Kasalath are essential to gene function. In Wuyunjing24, we predicted that mutants with a single base-pair frameshift insertion would experience premature termination and strong phenotypic defects, but instead these lines exhibited negligible phenotypic difference and normal fertility. Through RNA-seq, we show here that new mosaic transcripts of OsIAA23 were produced de novo, which circumvented the premature termination and thereby preserved the wild-type phenotype. This finding is a notable demonstration in plants that mutants can mask loss of function CRISPR/Cas9 editing of the target gene through de novo changes in alternative splicing.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Wrighton KH (2019)

Cytosine base editors go off-target.

Nature reviews. Genetics, 20(5):254-255.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Yu W, Z Wu (2019)

Use of AAV Vectors for CRISPR-Mediated In Vivo Genome Editing in the Retina.

Methods in molecular biology (Clifton, N.J.), 1950:123-139.

Degenerative retinal diseases such as retinitis pigmentosa (RP) and Leber's congenital amaurosis (LCA) may lead to blindness without effective treatment. With the rapid advancement of the CRISPR/Cas9 genome editing technology, in vivo application of CRISPR/Cas9 holds immense potential for treatment of these diseases. Adeno-associated virus (AAV) vectors are an ideal gene transfer tool for delivery of CRISPR components to the retina. Here, we describe a protocol for utilizing an AAV-based CRISPR/Cas9 system for in vivo genome editing in the retina.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Nomura W, Matsumoto D, Sugii T, et al (2018)

Efficient and Orthogonal Transcription Regulation by Chemically Inducible Artificial Transcription Factors.

Biochemistry, 57(45):6452-6459.

The DNA-binding specificity of genome editing tools can be applied to gene regulation. Recently, multiple artificial transcription factors (ATFs) were shown to synergistically and efficiently regulate gene expression. Chemically triggered protein associations are useful for functional regulation at specific timings. A combination of several inducible protein association systems could enable the regulation of multiple genes at different loci with independent timing. We applied the FKBP-rapamycin-FRB and GAI-gibberellin-GID systems for gene regulation using multiple TALEs and dCas9. By the combined use of currently available systems, reporter gene assays were performed; the results indicated that gene expression was regulated by rapamycin or gibberellin in the presence of the FRB or GAI effector domains, respectively. Furthermore, the activation of endogenous genes was differentially regulated by the system. This success suggests the usability of the chemically inducible multiple ATFs for the time-dependent regulation of multiple genes, such as the case for cellular phenomena that are dependent on the programmable timing of expression and the differential expression of multiple genes.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Rees HA, DR Liu (2018)

Base editing: precision chemistry on the genome and transcriptome of living cells.

Nature reviews. Genetics, 19(12):770-788.

RNA-guided programmable nucleases from CRISPR systems generate precise breaks in DNA or RNA at specified positions. In cells, this activity can lead to changes in DNA sequence or RNA transcript abundance. Base editing is a newer genome-editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks. DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors achieve analogous changes using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing by-products. In this Review, we summarize base-editing strategies to generate specific and precise point mutations in genomic DNA and RNA, highlight recent developments that expand the scope, specificity, precision and in vivo delivery of base editors and discuss limitations and future directions of base editing for research and therapeutic applications.

RevDate: 2019-07-16
CmpDate: 2019-07-16

George L, Indig FE, Abdelmohsen K, et al (2018)

Intracellular RNA-tracking methods.

Open biology, 8(10):.

RNA tracking allows researchers to visualize RNA molecules in cells and tissues, providing important spatio-temporal information regarding RNA dynamics and function. Methods such as fluorescent in situ hybridization (FISH) and molecular beacons rely on complementary oligonucleotides to label and view endogenous transcripts. Other methods create artificial chimeric transcripts coupled with bacteriophage-derived coat proteins (e.g. MS2, λN) to tag molecules in live cells. In other approaches, endogenous RNAs are recognized by complementary RNAs complexed with noncatalytic Cas proteins. Each technique has its own set of strengths and limitations that must be considered when planning an experiment. Here, we discuss the mechanisms, advantages, and weaknesses of in situ hybridization, molecular beacons, MS2 tagging and Cas-derived systems, as well as how RNA tracking can be employed to study various aspects of molecular biology.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Winters IP, Murray CW, MM Winslow (2018)

Towards quantitative and multiplexed in vivo functional cancer genomics.

Nature reviews. Genetics, 19(12):741-755.

Large-scale sequencing of human tumours has uncovered a vast array of genomic alterations. Genetically engineered mouse models recapitulate many features of human cancer and have been instrumental in assigning biological meaning to specific cancer-associated alterations. However, their time, cost and labour-intensive nature limits their broad utility; thus, the functional importance of the majority of genomic aberrations in cancer remains unknown. Recent advances have accelerated the functional interrogation of cancer-associated alterations within in vivo models. Specifically, the past few years have seen the emergence of CRISPR-Cas9-based strategies to rapidly generate increasingly complex somatic alterations and the development of multiplexed and quantitative approaches to ascertain gene function in vivo.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Sheth RU, HH Wang (2018)

DNA-based memory devices for recording cellular events.

Nature reviews. Genetics, 19(11):718-732.

Measuring biological data across time and space is critical for understanding complex biological processes and for various biosurveillance applications. However, such data are often inaccessible or difficult to directly obtain. Less invasive, more robust and higher-throughput biological recording tools are needed to profile cells and their environments. DNA-based cellular recording is an emerging and powerful framework for tracking intracellular and extracellular biological events over time across living cells and populations. Here, we review and assess DNA recorders that utilize CRISPR nucleases, integrases and base-editing strategies, as well as recombinase and polymerase-based methods. Quantitative characterization, modelling and evaluation of these DNA-recording modalities can guide their design and implementation for specific application areas.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Gavilan MP, Gandolfo P, Balestra FR, et al (2018)

The dual role of the centrosome in organizing the microtubule network in interphase.

EMBO reports, 19(11):.

Here, we address the regulation of microtubule nucleation during interphase by genetically ablating one, or two, of three major mammalian γ-TuRC-binding factors namely pericentrin, CDK5Rap2, and AKAP450. Unexpectedly, we find that while all of them participate in microtubule nucleation at the Golgi apparatus, they only modestly contribute at the centrosome where CEP192 has a more predominant function. We also show that inhibiting microtubule nucleation at the Golgi does not affect centrosomal activity, whereas manipulating the number of centrosomes with centrinone modifies microtubule nucleation activity of the Golgi apparatus. In centrosome-free cells, inhibition of Golgi-based microtubule nucleation triggers pericentrin-dependent formation of cytoplasmic-nucleating structures. Further depletion of pericentrin under these conditions leads to the generation of individual microtubules in a γ-tubulin-dependent manner. In all cases, a conspicuous MT network forms. Strikingly, centrosome loss increases microtubule number independently of where they were growing from. Our results lead to an unexpected view of the interphase centrosome that would control microtubule network organization not only by nucleating microtubules, but also by modulating the activity of alternative microtubule-organizing centers.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Doench JG (2018)

Am I ready for CRISPR? A user's guide to genetic screens.

Nature reviews. Genetics, 19(2):67-80.

Exciting new technologies are often self-limiting in their rollout, as access to state-of-the-art instrumentation or the need for years of hands-on experience, for better or worse, ensures slow adoption by the community. CRISPR technology, however, presents the opposite dilemma, where the simplicity of the system enabled the parallel development of many applications, improvements and derivatives, and new users are now presented with an almost paralyzing abundance of choices. This Review intends to guide users through the process of applying CRISPR technology to their biological problems of interest, especially in the context of discovering gene function at scale.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Guzzardo PM, Rashkova C, Dos Santos RL, et al (2017)

A small cassette enables conditional gene inactivation by CRISPR/Cas9.

Scientific reports, 7(1):16770.

The availability of CRISPR/Cas9 technology has enabled the rapid establishment of gene knockouts in many cell types and even whole organisms. However, conditional inactivation of essential genes remains a challenge. We devised an approach named DECAI (DEgradation based on Cre-regulated- Artificial Intron). It utilizes a small cassette of just 201 nucleotides that is inserted into the coding exon of a target gene using CRISPR/Cas9 technology and homology-directed repair. As its sequence is derived from an artificial intron, the cassette is removed by the splicing machinery and thus leaves no trace in the "off-state". Upon activation with Cre recombinase ("on-state"), the intron is crippled and the target gene is disrupted by a series of stop codons. We exemplify the utility of this approach on several non-essential and essential human genes. Clones bearing the conditional knockout cassette are recovered at frequencies above 5% and cassette function can be traced at the genomic DNA and the mRNA level. Importantly, cassette activation leads to loss of gene expression as judged by flow cytometry, Western blot or immunofluorescence. Altogether, this highlights the broad utility of the approach for conditional gene inactivation and suggests that this tool could be used to study the loss-of-function phenotypes of essential genes.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Nagy G, Szebenyi C, Csernetics Á, et al (2017)

Development of a plasmid free CRISPR-Cas9 system for the genetic modification of Mucor circinelloides.

Scientific reports, 7(1):16800.

