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19 Apr 2019 at 08:39
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Bibliography on: CRISPR-Cas


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RJR: Recommended Bibliography 19 Apr 2019 at 08:39 Created: 


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

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

Citations The Papers (from PubMed®)

RevDate: 2019-04-18

Tran MTN, Khalid MKNM, Pébay A, et al (2019)

Screening of CRISPR/Cas base editors to target the AMD high-risk Y402H complement factor H variant.

Molecular vision, 25:174-182.

Purpose: To evaluate the efficacy of using a CRISPR/Cas-mediated strategy to correct a common high-risk allele that is associated with age-related macular degeneration (AMD; rs1061170; NM_000186.3:c.1204T>C; NP_000177.2:p.His402Tyr) in the complement factor H (CFH) gene.

Methods: A human embryonic kidney cell line (HEK293A) was engineered to contain the pathogenic risk variant for AMD (HEK293A-CFH). Several different base editor constructs (BE3, SaBE3, SaKKH-BE3, VQR-BE3, and Target-AID) and their respective single-guide RNA (sgRNA) expression cassettes targeting either the pathogenic risk variant allele in the CFH locus or the LacZ gene, as a negative control, were evaluated head-to-head for the incidence of a cytosine-to-thymine nucleotide correction. The base editor construct that showed appreciable editing activity was selected for further assessment in which the base-edited region was subjected to next-generation deep sequencing to quantify on-target and off-target editing efficacy.

Results: The tandem use of the Target-AID base editor and its respective sgRNA demonstrated a base editing efficiency of facilitating a cytosine-to-thymine nucleotide correction in 21.5% of the total sequencing reads. Additionally, the incidence of insertions and deletions (indels) was detected in only 0.15% of the sequencing reads with virtually no off-target effects evident across the top 11 predicted off-target sites containing at least one cytosine in the activity window (n = 3, pooled amplicons).

Conclusions: CRISPR-mediated base editing can be used to facilitate a permanent and stably inherited cytosine-to-thymine nucleotide correction of the rs1061170 SNP in the CFH gene with minimal off-target effects.

RevDate: 2019-04-18

Molla KA, Y Yang (2019)

CRISPR/Cas-Mediated Base Editing: Technical Considerations and Practical Applications.

Trends in biotechnology pii:S0167-7799(19)30053-8 [Epub ahead of print].

Genome editing with CRISPR/Cas has rapidly gained popularity. Base editing, a new CRISPR/Cas-based approach, can precisely convert one nucleotide to another in DNA or RNA without inducing a double-strand DNA break (DSB). A combination of catalytically impaired nuclease variants with different deaminases has yielded diverse base-editing platforms that aim to address the key limitations such as specificity, protospacer adjacent motif (PAM) compatibility, editing window length, bystander editing, and sequence context preference. Because new base editors significantly reduce unintended editing in the genome, they hold great promise for treating genetic diseases and for developing superior agricultural crops. We review here the development of various base editors, assess their technical advantages and limitations, and discuss their broad applications in basic research, medicine, and agriculture.

RevDate: 2019-04-17

Grünewald J, Zhou R, Garcia SP, et al (2019)

Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.

Nature pii:10.1038/s41586-019-1161-z [Epub ahead of print].

CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications1,2. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex3,4. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells5,6. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38%-58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5' UTR, and 3' UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.

RevDate: 2019-04-17

Makarova KS, Karamycheva S, Shah SA, et al (2019)

Predicted highly derived class 1 CRISPR-Cas system in Haloarchaea containing diverged Cas5 and Cas7 homologs but no CRISPR array.

FEMS microbiology letters pii:5472869 [Epub ahead of print].

Screening of genomic and metagenomic databases for new variants of CRISPR-Cas systems increasingly results in the discovery of derived variants that do not seem to possess the interference capacity and are implicated in functions distinct from adaptive immunity. We describe an extremely derived putative class 1 CRISPR-Cas system that is present in many Halobacteria and consists of distant homologs of the Cas5 and Cas7 protein along with an uncharacterized conserved protein and various nucleases. We hypothesize that, although this system lacks typical CRISPR effectors or a CRISPR array, it functions as a RNA-dependent defense mechanism that, unlike other derived CRISPR-Cas, utilizes alternative nucleases to cleave invader genomes.