Mucor circinelloides and other members of Mucorales are filamentous fungi, widely used as model organisms in basic and applied studies. Although genetic manipulation methods have been described for some Mucoral fungi, construction of stable integrative transformants by homologous recombination has remained a great challenge in these organisms. In the present study, a plasmid free CRISPR-Cas9 system was firstly developed for the genetic modification of a Mucoral fungus. The described method offers a rapid but robust tool to obtain mitotically stable mutants of M. circinelloides via targeted integration of the desired DNA. It does not require plasmid construction and its expression in the recipient organism. Instead, it involves the direct introduction of the guide RNA and the Cas9 enzyme and, in case of homology directed repair (HDR), the template DNA into the recipient strain. Efficiency of the method for non-homologous end joining (NHEJ) and HDR was tested by disrupting two different genes, i.e. carB encoding phytoene dehydrogenase and hmgR2 encoding 3-hydroxy-3-methylglutaryl-CoA reductase, of M. circinelloides. Both NHEJ and HDR resulted in stable gene disruption mutants. While NHEJ caused extensive deletions upstream from the protospacer adjacent motif, HDR assured the integration of the deletion cassette at the targeted site.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Oesterle S, Gerngross D, Schmitt S, et al (2017)

Efficient engineering of chromosomal ribosome binding site libraries in mismatch repair proficient Escherichia coli.

Scientific reports, 7(1):12327.

Multiplexed gene expression optimization via modulation of gene translation efficiency through ribosome binding site (RBS) engineering is a valuable approach for optimizing artificial properties in bacteria, ranging from genetic circuits to production pathways. Established algorithms design smart RBS-libraries based on a single partially-degenerate sequence that efficiently samples the entire space of translation initiation rates. However, the sequence space that is accessible when integrating the library by CRISPR/Cas9-based genome editing is severely restricted by DNA mismatch repair (MMR) systems. MMR efficiency depends on the type and length of the mismatch and thus effectively removes potential library members from the pool. Rather than working in MMR-deficient strains, which accumulate off-target mutations, or depending on temporary MMR inactivation, which requires additional steps, we eliminate this limitation by developing a pre-selection rule of genome-library-optimized-sequences (GLOS) that enables introducing large functional diversity into MMR-proficient strains with sequences that are no longer subject to MMR-processing. We implement several GLOS-libraries in Escherichia coli and show that GLOS-libraries indeed retain diversity during genome editing and that such libraries can be used in complex genome editing operations such as concomitant deletions. We argue that this approach allows for stable and efficient fine tuning of chromosomal functions with minimal effort.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Martínez-Martínez E, Ibarrola J, Fernández-Celis A, et al (2017)

Differential Proteomics Identifies Reticulocalbin-3 as a Novel Negative Mediator of Collagen Production in Human Cardiac Fibroblasts.

Scientific reports, 7(1):12192.

Cardiac fibrosis is characterized by an excessive accumulation of extracellular matrix components, including collagens. Galectin-3 (Gal-3) and Cardiotrophin-1 (CT-1) are two profibrotic molecules that mediate Aldosterone (Aldo)-induced cardiac fibrosis. However the underlying mechanisms are not well defined. Our aim is to characterize changes in the proteome of human cardiac fibroblasts treated with Aldo, Gal-3 or CT-1 to identify new common proteins that might be new therapeutic targets in cardiac fibrosis. Using a quantitative proteomic approach in human cardiac fibroblasts, our results show that Aldo, Gal-3 and CT-1 modified the expression of 30, 17 and 89 proteins respectively, being common the reticulocalbin (RCN) family members. RCN-3 down-regulation triggered by Aldo, Gal-3 and CT-1 was verified. Treatment with recombinant RCN-3 decreased collagens expression in human cardiac fibroblasts through Akt phosphorylation. Interestingly, CRISPR/Cas9-mediated activation of RCN-3 decreased collagen production in human cardiac fibroblasts. In addition, recombinant RCN-3 blocked the profibrotic effects of Aldo, Gal-3 and CT-1. Interestingly, RCN-3 blunted the increase in collagens expression induced by other profibrotic stimuli, angiotensin II, in human cardiac fibroblasts. Our results suggest that RCN-3 emerges as a new potential negative regulator of collagen production and could represent a therapeutic target in the context of cardiac fibrosis.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Song J, Yang D, Ruan J, et al (2017)

Production of immunodeficient rabbits by multiplex embryo transfer and multiplex gene targeting.

Scientific reports, 7(1):12202.

Immunodeficient mice have been used predominantly in biomedical research. Realizing that large animal species may have an enhanced ability to predict clinical outcome relative to mice, we worked to develop immunodeficient rabbits by CRISPR/Cas9. We first demonstrated that multiplex embryo transfer efficiently produced multiple lines of single-gene mutant (SGM) founders. Embryos microinjected with single sgRNA targeting FOXN1, RAG2, IL2RG or PRKDC were pooled for embryo transfer. As few as three recipients were used to produce twenty SGM founders for four genes. We then demonstrated the powerful multiplex targeting capacity of CRISPR/Cas9. First, two genes on the same chromosome were targeted simultaneously, resulting in three RAG1/RAG2 double-gene mutant (DGM) founders. Next we microinjected forty-five embryos each with five sgRNAs targeting FOXN1, RAG1, RAG2, IL2RG and PRKDC, and transferred them to two recipients. Five founders were produced: one SGM, two DGM, one triple-gene mutant and one quadruple-gene mutant. The present work demonstrates that multiplex embryo transfer and multiplex gene targeting can be used to quickly and efficiently generate mutant rabbit founders. Four lines of SGM (e.g. FOXN1, RAG2, IL2RG, and PRKDC) immunodeficient rabbits, as well as multigenic mutant immunodeficient rabbits have been produced. These animals may prove useful for biomedical research.

RevDate: 2019-07-16
CmpDate: 2019-07-16

Burgess DJ (2017)

Genetic engineering: CREATE-ing genome-wide designed mutations.

Nature reviews. Genetics, 18(2):69.

RevDate: 2019-07-15

Krimsky S (2019)

Breaking the Germline Barrier in a Moral Vacuum.

Accountability in research [Epub ahead of print].

In November 2018 a Chinese scientist claimed to have used CRISPR/Cas 9 technology to genetically modify two human embryos that were then gestated in one adult woman through an IVF pregnancy and brought to term. The twin girls are allegedly the first babies born with their prenatal genomes edited. Using both English language and Chinese supporting documents, this paper discusses the background of this human experiment, the social context of Chinese science, and the alleged ethical transgressions of its principal scientist.

RevDate: 2019-07-15

Chen T, I Olsen (2019)

Porphyromonas gingivalis and its CRISPR-Cas system.

Journal of oral microbiology, 11(1):1638196 pii:1638196.

The clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated proteins (Cas) are immune systems in prokaryotes present in most Bacteria and Archaea. They provide adaptive immunity against foreign elements such as bacteriophages/viruses, plasmids and transposons. During immunization a small sequence of foreign DNA, a so-called spacer is integrated into the CRISPR locus in the host cell. Spacers are then transcribed into small RNA guides that direct cleavage of foreign DNA by Cas nucleases. Immunization through spacer acquisition is transferred vertically to the progeny. It is possible that this genetic immune system of bacteria participates in modulating the microbiome of 'chronic' periodontitis, in which Porphyromonas gingivalis has been identified as a keystone pathogen causing microbial dysbiosis. An in-depth review of our current knowledge on the CRISPR-Cas systems in P. gingivalis is given in this paper with the attempt to understand how this anaerobic bacterium may protect itself in the periodontal pocket where bacteriophages are abundant and even out-number bacteria.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Azvolinsky A (2019)

Molecular scissors cut in on stem cells.

Nature medicine, 25(6):864-866.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Rollins MF, Chowdhury S, Carter J, et al (2019)

Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry.

Molecular cell, 74(1):132-142.e5.

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.

RevDate: 2019-07-15
CmpDate: 2019-07-15

King MW, J Munger (2019)

Editing the human cytomegalovirus genome with the CRISPR/Cas9 system.

Virology, 529:186-194.

Human Cytomegalovirus (HCMV) is an opportunistic pathogen that causes substantial disease in neonates and immunocompromised individuals. Reverse genetic analysis of the HCMV genome is a powerful tool to dissect the roles that various viral genes play during infection. However, genetic engineering of HCMV is hampered by both the large size of the HCMV genome and HCMV's slow replication cycle. Currently, most laboratories that genetically engineer HCMV employ Bacterial Artificial Chromosome (BAC) mediated recombineering, which is a relatively lengthy process. We explored an alternative method of producing recombinant HCMV using the CRISPR/Cas9 system. We employed both homologous recombination (HR) and Non-homologous end-joining (NHEJ)-based methods, and find that each approach is capable of efficiently mutating the HCMV genome, with optimal efficiencies of 42% and 81% respectively. Our results suggest that CRISPR-mediated genomic engineering of HCMV is competitive with BAC-mediated recombineering and provide a framework for using CRISPR/Cas9 for mutational analysis of the HCMV genome.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Patil A, Manzano M, E Gottwein (2018)

CK1α and IRF4 are essential and independent effectors of immunomodulatory drugs in primary effusion lymphoma.

Blood, 132(6):577-586.