RevDate: 2019-04-16

Barkau CL, O'Reilly D, Rohilla KJ, et al (2019)

Rationally Designed Small Nucleic Acid-Based Inhibitors of CRISPR-Cas9.

Nucleic acid therapeutics [Epub ahead of print].

Clustered regularly interspaced short palindromic repeat (CRISPR) RNAs and their associated effector (Cas) enzymes are being developed into promising therapeutics to treat disease. However, CRISPR-Cas enzymes might produce unwanted gene editing or dangerous side effects. Drug-like molecules that can inactivate CRISPR-Cas enzymes could help facilitate safer therapeutic development. Based on the requirement of guide RNA and target DNA interaction by Cas enzymes, we rationally designed small nucleic acid-based inhibitors (SNuBs) of Streptococcus pyogenes (Sp) Cas9. Inhibitors were initially designed as 2'-O-methyl-modified oligonucleotides that bound the CRISPR RNA guide sequence (anti-guide) or repeat sequence (anti-tracr), or DNA oligonucleotides that bound the protospacer adjacent motif (PAM)-interaction domain (anti-PAM) of SpCas9. Coupling anti-PAM and anti-tracr modules together was synergistic and resulted in high binding affinity and efficient inhibition of Cas9 DNA cleavage activity. Incorporating 2'F-RNA and locked nucleic acid nucleotides into the anti-tracr module resulted in greater inhibition as well as dose-dependent suppression of gene editing in human cells. CRISPR SNuBs provide a platform for rational design of CRISPR-Cas enzyme inhibitors that should translate to other CRISPR effector enzymes and enable better control over CRISPR-based applications.

RevDate: 2019-04-16

Pasari N, Gupta M, Eqbal D, et al (2019)

Genome analysis of Paenibacillus polymyxa A18 gives insights into the features associated with its adaptation to the termite gut environment.

Scientific reports, 9(1):6091 pii:10.1038/s41598-019-42572-5.

Paenibacillus polymyxa A18 was isolated from termite gut and was identified as a potential cellulase and hemicellulase producer in our previous study. Considering that members belonging to genus Paenibacillus are mostly free-living in soil, we investigated here the essential genetic features that helped P. polymyxa A18 to survive in gut environment. Genome sequencing and analysis identified 4608 coding sequences along with several elements of horizontal gene transfer, insertion sequences, transposases and integrated phages, which add to its genetic diversity. Many genes coding for carbohydrate-active enzymes, including the enzymes responsible for woody biomass hydrolysis in termite gut, were identified in P. polymyxa A18 genome. Further, a series of proteins conferring resistance to 11 antibiotics and responsible for production of 4 antibiotics were also found to be encoded, indicating selective advantage for growth and colonization in the gut environment. To further identify genomic regions unique to this strain, a BLAST-based comparative analysis with the sequenced genomes of 47 members belonging to genus Paenibacillus was carried out. Unique regions coding for nucleic acid modifying enzymes like CRISPR/Cas and Type I Restriction-Modification enzymes were identified in P. polymyxa A18 genome suggesting the presence of defense mechanism to combat viral infections in the gut. In addition, genes responsible for the formation of biofilms, such as Type IV pili and adhesins, which might be assisting P. polymyxa A18 in colonizing the gut were also identified in its genome. In situ colonization experiment further confirmed the ability of P. polymyxa A18 to colonize the gut of termite.

RevDate: 2019-04-15

Hoffmann MD, Aschenbrenner S, Grosse S, et al (2019)

Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins.

Nucleic acids research pii:5458120 [Epub ahead of print].

The rapid development of CRISPR-Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR-Cas orthologue, for which a potent anti-CRISPR protein is known.

RevDate: 2019-04-13

Zirpel H, J Clos (2019)

Gene Replacement by Homologous Recombination.

Methods in molecular biology (Clifton, N.J.), 1971:169-188.

While homologous recombination-based gene replacement is about to be supplanted by more modern approaches, it is still retaining usefulness for genes that prove to be poor targets for CRISPR/cas-based approaches. Homologous recombination has proven to be relatively robust to minor sequence mismatches between GOI-flanking sequences and the gene replacement constructs, and the faithfulness of recombination events is easily verified by whole-genome sequencing. Moreover, the availability of custom synthetic gene production by numerous service providers should allow for a relatively quick generation of null mutants without the need to introduce additional protein-coding genes beyond the selection markers.