Primary effusion lymphoma (PEL) is an aggressive cancer with few treatment options. The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide have recently been shown to kill PEL cell lines, and lenalidomide is in clinical trials against PEL. IMiDs bind to the CRL4CRBN E3 ubiquitin ligase complex, leading to the acquisition of the Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3), casein kinase 1 α (CK1α), and zinc finger protein 91 (ZFP91) as neosubstrates. IMiDs are effective against multiple myeloma because of degradation of IKZF1 and IKZF3 and the consequent loss of interferon regulatory factor 4 (IRF4) and MYC expression. Lenalidomide is also effective in chromosome 5q deletion-associated myelodysplastic syndrome as a result of degradation of CK1α. An essential IKZF1-IRF4-MYC axis has recently been proposed to underlie the toxicity of IMiDs in PEL. Here, we further investigate IMiD effectors in PEL cell lines, based on genome-wide CRISPR/Cas9 screens for essential human genes. These screens and extensive validation experiments show that, of the 4 neosubstrates, only CK1α is essential for the survival of PEL cell lines. In contrast, IKZF1 and IKZF3 are dispensable, individually or in combination. IRF4 was critical in all 8 PEL cell lines tested, and surprisingly, IMiDs triggered downregulation of IRF4 expression independently of both IKZF1 and IKZF3. Reexpression of CK1α and/or IRF4 partially rescued PEL cell lines from IMiD-mediated toxicity. In conclusion, IMiD toxicity in PEL cell lines is independent of IKZF1 and IKZF3 but proceeds through degradation of the neosubstrate CK1α and downregulation of IRF4.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Kahn JD, Miller PG, Silver AJ, et al (2018)

PPM1D-truncating mutations confer resistance to chemotherapy and sensitivity to PPM1D inhibition in hematopoietic cells.

Blood, 132(11):1095-1105.

Truncating mutations in the terminal exon of protein phosphatase Mg2+/Mn2+ 1D (PPM1D) have been identified in clonal hematopoiesis and myeloid neoplasms, with a striking enrichment in patients previously exposed to chemotherapy. In this study, we demonstrate that truncating PPM1D mutations confer a chemoresistance phenotype, resulting in the selective expansion of PPM1D-mutant hematopoietic cells in the presence of chemotherapy in vitro and in vivo. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease mutational profiling of PPM1D in the presence of chemotherapy selected for the same exon 6 mutations identified in patient samples. These exon 6 mutations encode for a truncated protein that displays elevated expression and activity due to loss of a C-terminal degradation domain. Global phosphoproteomic profiling revealed altered phosphorylation of target proteins in the presence of the mutation, highlighting multiple pathways including the DNA damage response (DDR). In the presence of chemotherapy, PPM1D-mutant cells have an abrogated DDR resulting in altered cell cycle progression, decreased apoptosis, and reduced mitochondrial priming. We demonstrate that treatment with an allosteric, small molecule inhibitor of PPM1D reverts the phosphoproteomic, DDR, apoptotic, and mitochondrial priming changes observed in PPM1D-mutant cells. Finally, we show that the inhibitor preferentially kills PPM1D-mutant cells, sensitizes the cells to chemotherapy, and reverses the chemoresistance phenotype. These results provide an explanation for the enrichment of truncating PPM1D mutations in the blood of patients exposed to chemotherapy and in therapy-related myeloid neoplasms, and demonstrate that PPM1D can be a targeted in the prevention of clonal expansion of PPM1D-mutant cells and the treatment of PPM1D-mutant disease.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Leibman-Markus M, Pizarro L, Schuster S, et al (2018)

The intracellular nucleotide-binding leucine-rich repeat receptor (SlNRC4a) enhances immune signalling elicited by extracellular perception.

Plant, cell & environment, 41(10):2313-2327.

Plant recognition and defence against pathogens employs a two-tiered perception system. Surface-localized pattern recognition receptors (PRRs) act to recognize microbial features, whereas intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) directly or indirectly recognize pathogen effectors inside host cells. Employing the tomato PRR LeEIX2/EIX model system, we explored the molecular mechanism of signalling pathways. We identified an NLR that can associate with LeEIX2, termed SlNRC4a (NB-LRR required for hypersensitive response-associated cell death-4). Co-immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR-mediated responses. SlNRC4a overexpression enhances defence responses, whereas silencing SlNRC4 reduces plant immunity. Moreover, the coiled-coil domain of SlNRC4a is able to associate with LeEIX2 and is sufficient to enhance responses upon EIX perception. On the basis of these findings, we propose that SlNRC4a acts as a noncanonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perceptions.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Li C, Psatha N, Sova P, et al (2018)

Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing.

Blood, 131(26):2915-2928.

Disorders involving β-globin gene mutations, primarily β-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes CRISPR/Cas9-mediated genome editing approaches in adult CD34+ cells aimed toward the reactivation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34+ cells have limitations in assessing γ-globin reactivation, we focused on human β-globin locus-transgenic (β-YAC) mice. We used a helper-dependent human CD46-targeting adenovirus vector expressing CRISPR/Cas9 (HDAd-HBG-CRISPR) to disrupt a repressor binding region within the γ-globin promoter. We transduced HSPCs from β-YAC/human CD46-transgenic mice ex vivo and subsequently transplanted them into irradiated recipients. Furthermore, we used an in vivo HSPC transduction approach that involves HSPC mobilization and the intravenous injection of HDAd-HBG-CRISPR into β-YAC/CD46-transgenic mice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human β- to γ-globin expression in red blood cells of adult mice that was maintained after secondary transplantation of HSPCs. In long-term follow-up studies, we did not detect hematological abnormalities, indicating that HBG promoter editing does not negatively affect hematopoiesis. This is the first study that shows successful in vivo HSPC genome editing by CRISPR/Cas9.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Luo H, Wang F, Zha J, et al (2018)

CTCF boundary remodels chromatin domain and drives aberrant HOX gene transcription in acute myeloid leukemia.

Blood, 132(8):837-848.

HOX gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant HOX gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal HOX gene-expression patterns within confined chromatin domains for normal development. Here, we used targeted pooled CRISPR-Cas9-knockout library screening to interrogate the function of CTCF boundaries in the HOX gene loci. We discovered that the CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) is critical for establishing and maintaining aberrant HOXA9-HOXA13 gene expression in AML. Disruption of the CBS7/9 boundary resulted in spreading of repressive H3K27me3 into the posterior active HOXA chromatin domain that subsequently impaired enhancer/promoter chromatin accessibility and disrupted ectopic long-range interactions among the posterior HOXA genes. Consistent with the role of the CBS7/9 boundary in HOXA locus chromatin organization, attenuation of the CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid-specific transcriptome profiles important for pathogenesis of myeloid malignancies. Furthermore, heterozygous deletion of the CBS7/9 chromatin boundary in the HOXA locus reduced human leukemic blast burden and enhanced survival of transplanted AML cell xenograft and patient-derived xenograft mouse models. Thus, the CTCF boundary constrains the normal gene-expression program, as well as plays a role in maintaining the oncogenic transcription program for leukemic transformation. The CTCF boundaries may serve as novel therapeutic targets for the treatment of myeloid malignancies.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Zhou Z, Wang L, Ge F, et al (2018)

Pold3 is required for genomic stability and telomere integrity in embryonic stem cells and meiosis.

Nucleic acids research, 46(7):3468-3486.

Embryonic stem cells (ESCs) and meiosis are featured by relatively higher frequent homologous recombination associated with DNA double strand breaks (DSB) repair. Here, we show that Pold3 plays important roles in DSB repair, telomere maintenance and genomic stability of both ESCs and spermatocytes in mice. By attempting to generate Pold3 deficient mice using CRISPR/Cas9 or transcription activator-like effector nucleases, we show that complete loss of Pold3 (Pold3-/-) resulted in early embryonic lethality at E6.5. Rapid DNA damage response and massive apoptosis occurred in both outgrowths of Pold3-null (Pold3-/-) blastocysts and Pold3 inducible knockout (iKO) ESCs. While Pold3-/- ESCs were not achievable, Pold3 iKO led to increased DNA damage response, telomere loss and chromosome breaks accompanied by extended S phase. Meanwhile, loss of Pold3 resulted in replicative stress, micronucleation and aneuploidy. Also, DNA repair was impaired in Pold3+/- or Pold3 knockdown ESCs. Moreover, Pold3 mediates DNA replication and repair by regulating 53BP1, RIF1, ATR and ATM pathways. Furthermore, spermatocytes of Pold3 haploinsufficient (Pold3+/-) mice with increasing age displayed impaired DSB repair, telomere shortening and loss, and chromosome breaks, like Pold3 iKO ESCs. These data suggest that Pold3 maintains telomere integrity and genomic stability of both ESCs and meiosis by suppressing replicative stress.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Wang Y, Ji T, Nelson AD, et al (2018)

Critical roles of αII spectrin in brain development and epileptic encephalopathy.

The Journal of clinical investigation, 128(2):760-773.

The nonerythrocytic α-spectrin-1 (SPTAN1) gene encodes the cytoskeletal protein αII spectrin. Mutations in SPTAN1 cause early infantile epileptic encephalopathy type 5 (EIEE5); however, the role of αII spectrin in neurodevelopment and EIEE5 pathogenesis is unknown. Prior work suggests that αII spectrin is absent in the axon initial segment (AIS) and contributes to a diffusion barrier in the distal axon. Here, we have shown that αII spectrin is expressed ubiquitously in rodent and human somatodendritic and axonal domains. CRISPR-mediated deletion of Sptan1 in embryonic rat forebrain by in utero electroporation caused altered dendritic and axonal development, loss of the AIS, and decreased inhibitory innervation. Overexpression of human EIEE5 mutant SPTAN1 in embryonic rat forebrain and mouse hippocampal neurons led to similar developmental defects that were also observed in EIEE5 patient-derived neurons. Additionally, patient-derived neurons displayed aggregation of spectrin complexes. Taken together, these findings implicate αII spectrin in critical aspects of dendritic and axonal development and synaptogenesis, and support a dominant-negative mechanism of SPTAN1 mutations in EIEE5.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Ikeda M, Matsuyama S, Akagi S, et al (2017)

Correction of a Disease Mutation using CRISPR/Cas9-assisted Genome Editing in Japanese Black Cattle.

Scientific reports, 7(1):17827.

Isoleucyl-tRNA synthetase (IARS) syndrome is a recessive disease of Japanese Black cattle caused by a single nucleotide substitution. To repair the mutated IARS gene, we designed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create a double-strand break near the mutation site. CRISPR/Cas9 and donor DNA that contained a synonymous codon for the correct amino acid and an Aequorea coerulescens Green Fluorescent Protein (AcGFP) cassette with a piggyBac transposase recognition site at both ends were introduced into bovine fetal fibroblast (BFF) cells isolated from a homozygous mutant calf. Recombinant cells were enriched on the basis of expression of AcGFP, and two cell lines that contained the repaired allele were subcloned. We generated somatic cell nuclear transfer (SCNT) embryos from the repaired cells and transferred 22 blastocysts to recipient cows. In total, five viable fetuses were retrieved at Days 34 and 36. PiggyBac transposase mRNA was introduced into BFF cells isolated from cloned foetuses and AcGFP-negative cells were used for second round of cloning. We transferred nine SCNT embryos to recipient cows and retrieved two fetuses at Day 34. Fetal genomic DNA analysis showed correct repair of the IARS mutation without any additional DNA footprint.

RevDate: 2019-07-15
CmpDate: 2019-07-15

le Sage C, Lawo S, Panicker P, et al (2017)

Dual direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance.

Scientific reports, 7(1):17693.

Pooled CRISPR-Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted to amplified loci, the resulting multiple DNA cleavage events can be a cause of false positive hit identification. The generation of nuclease deficient versions of Cas9 has enabled the development of two additional techniques - CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) - that enable the repression or overexpression, respectively, of target genes. Here we report the first direct combination of all three approaches (CRISPRko, CRISPRi and CRISPRa) in the context of genome-wide screens to identify components that influence resistance and sensitivity to the BRAF inhibitor, vemurafenib. The pairing of both loss- and gain-of-function datasets reveals complex gene networks which control drug response and illustrates how such data can add substantial confidence to target identification and validation analyses.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Kook S, Qi A, Wang P, et al (2018)

Gene-edited MLE-15 Cells as a Model for the Hermansky-Pudlak Syndromes.

American journal of respiratory cell and molecular biology, 58(5):566-574.

Defining the mechanisms of cellular pathogenesis in rare lung diseases such as Hermansky-Pudlak syndrome (HPS) is often complicated by loss of the differentiated phenotype of cultured primary alveolar type 2 (AT2) cells, as well as by a lack of durable cell lines that are faithful to both AT2-cell and rare disease phenotypes. We used CRISPR/Cas9 gene editing to generate a series of HPS-specific mutations in the MLE-15 cell line. The resulting MLE-15/HPS cell lines exhibit preservation of AT2 cellular functions, including formation of lamellar body-like organelles, complete processing of surfactant protein B, and known features of HPS specific to each trafficking complex, including loss of protein targeting to lamellar bodies. MLE-15/HPS1 and MLE-15/HPS2 (with a mutation in Ap3β1) express increased macrophage chemotactic protein-1, a well-described mediator of alveolitis in patients with HPS and in mouse models. We show that MLE-15/HPS9 and pallid AT2 cells (with a mutation in Bloc1s6) also express increased macrophage chemotactic protein-1, suggesting that mice and humans with BLOC-1 mutations may also be susceptible to alveolitis. In addition to providing a flexible platform to examine the role of HPS-specific mutations in trafficking AT2 cells, MLE-15/HPS cell lines provide a durable resource for high-throughput screening and studies of cellular pathophysiology that are likely to accelerate progress toward developing novel therapies for this rare lung disease.

RevDate: 2019-07-15
CmpDate: 2019-07-15

Leung TH, Tang HW, Siu MK, et al (2018)

Human papillomavirus E6 protein enriches the CD55(+) population in cervical cancer cells, promoting radioresistance and cancer aggressiveness.

The Journal of pathology, 244(2):151-163.

Accumulating evidence indicates that the human papillomavirus (HPV) E6 protein plays a crucial role in the development of cervical cancer. Subpopulations of cells that reside within tumours are responsible for tumour resistance to cancer therapy and recurrence. However, the identity of such cells residing in cervical cancer and their relationship with the HPV-E6 protein have not been identified. Here, we isolated sphere-forming cells, which showed self-renewal ability, from primary cervical tumours. Gene expression profiling revealed that cluster of differentiation (CD) 55 was upregulated in primary cervical cancer sphere cells. Flow-cytometric analysis detected abundant CD55(+) populations among a panel of HPV-positive cervical cancer cell lines, whereas few CD55(+) cells were found in HPV-negative cervical cancer and normal cervical epithelial cell lines. The CD55(+) subpopulation isolated from the C33A cell line showed significant sphere-forming ability and enhanced tumourigenicity, cell migration, and radioresistance. In contrast, the suppression of CD55 in HPV-positive CaSki cells inhibited tumourigenicity both in vitro and in vivo, and sensitized cells to radiation treatment. In addition, ectopic expression of the HPV-E6 protein in HPV-negative cervical cancer cells dramatically enriched the CD55(+) subpopulation. CRISPR/Cas9 knockout of CD55 in an HPV-E6-overexpressing stable clone abolished the tumourigenic effects of the HPV-E6 protein. Taken together, our data suggest that HPV-E6 protein expression enriches the CD55(+) population, which contributes to tumourigenicity and radioresistance in cervical cancer cells. Targeting CD55 via CRISPR/Cas9 may represent a novel avenue for developing new strategies and effective therapies for the treatment of cervical cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

RevDate: 2019-07-12

Guo T, Zheng F, Zeng Z, et al (2019)

Cmr3 regulates the suppression on cyclic oligoadenylate synthesis by tag complementarity in a Type III-B CRISPR-Cas system.

RNA biology [Epub ahead of print].

Type III CRISPR-Cas systems code for a multi-subunit ribonucleoprotein (RNP) complex that mediates DNA cleavage and synthesizes cyclic oligoadenylate (cOA) second messenger to confer anti-viral immunity. Both immune activities are to be activated upon binding to target RNA transcripts by their complementarity to crRNA, and autoimmunity avoidance is determined by extended complementarity between the 5'-repeat tag of crRNA and 3'-flanking sequences of target transcripts (anti-tag). However, as to how the strategy could achieve stringent autoimmunity avoidance remained elusive. In this study, we systematically investigated how the complementarity of the crRNA 5'-tag and anti-tag (i.e., tag complementarity) could affect the interference activities (DNA cleavage activity and cOA synthesis activity) of Cmr-α, a type III-B system in Sulfolobus islandicus Rey15A. The results revealed an increasing suppression on both activities by increasing degrees of tag complementarity and a critical function of the 7th nucleotide of crRNA in avoiding autoimmunity. More importantly, mutagenesis of Cmr3α exerts either positive or negative effects on the cOA synthesis activity depending on the degrees of tag complementarity, suggesting that the subunit, coupling with the interaction between crRNA tag and anti-tag, function in facilitating immunity and avoiding autoimmunity in Type III-B systems.

RevDate: 2019-07-11

Abedon ST (2017)

Phage "delay" towards enhancing bacterial escape from biofilms: a more comprehensive way of viewing resistance to bacteriophages.

AIMS microbiology, 3(2):186-226 pii:microbiol-03-02-186.

In exploring bacterial resistance to bacteriophages, emphasis typically is placed on those mechanisms which completely prevent phage replication. Such resistance can be detected as extensive reductions in phage ability to form plaques, that is, reduced efficiency of plating. Mechanisms include restriction-modification systems, CRISPR/Cas systems, and abortive infection systems. Alternatively, phages may be reduced in their "vigor" when infecting certain bacterial hosts, that is, with phages displaying smaller burst sizes or extended latent periods rather than being outright inactivated. It is well known, as well, that most phages poorly infect bacteria that are less metabolically active. Extracellular polymers such as biofilm matrix material also may at least slow phage penetration to bacterial surfaces. Here I suggest that such "less-robust" mechanisms of resistance to bacteriophages could serve bacteria by slowing phage propagation within bacterial biofilms, that is, delaying phage impact on multiple bacteria rather than necessarily outright preventing such impact. Related bacteria, ones that are relatively near to infected bacteria, e.g., roughly 10+ µm away, consequently may be able to escape from biofilms with greater likelihood via standard dissemination-initiating mechanisms including erosion from biofilm surfaces or seeding dispersal/central hollowing. That is, given localized areas of phage infection, so long as phage spread can be reduced in rate from initial points of contact with susceptible bacteria, then bacterial survival may be enhanced due to bacteria metaphorically "running away" to more phage-free locations. Delay mechanisms-to the extent that they are less specific in terms of what phages are targeted-collectively could represent broader bacterial strategies of phage resistance versus outright phage killing, the latter especially as require specific, evolved molecular recognition of phage presence. The potential for phage delay should be taken into account when developing protocols of phage-mediated biocontrol of biofilm bacteria, e.g., as during phage therapy of chronic bacterial infections.