RevDate: 2019-04-12

Zhang H, Dong C, Li L, et al (2019)

Structural insights into the modulatory role of the accessory protein WYL1 in the Type VI-D CRISPR-Cas system.

Nucleic acids research pii:5446253 [Epub ahead of print].

The Type VI-D CRISPR-Cas system employs an RNA-guided RNase Cas13d with minimal targeting constraints to combat viral infections. This CRISPR system contains RspWYL1 as a unique accessory protein that plays a key role in boosting its effector function on target RNAs, but the mechanism behind this RspWYL1-mediated stimulation remains completely unexplored. Through structural and biophysical approaches, we reveal that the full-length RspWYL1 possesses a novel three-domain architecture and preferentially binds ssRNA with high affinity. Specifically, the N-terminus of RspWYL1 harbors a ribbon-helix-helix motif reminiscent of transcriptional regulators; the central WYL domain of RspWYL1 displays a Sm-like β-barrel fold; and the C-terminal domain of RspWYL1 primarily contributes to the dimerization of RspWYL1 and may regulate the RspWYL1 function via a large conformational change. Collectively, this study provides a first glimpse into the complex mechanism behind the RspWYL1-dictated boosting of target ssRNA cleavage in the Type VI-D CRISPR-Cas system.

RevDate: 2019-04-16

Dolan AE, Hou Z, Xiao Y, et al (2019)

Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas.

Molecular cell pii:S1097-2765(19)30217-5 [Epub ahead of print].

CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicase-nuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%-60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes.

RevDate: 2019-04-11

Naduthodi MIS, Mohanraju P, Südfeld C, et al (2019)

CRISPR-Cas ribonucleoprotein mediated homology-directed repair for efficient targeted genome editing in microalgae Nannochloropsis oceanica IMET1.

Biotechnology for biofuels, 12:66 pii:1401.

Background: Microalgae are considered as a sustainable feedstock for the production of biofuels and other value-added compounds. In particular, Nannochloropsis spp. stand out from other microalgal species due to their capabilities to accumulate both triacylglycerol (TAG) and polyunsaturated fatty acids (PUFAs). However, the commercialization of microalgae-derived products is primarily hindered by the high production costs compared to less sustainable alternatives. Efficient genome editing techniques leading to effective metabolic engineering could result in strains with enhanced productivities of interesting metabolites and thereby reduce the production costs. Competent CRISPR-based genome editing techniques have been reported in several microalgal species, and only very recently in Nannochloropsis spp. (2017). All the reported CRISPR-Cas-based systems in Nannochloropsis spp. rely on plasmid-borne constitutive expression of Cas9 and a specific guide, combined with repair of double-stranded breaks (DSB) by non-homologous end joining (NHEJ) for the target gene knockout.

Results: In this study, we report for the first time an alternative approach for CRISPR-Cas-mediated genome editing in Nannochloropsis sp.; the Cas ribonucleoproteins (RNP) and an editing template were directly delivered into microalgal cells via electroporation, making Cas expression dispensable and homology-directed repair (HDR) possible with high efficiency. Apart from widely used SpCas9, Cas12a variants from three different bacterium were used for this approach. We observed that FnCas12a from Francisella novicida generated HDR-based targeted mutants with highest efficiency (up to 93% mutants among transformants) while AsCas12a from Acidaminococcus sp. resulted in the lowest efficiency. We initially show that the native homologous recombination (HR) system in N. oceanica IMET1 is not efficient for easy isolation of targeted mutants by HR. Cas9/sgRNA RNP delivery greatly enhanced HR at the target site, generating around 70% of positive mutant lines.

Conclusion: We show that the delivery of Cas RNP by electroporation can be an alternative approach to the presently reported plasmid-based Cas9 method for generating mutants of N. oceanica. The co-delivery of Cas-RNPs along with a dsDNA repair template efficiently enhanced HR at the target site, resulting in a remarkable higher percentage of positive mutant lines. Therefore, this approach can be used for efficient generation of targeted mutants in Nannochloropsis sp. In addition, we here report the activity of several Cas12a homologs in N. oceanica IMET1, identifying FnCas12a as the best performer for high efficiency targeted genome editing.