RevDate: 2019-07-11

Gupta D, Bhattacharjee O, Mandal D, et al (2019)

CRISPR-Cas9 system: A new-fangled dawn in gene editing.

Life sciences pii:S0024-3205(19)30562-4 [Epub ahead of print].

Till date, only three techniques namely Zinc Finger Nuclease (ZFN), Transcription-Activator Like Effector Molecules (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9) are available for targeted genome editing. CRISPR-Cas system is very efficient, fast, easy and cheap technique for achieving knock-out gene in the cell. CRISPR-Cas9 system refurbishes the targeted genome editing approach into a more expedient and competent way, thus facilitating proficient genome editing through embattled double-strand breaks in approximately any organism and cell type. The off-target effects of CRISPR Cas system has been circumnavigated by using paired nickases. Moreover, CRISPR-Cas9 has been used effectively for numerous purposes, like knock-out of a gene, regulation of endogenous gene expression, live-cell labelling of chromosomal loci, edition of single-stranded RNA and high-throughput gene screening. The execution of the CRISPR-Cas9 system has amplified the number of accessible scientific substitutes for studying gene function, thus enabling generation of CRISPR-based disease models. Even though many mechanistic questions are left behind to be answered and the system is not yet fool-proof i.e., a number of challenges are yet to be addressed, the employment of CRISPR-Cas9-based genome engineering technologies will increase our understanding to disease processes and their treatment in the near future. In this review we have discussed the history of CRISPR-Cas9, its mechanism for genome editing and its application in animal, plant and protozoan parasites. Additionally, the pros and cons of CRISPR-Cas9 and its potential in therapeutic application have also been detailed here.

RevDate: 2019-07-11

Ashari KS, Roslan NS, Omar AR, et al (2019)

Genome sequencing and analysis of Salmonella enterica subsp. enterica serovar Stanley UPM 517: Insights on its virulence-associated elements and their potentials as vaccine candidates.

PeerJ, 7:e6948 pii:6948.

Salmonella enterica subsp. enterica serovar Stanley (S. Stanley) is a pathogen that contaminates food, and is related to Salmonella outbreaks in a variety of hosts such as humans and farm animals through products like dairy items and vegetables. Despite the fact that several vaccines of Salmonella strains had been constructed, none of them were developed according to serovar Stanley up to this day. This study presents results of genome sequencing and analysis on our S. Stanley UPM 517 strain taken from fecal swabs of 21-day-old healthy commercial chickens in Perak, Malaysia and used Salmonella enterica subsp. enterica serovar Typhimurium LT2 (S. Typhimurium LT2) as a reference to be compared with. First, sequencing and assembling of the Salmonella Stanley UPM 517 genome into a contiguous form were done. The work was then continued with scaffolding and gap filling. Annotation and alignment of the draft genome was performed with S. Typhimurium LT2. The other elements of virulence estimated in this study included Salmonella pathogenicity islands, resistance genes, prophages, virulence factors, plasmid regions, restriction-modification sites and the CRISPR-Cas system. The S. Stanley UPM 517 draft genome had a length of 4,736,817 bp with 4,730 coding sequence and 58 RNAs. It was discovered via genomic analysis on this strain that there were antimicrobial resistance properties toward a wide variety of antibiotics. Tcf and ste, the two fimbrial virulence clusters related with human and broiler intestinal colonizations which were not found in S. Typhimurium LT2, were atypically discovered in the S. Stanley UPM 517 genome. These clusters are involved in the intestinal colonization of human and broilers, respectively. There were seven Salmonella pathogenicity islands (SPIs) within the draft genome, which contained the virulence factors associated with Salmonella infection (except SPI-14). Five intact prophage regions, mostly comprising of the protein encoding Gifsy-1, Fels-1, RE-2010 and SEN34 prophages, were also encoded in the draft genome. Also identified were Type I-III restriction-modification sites and the CRISPR-Cas system of the Type I-E subtype. As this strain exhibited resistance toward numerous antibiotics, we distinguished several genes that had the potential for removal in the construction of a possible vaccine candidate to restrain and lessen the pervasiveness of salmonellosis and to function as an alternative to antibiotics.

RevDate: 2019-07-11

Lima R, Del Fiol FS, VM Balcão (2019)

Prospects for the Use of New Technologies to Combat Multidrug-Resistant Bacteria.

Frontiers in pharmacology, 10:692.

The increasing use of antibiotics is being driven by factors such as the aging of the population, increased occurrence of infections, and greater prevalence of chronic diseases that require antimicrobial treatment. The excessive and unnecessary use of antibiotics in humans has led to the emergence of bacteria resistant to the antibiotics currently available, as well as to the selective development of other microorganisms, hence contributing to the widespread dissemination of resistance genes at the environmental level. Due to this, attempts are being made to develop new techniques to combat resistant bacteria, among them the use of strictly lytic bacteriophage particles, CRISPR-Cas, and nanotechnology. The use of these technologies, alone or in combination, is promising for solving a problem that humanity faces today and that could lead to human extinction: the domination of pathogenic bacteria resistant to artificial drugs. This prospective paper discusses the potential of bacteriophage particles, CRISPR-Cas, and nanotechnology for use in combating human (bacterial) infections.

RevDate: 2019-07-11
CmpDate: 2019-07-11

Léger S, Costa MBW, D Tulpan (2019)

Pairwise visual comparison of small RNA secondary structures with base pair probabilities.

BMC bioinformatics, 20(1):293 pii:10.1186/s12859-019-2902-6.

BACKGROUND: Predicted RNA secondary structures are typically visualized using dot-plots for base pair binding probabilities and planar graphs for unique structures, such as the minimum free energy structure. These are however difficult to analyze simultaneously.

RESULTS: This work introduces a compact unified view of the most stable conformation of an RNA secondary structure and its base pair probabilities, which is called the Circular Secondary Structure Base Pairs Probabilities Plot (CS2BP2-Plot). Along with our design we provide access to a web server implementation of our solution that facilitates pairwise comparison of short RNA (and DNA) sequences up to 200 base pairs. The web server first calculates the minimum free energy secondary structure and the base pair probabilities for up to 10 RNA or DNA sequences using RNAfold and then provides a two panel comparative view that includes CS2BP2-Plots along with the traditional graph, planar and circular diagrams obtained with VARNA. The CS2BP2-Plots include highlighting of the nucleotide differences between two selected sequences using ClustalW local alignments. We also provide descriptive statistics, dot-bracket secondary structure representations and ClustalW local alignments for compared sequences.

CONCLUSIONS: Using circular diagrams and colour and weight-coded arcs, we demonstrate how a single image can replace the state-of-the-art dual representations (dot-plots and minimum free energy structures) for base-pair probabilities of RNA secondary structures while allowing efficient exploration and comparison of different RNA conformations via a web server front end. With that, we provide the community, especially the biologically oriented, with an intuitive tool for ncRNA visualization. Web-server: https://nrcmonsrv01.nrc.ca/cs2bp2plot.

RevDate: 2019-07-12
CmpDate: 2019-07-12

Huang JM, Chang YT, Shih MH, et al (2019)

Identification and characterization of a secreted M28 aminopeptidase protein in Acanthamoeba.

Parasitology research, 118(6):1865-1874.

Acanthamoeba is a free-living pathogenic protozoan that is distributed in different environmental reservoirs, including lakes and soil. Pathogenic Acanthamoeba can cause severe human diseases, such as blinding keratitis and granulomatous encephalitis. Therefore, it is important to understand the pathogenic relationship between humans and Acanthamoeba. By comparison of systemic analysis results for Acanthamoeba isolates, we identified a novel secreted protein of Acanthamoeba, an M28 aminopeptidase (M28AP), which targets of the human innate immune defense. We investigated the molecular functions and characteristics of the M28AP protein by anti-M28 antibodies and a M28AP mutant strain generated by the CRISPR/Cas9 system. Human complement proteins such as C3b and iC3b were degraded by Acanthamoeba M28AP. We believe that M28AP is an important factor in human innate immunity. This study provides new insight for the development of more efficient medicines to treat Acanthamoeba infection.

RevDate: 2019-07-11
CmpDate: 2019-07-11

Mimitou EP, Cheng A, Montalbano A, et al (2019)

Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells.

Nature methods, 16(5):409-412.

Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct single-guide RNA capture and to clonotype-aware multimodal phenotyping of cancer samples.

RevDate: 2019-07-11
CmpDate: 2019-07-11

Chow RD, Wang G, Ye L, et al (2019)

In vivo profiling of metastatic double knockouts through CRISPR-Cpf1 screens.

Nature methods, 16(5):405-408.

Systematic investigation of the genetic interactions that influence metastatic potential has been challenging. Here we developed massively parallel CRISPR-Cpf1/Cas12a crRNA array profiling (MCAP), an approach for combinatorial interrogation of double knockouts in vivo. We designed an MCAP library of 11,934 arrays targeting 325 pairwise combinations of genes implicated in metastasis. By assessing the metastatic potential of the double knockouts in mice, we unveiled a quantitative landscape of genetic interactions that drive metastasis.

RevDate: 2019-07-11
CmpDate: 2019-07-11

Sibbritt T, Osteil P, Fan X, et al (2019)

Gene Editing of Mouse Embryonic and Epiblast Stem Cells.

Methods in molecular biology (Clifton, N.J.), 1940:77-95.

Efficient and reliable methods for gene editing are critical for the generation of loss-of-gene function stem cells and genetically modified mice. Here, we outline the application of CRISPR-Cas9 technology for gene editing in mouse embryonic stem cells (mESCs) to generate knockout ESC chimeras for the fast-tracked analysis of gene function. Furthermore, we describe the application of gene editing directly to mouse epiblast stem cells (mEpiSCs) for modelling germ layer differentiation in vitro.

RevDate: 2019-07-12
CmpDate: 2019-07-12

Burgess DJ (2019)

Genome editing for disease locus dissection.

Nature reviews. Genetics, 20(2):67.

RevDate: 2019-07-12
CmpDate: 2019-07-12

Cloney R (2019)

The oracle of inDelphi predicts Cas9 repair outcomes.

Nature reviews. Genetics, 20(1):4-5.

RevDate: 2019-07-11
CmpDate: 2019-07-11

Maguire JA, Cardenas-Diaz FL, Gadue P, et al (2019)

Highly Efficient CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.

Current protocols in stem cell biology, 48(1):e64.

Human PSCs offer tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR-Cas9 technology has expanded this potential by providing a valuable tool for engineering or correcting disease-associated mutations. Because of the high efficiency with which CRISPR-Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele, without indel formation on the non-targeted allele. To overcome this obstacle, we describe the use of two oligonucleotides, one expressing the sequence change, with the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodology to make this protocol efficient with small numbers of cells and to limit the amount of labor-intensive clone passaging. This protocol provides a streamlined and technically simple approach for generating valuable tools to model human disease in stem cells. © 2018 by John Wiley & Sons, Inc.

RevDate: 2019-07-11
CmpDate: 2019-07-11

Yang Z, Wang H, Wang Y, et al (2018)

Manufacturing Multienzymatic Complex Reactors In Vivo by Self-Assembly To Improve the Biosynthesis of Itaconic Acid in Escherichia coli.

ACS synthetic biology, 7(5):1244-1250.

The self-assembly of multienzyme into bioreactors is of extensive interest to spatially regulate valuable reactions. Despite the important progresses achieved, methods to precisely manufacture multienzymatic complex reactors (MECRs) are still poorly proposed both in vivo and in vitro, particularly for more than three biocatalytically relevant enzymes. Here, we developed a sequential self-assembly system to form multitude MECRs involving three enzymes in the itaconic acid (IA) pathway with two pairs of protein-peptide interactions. The MECRs were identified as nanoscale particle-like structures when self-assembled in vitro and produced higher IA production than the unassembled and linearly assembled systems when applied in vivo coupling with CRISPR-Cas9 based metabolic engineering. This work provides novel insights into the construction of multifarious multienzyme complex into bioreactors by the self-assembly strategy for multistep cascades to sequentially control metabolic fluxes inside cells.

RevDate: 2019-07-11
CmpDate: 2019-07-11

Agrawal DK, Tang X, Westbrook A, et al (2018)

Mathematical Modeling of RNA-Based Architectures for Closed Loop Control of Gene Expression.

ACS synthetic biology, 7(5):1219-1228.

Feedback allows biological systems to control gene expression precisely and reliably, even in the presence of uncertainty, by sensing and processing environmental changes. Taking inspiration from natural architectures, synthetic biologists have engineered feedback loops to tune the dynamics and improve the robustness and predictability of gene expression. However, experimental implementations of biomolecular control systems are still far from satisfying performance specifications typically achieved by electrical or mechanical control systems. To address this gap, we present mathematical models of biomolecular controllers that enable reference tracking, disturbance rejection, and tuning of the temporal response of gene expression. These controllers employ RNA transcriptional regulators to achieve closed loop control where feedback is introduced via molecular sequestration. Sensitivity analysis of the models allows us to identify which parameters influence the transient and steady state response of a target gene expression process, as well as which biologically plausible parameter values enable perfect reference tracking. We quantify performance using typical control theory metrics to characterize response properties and provide clear selection guidelines for practical applications. Our results indicate that RNA regulators are well-suited for building robust and precise feedback controllers for gene expression. Additionally, our approach illustrates several quantitative methods useful for assessing the performance of biomolecular feedback control systems.

RevDate: 2019-07-11
CmpDate: 2019-07-11

Li L, Hu S, X Chen (2018)

Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities.

Biomaterials, 171:207-218.

In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed.

RevDate: 2019-07-11
CmpDate: 2019-07-11

Wang W, Zheng W, Hu F, et al (2018)

Enhanced Biosynthesis Performance of Heterologous Proteins in CHO-K1 Cells Using CRISPR-Cas9.

ACS synthetic biology, 7(5):1259-1268.

Chinese hamster ovary (CHO) cells are the famous expression system for industrial production of recombinant proteins, such as therapeutic antibodies. However, there still remain bottlenecks in protein quality and weakness in expression efficiency because of the intrinsic genetic properties of the cell. Here we have enhanced biosynthesis performance of heterologous proteins in CHO-K1 cells using CRISPR-Cas9 by editing the genome precisely with two genes for improving ER microenvironment and reinforcing antiapoptotic ability. A linear donor plasmid harboring eGFP-HsQSOX1b and Survivin genes was knocked in specific locus in CHO-K1 genome by the CRISPR-Cas9 RNA guided nucleases via NHEJ with efficiencies of up to 3.85% in the CHO-K1 cell pools following FACS, and the hQSOX1 and hSurvivin genes were integrated into expected genome locus successfully. Compared with control, the antiapoptotic viability of edited CHO-K1 cells was increased by 6.40 times, and the yield has been raised by 5.55 times with GLuc as model protein. The possible molecular mechanisms and pathways of remarkable antiapoptotic ability and protein biosynthesis in modified CHO-K1 cells have been elucidated reasonably. In conclusion, the novel ideas and reliable techniques for obtaining foreign proteins more efficiently in engineered animal cells were very valuable to meet large clinical needs.

RevDate: 2019-07-11
CmpDate: 2019-07-11

Gao W, Yin J, Bao L, et al (2018)

Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State.

ACS synthetic biology, 7(5):1291-1302.

Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.

RevDate: 2019-07-11
CmpDate: 2019-07-11

Liu Y, Wei WP, BC Ye (2018)

High GC Content Cas9-Mediated Genome-Editing and Biosynthetic Gene Cluster Activation in Saccharopolyspora erythraea.

ACS synthetic biology, 7(5):1338-1348.

The overexpression of bacterial secondary metabolite biosynthetic enzymes is the basis for industrial overproducing strains. Genome editing tools can be used to further improve gene expression and yield. Saccharopolyspora erythraea produces erythromycin, which has extensive clinical applications. In this study, the CRISPR-Cas9 system was used to edit genes in the S. erythraea genome. A temperature-sensitive plasmid containing the PermE promoter, to drive Cas9 expression, and the Pj23119 and PkasO promoters, to drive sgRNAs, was designed. Erythromycin esterase, encoded by S. erythraea SACE_1765, inactivates erythromycin by hydrolyzing the macrolactone ring. Sequencing and qRT-PCR confirmed that reporter genes were successfully inserted into the SACE_1765 gene. Deletion of SACE_1765 in a high-producing strain resulted in a 12.7% increase in erythromycin levels. Subsequent PermE- egfp knock-in at the SACE_0712 locus resulted in an 80.3% increase in erythromycin production compared with that of wild type. Further investigation showed that PermE promoter knock-in activated the erythromycin biosynthetic gene clusters at the SACE_0712 locus. Additionally, deletion of indA (SACE_1229) using dual sgRNA targeting without markers increased the editing efficiency to 65%. In summary, we have successfully applied Cas9-based genome editing to a bacterial strain, S. erythraea, with a high GC content. This system has potential application for both genome-editing and biosynthetic gene cluster activation in Actinobacteria.

RevDate: 2019-07-12
CmpDate: 2019-07-12

Wrighton KH (2018)

Genetic engineering: Expanding the reach of Cas9.

Nature reviews. Genetics, 19(5):250-251.

RevDate: 2019-07-12
CmpDate: 2019-07-12

Burgess DJ (2018)

Translational genetics: CRISPR therapies - making the grade not the cut.

Nature reviews. Genetics, 19(2):63.

RevDate: 2019-07-12
CmpDate: 2019-07-12

Zhao D, Feng X, Zhu X, et al (2017)

CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency.

Scientific reports, 7(1):16624.

The CRISPR/Cas9 system is a powerful, revolutionary tool for genome editing. However, it is not without limitations. There are PAM-free and CRISPR-tolerant regions that cannot be modified by the standard CRISPR/Cas9 system, and off-target activity impedes its broader applications. To avoid these drawbacks, we developed a very simple CRISPR/Cas9-assisted gRNA-free one-step (CAGO) genome editing technique which does not require the construction of a plasmid to express a specific gRNA. Instead, a universal N20 sequence with a very high targeting efficiency is inserted into the E. coli chromosome by homologous recombination, which in turn undergoes a double-stranded break by CRISPR/Cas9 and induces an intra-chromosomal recombination event to accomplish the editing process. This technique was shown to be able to edit PAM-free and CRISPR-tolerant regions with no off-target effects in Escherichia coli. When applied to multi-locus editing, CAGO was able to modify one locus in two days with a near 100% editing efficiency. Furthermore, modified CAGO was used to edit large regions of up to 100 kbp with at least 75% efficiency. Finally, genome editing by CAGO only requires a transformation procedure and the construction of a linear donor DNA cassette, which was further simplified by applying a modular design strategy. Although the technique was established in E. coli, it should be applicable to other organisms with only minor modifications.