RevDate: 2019-04-15
CmpDate: 2019-04-15

Bao A, Chen H, Chen L, et al (2019)

CRISPR/Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean.

BMC plant biology, 19(1):131 pii:10.1186/s12870-019-1746-6.

BACKGROUND: The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants.

RESULTS: In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean.

CONCLUSIONS: Our results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.

RevDate: 2019-04-12

Sunwoo IY, Sukwong P, Jeong DY, et al (2019)

Enhancement of galactose consumption rate in Saccharomyces cerevisiae CEN.PK2-1 by CRISPR Cas9 and adaptive evolution for fermentation of Kappaphycus alvarezii hydrolysate.

Journal of biotechnology, 297:78-84 pii:S0168-1656(19)30093-8 [Epub ahead of print].

Ethanol ferrmentation of Kappaphycus alvarezii hydrolysates was performed using wild-type (WT) Saccharomyces cerevisiae CEN.PK2-1, hexokinase 2 deleted (Δhxk2) and adapted strain on high galactose concentrations. The WT and Δhxk2 strains produced 8.9 and 14.67 g/L of ethanol with yield coefficient (YEtOH) of 0.20 and 0.33 (g/g), respectively. However, neither the WT nor Δhxk2strain could utilize all of the galactose, leaving 16.4 and 6.2 g/L of galactose in the fermentation broth, respectively. Therefore, fermentation with S. cerevisiae CEN.PK2-1 adapted to galactose was carried out to increase the ethanol yield coefficient (YEtOH), producing a maximum ethanol concentration of 20.0 g/L with a YEtOH of 0.44 (g/g). Ethanol concentration of adapted strain was 1.36-2.25 times higher than WT and Δhxk2 strains. The adapted yeast exhibited the highest transcript levels of GAL genes. The yeast strain via adaptive yeast strain produced ethanol with a higher titer and yield due to a modular activation of GAL genes than WT or the hxk2 deleted strains.

RevDate: 2019-04-08

Fineran PC (2019)

Resistance is not futile: bacterial 'innate' and CRISPR-Cas 'adaptive' immune systems.

Microbiology (Reading, England) [Epub ahead of print].

Bacteria are under a constant pressure from their viruses (phages) and other mobile genetic elements. They protect themselves through a range of defence strategies, which can be broadly classified as 'innate' and 'adaptive'. The bacterial innate immune systems include defences provided by restriction modification and abortive infection, among others. Bacterial adaptive immunity is elicited by a diverse range of CRISPR-Cas systems. Here, I discuss our research on both innate and adaptive phage resistance mechanisms and some of the evasion strategies employed by phages.

RevDate: 2019-04-12

Koujah L, Shukla D, AR Naqvi (2019)

CRISPR-Cas based targeting of host and viral genes as an antiviral strategy.

Seminars in cell & developmental biology pii:S1084-9521(18)30108-3 [Epub ahead of print].

Viral infections in human are leading cause of mortality and morbidity across the globe. Several viruses (including HIV and Herpesvirus), have evolved ingenious strategies to evade host-immune system and persist life-long. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) is an ancient antiviral system recently discovered in bacteria that has shown tremendous potential as a precise, invariant genome editing tool. Using CRISPR-Cas based system to activate host defenses or genetic modification of viral genome can provide novel, exciting and successful antiviral mechanisms and treatment modalities. In this review, we will provide progress on the CRISPR-Cas based antiviral approaches that facilitate clearance of virus-infected cells and/or prohibit virus infection or replication. We will discuss on the possibilities of CRIPSR-Cas as prophylaxis and therapy in viral infections and review the challenges of this potent gene editing technology.

RevDate: 2019-04-06

He S, Cuentas-Condori A, DM Miller (2019)

NATF (Native And Tissue-Specific Fluorescence): A Strategy for Bright, Tissue-Specific GFP Labeling of Native Proteins in Caenorhabditis elegans.

Genetics pii:genetics.119.302063 [Epub ahead of print].

GFP labeling by genome editing can reveal the authentic location of a native protein but is frequently hampered by weak GFP signals and broad expression across a range of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy which combines CRISPR/Cas-9 and split-GFP to yield bright, cell-specific protein labeling. We use CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 ß-strand (gfp11x7) at the genomic locus of each target protein. The resultant gfp11x7 knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment (gfp1-10) in specific cell types thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein, OIG-1, and demarcates the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the C. elegans nervous system.