RevDate: 2019-07-12
CmpDate: 2019-07-12

Musunuru K, Lagor WR, JM Miano (2017)

What Do We Really Think About Human Germline Genome Editing, and What Does It Mean for Medicine?.

Circulation. Cardiovascular genetics, 10(5):.

RevDate: 2019-07-12
CmpDate: 2019-07-12

Hennessy EJ (2017)

Cardiovascular Disease and Long Noncoding RNAs: Tools for Unraveling the Mystery Lnc-ing RNA and Phenotype.

Circulation. Cardiovascular genetics, 10(4):e001556.

RevDate: 2019-07-12
CmpDate: 2019-07-12

Burgess DJ (2017)

Genetic screens: Combining CRISPR perturbations and RNA-seq.

Nature reviews. Genetics, 18(2):67.

RevDate: 2019-07-12
CmpDate: 2019-07-12

Cloney R (2017)

Genetic engineering: A genome-editing off switch.

Nature reviews. Genetics, 18(2):68-69.

RevDate: 2019-07-03
CmpDate: 2019-07-03

McMahon MA, DW Cleveland (2017)

Gene therapy: Gene-editing therapy for neurological disease.

Nature reviews. Neurology, 13(1):7-9.

RevDate: 2019-07-10

Steinecke A, Kurabayashi N, Hayano Y, et al (2019)

In Vivo Single-Cell Genotyping of Mouse Cortical Neurons Transfected with CRISPR/Cas9.

Cell reports, 28(2):325-331.e4.

CRISPR/Cas-based technologies have revolutionized genetic approaches to addressing a wide range of neurobiological questions. The ability of CRISPR/Cas to introduce mutations into target genes allows us to perform in vivo loss-of-function experiments without generating genetically engineered mice. However, the lack of a reliable method to determine genotypes of individual CRISPR/Cas-transfected cells has made it impossible to unambiguously identify the genetic cause of their phenotypes in vivo. Here, we report a strategy for single-cell genotyping in CRISPR/Cas-transfected neurons that were phenotypically characterized in vivo. We show that re-sectioning of cortical slices and subsequent laser microdissection allow us to isolate individual CRISPR/Cas-transfected neurons. Sequencing of PCR products containing a CRISPR/Cas-targeted genomic region in single reference neurons provided genotypes that completely correspond with those deduced from their target protein expression and phenotypes. Thus, our study establishes a powerful strategy to determine the causality between genotypes and phenotypes in CRISPR/Cas-transfected neurons.

RevDate: 2019-07-10

Jin M, Garreau de Loubresse N, Kim Y, et al (2019)

Programmable CRISPR-Cas Repression, Activation, and Computation with Sequence-Independent Targets and Triggers.

ACS synthetic biology [Epub ahead of print].

The programmability of CRISPR-derived Cas9 as a sequence-specific DNA-targeting protein has made it a powerful tool for genomic manipulation in biological research and translational applications. Cas9 activity can be programmably engineered to respond to nucleic acids, but these efforts have focused primarily on single-input control of Cas9, and until recently, they were limited by sequence dependence between parts of the guide RNA and the sequence to be detected. Here, we not only design and present DNA- and RNA-sensing conditional guide RNA (cgRNA) that have no such sequence constraints, but also demonstrate a complete set of logical computations using these designs on DNA and RNA sequence inputs, including AND, OR, NAND, and NOR. The development of sequence-independent nucleic acid-sensing CRISPR-Cas9 systems with multi-input logic computation capabilities could lead to improved genome engineering and regulation as well as the construction of synthetic circuits with broader functionality.

RevDate: 2019-07-10

Lam TJ, Y Ye (2019)

Long reads reveal the diversification and dynamics of CRISPR reservoir in microbiomes.

BMC genomics, 20(1):567 pii:10.1186/s12864-019-5922-8.

BACKGROUND: Sequencing of microbiomes has accelerated the characterization of the diversity of CRISPR-Cas immune systems. However, the utilization of next generation short read sequences for the characterization of CRISPR-Cas dynamics remains limited due to the repetitive nature of CRISPR arrays. CRISPR arrays are comprised of short spacer segments (derived from invaders' genomes) interspaced between flanking repeat sequences. The repetitive structure of CRISPR arrays poses a computational challenge for the accurate assembly of CRISPR arrays from short reads. In this paper we evaluate the use of long read sequences for the analysis of CRISPR-Cas system dynamics in microbiomes.

RESULTS: We analyzed a dataset of Illumina's TruSeq Synthetic Long-Reads (SLR) derived from a gut microbiome. We showed that long reads captured CRISPR spacers at a high degree of redundancy, which highlights the spacer conservation of spacer sharing CRISPR variants, enabling the study of CRISPR array dynamics in ways difficult to achieve though short read sequences. We introduce compressed spacer graphs, a visual abstraction of spacer sharing CRISPR arrays, to provide a simplified view of complex organizational structures present within CRISPR array dynamics. Utilizing compressed spacer graphs, several key defining characteristics of CRISPR-Cas system dynamics were observed including spacer acquisition and loss events, conservation of the trailer end spacers, and CRISPR arrays' directionality (transcription orientation). Other result highlights include the observation of intense array contraction and expansion events, and reconstruction of a full-length genome for a potential invader (Faecalibacterium phage) based on identified spacers.

CONCLUSION: We demonstrate in an in silico system that long reads provide the necessary context for characterizing the organization of CRISPR arrays in a microbiome, and reveal dynamic and evolutionary features of CRISPR-Cas systems in a microbial population.

RevDate: 2019-07-10
CmpDate: 2019-07-10

Hou Z, Y Zhang (2019)

Inserting DNA with CRISPR.

Science (New York, N.Y.), 365(6448):25-26.

RevDate: 2019-07-10
CmpDate: 2019-07-10

Pei W, Wang X, Rössler J, et al (2019)

Using Cre-recombinase-driven Polylox barcoding for in vivo fate mapping in mice.

Nature protocols, 14(6):1820-1840.

Fate mapping is a powerful genetic tool for linking stem or progenitor cells with their progeny, and hence for defining cell lineages in vivo. The resolution of fate mapping depends on the numbers of distinct markers that are introduced in the beginning into stem or progenitor cells; ideally, numbers should be sufficiently large to allow the tracing of output from individual cells. Highly diverse genetic barcodes can serve this purpose. We recently developed an endogenous genetic barcoding system, termed Polylox. In Polylox, random DNA recombination can be induced by transient activity of Cre recombinase in a 2.1-kb-long artificial recombination substrate that has been introduced into a defined locus in mice (Rosa26Polylox reporter mice). Here, we provide a step-by-step protocol for the use of Polylox, including barcode induction and estimation of induction efficiency, barcode retrieval with single-molecule real-time (SMRT) DNA sequencing followed by computational barcode identification, and the calculation of barcode-generation probabilities, which is key for estimations of single-cell labeling for a given number of stem cells. Thus, Polylox barcoding enables high-resolution fate mapping in essentially all tissues in mice for which inducible Cre driver lines are available. Alternative methods include ex vivo cell barcoding, inducible transposon insertion and CRISPR-Cas9-based barcoding; Polylox currently allows combining non-invasive and cell-type-specific labeling with high label diversity. The execution time of this protocol is ~2-3 weeks for experimental data generation and typically <2 d for computational Polylox decoding and downstream analysis.

RevDate: 2019-07-10
CmpDate: 2019-07-10

Wu Y, Zeng J, Roscoe BP, et al (2019)

Highly efficient therapeutic gene editing of human hematopoietic stem cells.

Nature medicine, 25(5):776-783.

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2-6. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.

RevDate: 2019-07-10
CmpDate: 2019-07-10

Rui Y, Wilson DR, Sanders K, et al (2019)

Reducible Branched Ester-Amine Quadpolymers (rBEAQs) Codelivering Plasmid DNA and RNA Oligonucleotides Enable CRISPR/Cas9 Genome Editing.

ACS applied materials & interfaces, 11(11):10472-10480.

Functional codelivery of plasmid DNA and RNA oligonucleotides in the same nanoparticle system is challenging due to differences in their physical properties as well as their intracellular locations of function. In this study, we synthesized a series of reducible branched ester-amine quadpolymers (rBEAQs) and investigated their ability to coencapsulate and deliver DNA plasmids and RNA oligos. The rBEAQs are designed to leverage polymer branching, reducibility, and hydrophobicity to successfully cocomplex DNA and RNA in nanoparticles at low polymer to nucleic acid w/w ratios and enable high delivery efficiency. We validate the synthesis of this new class of biodegradable polymers, characterize the self-assembled nanoparticles that these polymers form with diverse nucleic acids, and demonstrate that the nanoparticles enable safe, effective, and efficient DNA-siRNA codelivery as well as nonviral CRISPR-mediated gene editing utilizing Cas9 DNA and sgRNA codelivery.

RevDate: 2019-07-10
CmpDate: 2019-07-10

Montaño A, Forero-Castro M, Hernández-Rivas JM, et al (2018)

Targeted genome editing in acute lymphoblastic leukemia: a review.

BMC biotechnology, 18(1):45.

BACKGROUND: Genome editing technologies offers new opportunities for tackling diseases such as acute lymphoblastic leukemia (ALL) that have been beyond the reach of previous therapies.

RESULTS: We show how the recent availability of genome-editing tools such as CRISPR-Cas9 are an important means of advancing functional studies of ALL through the incorporation, elimination and modification of somatic mutations and fusion genes in cell lines and mouse models. These tools not only broaden the understanding of the involvement of various genetic alterations in the pathogenesis of the disease but also identify new therapeutic targets for future clinical trials.

CONCLUSIONS: New approaches including CRISPR-Cas9 are crucial for functional studies of genetic aberrations driving cancer progression, and that may be responsible for treatment resistance and relapses. By using this approach, diseases can be more faithfully reproduced and new therapeutic targets and approaches found.

RevDate: 2019-07-10
CmpDate: 2019-07-10

Bleijenberg A, E Dekker (2018)

Reverse-engineering the serrated neoplasia pathway using CRISPR-Cas9.

Nature reviews. Gastroenterology & hepatology, 15(9):522-524.

RevDate: 2019-07-10
CmpDate: 2019-07-10

Paulo JA, SP Gygi (2018)

Isobaric Tag-Based Protein Profiling of a Nicotine-Treated Alpha7 Nicotinic Receptor-Null Human Haploid Cell Line.

Proteomics, 18(11):e1700475.

Nicotinic acetylcholine receptors (nAChR), the primary cell surface targets of nicotine, have implications in various neurological disorders. Here we investigate the proteome-wide effects of nicotine on human haploid cell lines (wildtype HAP1 and α7KO-HAP1) to address differences in nicotine-induced protein abundance profiles between these cell lines. We performed an SPS-MS3-based TMT10-plex experiment arranged in a 2-3-2-3 design with two replicates of the untreated samples and three of the treated samples for each cell line. We quantified 8775 proteins across all ten samples, of which several hundred differed significantly in abundance. Comparing α7KO-HAP1 and HAP1wt cell lines to each other revealed significant protein abundance alterations; however, we also measured differences resulting from nicotine treatment in both cell lines. Among proteins with increased abundance levels due to nicotine treatment included those previously identified: APP, APLP2, and ITM2B. The magnitude of these changes was greater in HAP1wt compared to the α7KO-HAP1 cell line, implying a potential role for the α7 nAChR in HAP1 cells. Moreover, the data revealed that membrane proteins and proteins commonly associated with neurons were predominant among those with altered abundance. This study, which is the first TMT-based proteome profiling of HAP1 cells, defines further the effects of nicotine on non-neuronal cellular proteomes.

RevDate: 2019-07-10
CmpDate: 2019-07-10

Phan QV, Contzen J, Seemann P, et al (2017)

Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease.

Scientific reports, 7(1):17771.

Site-specific recombination systems like those based on the Flp recombinase proved themselves as efficient tools for cell line engineering. The recent emergence of designer nucleases, especially RNA guided endonucleases like Cas9, has considerably broadened the available toolbox for applications like targeted transgene insertions. Here we established a recombinase-mediated cassette exchange (RMCE) protocol for the fast and effective, drug-free isolation of recombinant cells. Distinct fluorescent protein patterns identified the recombination status of individual cells. In derivatives of a CHO master cell line the expression of the introduced transgene of interest could be dramatically increased almost 20-fold by subsequent deletion of the fluorescent protein gene that provided the initial isolation principle. The same master cell line was employed in a comparative analysis using CRISPR/Cas9 for transgene integration in identical loci. Even though the overall targeting efficacy was comparable, multi-loci targeting was considerably more effective for Cas9-mediated transgene insertion when compared to RMCE. While Cas9 is inherently more flexible, our results also alert to the risk of aberrant recombination events around the cut site. Together, this study points at the individual strengths in performance of both systems and provides guidance for their appropriate use.

RevDate: 2019-07-10
CmpDate: 2019-07-10

Jacobs EZ, Warrier S, Volders PJ, et al (2017)

CRISPR/Cas9-mediated genome editing in naïve human embryonic stem cells.

Scientific reports, 7(1):16650.

The combination of genome-edited human embryonic stem cells (hESCs) and subsequent neural differentiation is a powerful tool to study neurodevelopmental disorders. Since the naïve state of pluripotency has favourable characteristics for efficient genome-editing, we optimized a workflow for the CRISPR/Cas9 system in these naïve stem cells. Editing efficiencies of respectively 1.3-8.4% and 3.8-19% were generated with the Cas9 nuclease and the D10A Cas9 nickase mutant. Next to this, wildtype and genome-edited naïve hESCs were successfully differentiated to neural progenitor cells. As a proof-of-principle of our workflow, two monoclonal genome-edited naïve hESCs colonies were obtained for TUNA, a long non-coding RNA involved in pluripotency and neural differentiation. In these genome-edited hESCs, an effect was seen on expression of TUNA, although not on neural differentiation potential. In conclusion, we optimized a genome-editing workflow in naïve hESCs that can be used to study candidate genes involved in neural differentiation and/or functioning.

RevDate: 2019-07-09

Goulin EH, Galdeano DM, Granato LM, et al (2019)

RNA interference and CRISPR: Promising approaches to better understand and control citrus pathogens.

Microbiological research, 226:1-9.

Citrus crops have great economic importance worldwide. However, citrus production faces many diseases caused by different pathogens, such as bacteria, oomycetes, fungi and viruses. To overcome important plant diseases in general, new technologies have been developed and applied to crop protection, including RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. RNAi has been demonstrated to be a powerful tool for application in plant defence mechanisms against different pathogens as well as their respective vectors, and CRISPR/Cas system has become widely used in gene editing or reprogramming or knocking out any chosen DNA/RNA sequence. In this article, we provide an overview of the use of RNAi and CRISPR/Cas technologies in management strategies to control several plants diseases, and we discuss how these strategies can be potentially used against citrus pathogens.

RevDate: 2019-07-09
CmpDate: 2019-07-09

Bell CC, Fennell KA, Chan YC, et al (2019)

Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.

Nature communications, 10(1):2723 pii:10.1038/s41467-019-10652-9.

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.

RevDate: 2019-07-09
CmpDate: 2019-07-09

Naseri G, Behrend J, Rieper L, et al (2019)

COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors.

Nature communications, 10(1):2615 pii:10.1038/s41467-019-10224-x.

Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing β-carotene and co-producing β-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.

RevDate: 2019-07-09
CmpDate: 2019-07-09

Hojo MA, Masuda K, Hojo H, et al (2019)

Identification of a genomic enhancer that enforces proper apoptosis induction in thymic negative selection.

Nature communications, 10(1):2603 pii:10.1038/s41467-019-10525-1.

During thymic negative selection, autoreactive thymocytes carrying T cell receptor (TCR) with overtly strong affinity to self-MHC/self-peptide are removed by Bim-dependent apoptosis, but how Bim is specifically regulated to link TCR activation and apoptosis induction is unclear. Here we identify a murine T cell-specific genomic enhancer EBAB (Bub1-Acoxl-Bim), whose deletion leads to accumulation of thymocytes expressing high affinity TCRs. Consistently, EBAB knockout mice have defective negative selection and fail to delete autoreactive thymocytes in various settings, with this defect accompanied by reduced Bim expression and apoptosis induction. By contrast, EBAB is dispensable for maintaining peripheral T cell homeostasis via Bim-dependent pathways. Our data thus implicate EBAB as an important, developmental stage-specific regulator of Bim expression and apoptosis induction to enforce thymic negative selection and suppress autoimmunity. Our study unravels a part of genomic enhancer codes that underlie complex and context-dependent gene regulation in TCR signaling.

RevDate: 2019-07-09
CmpDate: 2019-07-09

Chen Z, Y Zhang (2019)

Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development.

Nature genetics, 51(6):947-951.

How maternal factors in oocytes trigger zygotic genome activation (ZGA) is a long-standing question in developmental biology. Recent studies in 2-cell-like embryonic stem cells (2C-like cells) suggest that transcription factors of the DUX family are key regulators of ZGA in placental mammals1,2. To characterize the role of DUX in ZGA, we generated Dux cluster knockout (KO) mouse lines. Unexpectedly, we found that both Dux zygotic KO (Z-KO) and maternal and zygotic KO (MZ-KO) embryos can survive to adulthood despite showing reduced developmental potential. Furthermore, transcriptome profiling of the MZ-KO embryos revealed that loss of DUX has minimal effects on ZGA and most DUX targets in 2C-like cells are normally activated in MZ-KO embryos. Thus, contrary to the key function of DUX in inducing 2C-like cells, our data indicate that DUX has only a minor role in ZGA and that loss of DUX is compatible with mouse development.

RevDate: 2019-07-08
CmpDate: 2019-07-08

Soriano V (2019)

Gene Editing for HIV Cure at the Edge.

AIDS reviews, 21(1):50a-51.

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ESP Quick Facts

ESP Origins

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

ESP Support

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

ESP Rationale

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

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

ESP Usage

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

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

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

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

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CRISPR-Cas

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

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Papers in Classical Genetics

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

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

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