RevDate: 2019-04-09

Li L, Liu X, Wei K, et al (2019)

Synthetic biology approaches for chromosomal integration of genes and pathways in industrial microbial systems.

Biotechnology advances pii:S0734-9750(19)30061-8 [Epub ahead of print].

Industrial biotechnology is reliant on native pathway engineering or foreign pathway introduction for efficient biosynthesis of target products. Chromosomal integration, with intrinsic genetic stability, is an indispensable step for reliable expression of homologous or heterologous genes and pathways in large-scale and long-term fermentation. With advances in synthetic biology and CRISPR-based genome editing approaches, a wide variety of novel enabling technologies have been developed for single-step, markerless, multi-locus genomic integration of large biochemical pathways, which significantly facilitate microbial overproduction of chemicals, pharmaceuticals and other value-added biomolecules. Notably, the newly discovered homology-mediated end joining strategy could be widely applicable for high-efficiency genomic integration in a number of homologous recombination-deficient microbes. In this review, we explore the fundamental principles and characteristics of genomic integration, and highlight the development and applications of targeted integration approaches in the three representative industrial microbial systems, including Escherichia coli, actinomycetes and yeasts.

RevDate: 2019-04-17

Patsali P, Kleanthous M, CW Lederer (2019)

Disruptive Technology: CRISPR/Cas-Based Tools and Approaches.

Molecular diagnosis & therapy, 23(2):187-200.

Designer nucleases are versatile tools for genome modification and therapy development and have gained widespread accessibility with the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology. Prokaryotic RNA-guided nucleases of CRISPR/Cas type, since first being adopted as editing tools in eukaryotic cells, have experienced rapid uptake and development. Diverse modes of delivery by viral and non-viral vectors and ongoing discovery and engineering of new CRISPR/Cas-type tools with alternative target site requirements, cleavage patterns and DNA- or RNA-specific action continue to expand the versatility of this family of nucleases. CRISPR/Cas-based molecules may also act without double-strand breaks as DNA base editors or even without single-stranded cleavage, be it as epigenetic regulators, transcription factors or RNA base editors, with further scope for discovery and development. For many potential therapeutic applications of CRISPR/Cas-type molecules and their derivatives, efficiencies still need to be improved and safety issues addressed, including those of preexisting immunity against Cas molecules, off-target activity and recombination and sequence alterations relating to double-strand-break events. This review gives a concise overview of current CRISPR/Cas tools, applications, concerns and trends.

RevDate: 2019-04-17

Papasavva P, Kleanthous M, CW Lederer (2019)

Rare Opportunities: CRISPR/Cas-Based Therapy Development for Rare Genetic Diseases.

Molecular diagnosis & therapy, 23(2):201-222.

Rare diseases pose a global challenge, in that their collective impact on health systems is considerable, whereas their individually rare occurrence impedes research and development of efficient therapies. In consequence, patients and their families are often unable to find an expert for their affliction, let alone a cure. The tide is turning as pharmaceutical companies embrace gene therapy development and as serviceable tools for the repair of primary mutations separate the ability to create cures from underlying disease expertise. Whereas gene therapy by gene addition took decades to reach the clinic by incremental disease-specific refinements of vectors and methods, gene therapy by genome editing in its basic form merely requires certainty about the causative mutation. Suddenly we move from concept to trial in 3 years instead of 30: therapy development in the fast lane, with all the positive and negative implications of the phrase. Since their first application to eukaryotic cells in 2013, the proliferation and refinement in particular of tools based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) prokaryotic RNA-guided nucleases has prompted a landslide of therapy-development studies for rare diseases. An estimated thousands of orphan diseases are up for adoption, and legislative, entrepreneurial, and research initiatives may finally conspire to find many of them a good home. Here we summarize the most significant recent achievements and remaining hurdles in the application of CRISPR/Cas technology to rare diseases and take a glimpse at the exciting road ahead.

RevDate: 2019-04-16

Xu J, Pei D, Nicholson A, et al (2019)

In Silico Identification of Three Types of Integrative and Conjugative Elements in Elizabethkingia anophelis Strains Isolated from around the World.

mSphere, 4(2): pii:4/2/e00040-19.

Elizabethkingia anophelis is an emerging global multidrug-resistant opportunistic pathogen. We assessed the diversity among 13 complete genomes and 23 draft genomes of E. anophelis strains derived from various environmental settings and human infections from different geographic regions around the world from 1950s to the present. Putative integrative and conjugative elements (ICEs) were identified in 31/36 (86.1%) strains in the study. A total of 52 putative ICEs (including eight degenerated elements lacking integrases) were identified and categorized into three types based on the architecture of the conjugation module and the phylogeny of the relaxase, coupling protein, TraG, and TraJ protein sequences. The type II and III ICEs were found to integrate adjacent to tRNA genes, while type I ICEs integrate into intergenic regions or into a gene. The ICEs carry various cargo genes, including transcription regulator genes and genes conferring antibiotic resistance. The adaptive immune CRISPR-Cas system was found in nine strains, including five strains in which CRISPR-Cas machinery and ICEs coexist at different locations on the same chromosome. One ICE-derived spacer was present in the CRISPR locus in one strain. ICE distribution in the strains showed no geographic or temporal patterns. The ICEs in E. anophelis differ in architecture and sequence from CTnDOT, a well-studied ICE prevalent in Bacteroides spp. The categorization of ICEs will facilitate further investigations of the impact of ICE on virulence, genome epidemiology, and adaptive genomics of E. anophelisIMPORTANCEElizabethkingia anophelis is an opportunistic human pathogen, and the genetic diversity between strains from around the world becomes apparent as more genomes are sequenced. Genome comparison identified three types of putative ICEs in 31 of 36 strains. The diversity of ICEs suggests that they had different origins. One of the ICEs was discovered previously from a large E. anophelis outbreak in Wisconsin in the United States; this ICE has integrated into the mutY gene of the outbreak strain, creating a mutator phenotype. Similar to ICEs found in many bacterial species, ICEs in E. anophelis carry various cargo genes that enable recipients to resist antibiotics and adapt to various ecological niches. The adaptive immune CRISPR-Cas system is present in nine of 36 strains. An ICE-derived spacer was found in the CRISPR locus in a strain that has no ICE, suggesting a past encounter and effective defense against ICE.

RevDate: 2019-04-07

Chou-Zheng L, A Hatoum-Aslan (2019)

A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity.

eLife, 8: pii:45393.

CRISPR-Cas systems provide sequence-specific immunity against phages and mobile genetic elements using CRISPR-associated nucleases guided by short CRISPR RNAs (crRNAs). Type III systems exhibit a robust immune response that can lead to the extinction of a phage population, a feat coordinated by a multi-subunit effector complex that destroys invading DNA and RNA. Here, we demonstrate that a model type III system in Staphylococcus epidermidis relies upon the activities of two degradosome-associated nucleases, PNPase and RNase J2, to mount a successful defense. Genetic, molecular, and biochemical analyses reveal that PNPase promotes crRNA maturation, and both nucleases are required for efficient clearance of phage-derived nucleic acids. Furthermore, functional assays show that RNase J2 is essential for immunity against diverse mobile genetic elements originating from plasmid and phage. Altogether, our observations reveal the evolution of a critical collaboration between two nucleic acid degrading machines which ensures cell survival when faced with phage attack.

RevDate: 2019-04-15
CmpDate: 2019-04-15

Cai P, Gao J, Y Zhou (2019)

CRISPR-mediated genome editing in non-conventional yeasts for biotechnological applications.

Microbial cell factories, 18(1):63 pii:10.1186/s12934-019-1112-2.

Non-conventional yeasts are playing important roles as cell factories for bioproduction of biofuels, food additives and proteins with outstanding natural characteristics. However, the precise genome editing is challenging in non-conventional yeasts due to lack of efficient genetic tools. In the past few years, CRISPR-based genome editing worked as a revolutionary tool for genetic engineering and showed great advantages in cellular metabolic engineering. Here, we review the current advances and barriers of CRISPR-Cas9 for genome editing in non-conventional yeasts and propose the possible solutions in enhancing its efficiency for precise genetic engineering.

RevDate: 2019-04-02

Almendros C, Nobrega FL, McKenzie RE, et al (2019)

Cas4-Cas1 fusions drive efficient PAM selection and control CRISPR adaptation.

Nucleic acids research pii:5425342 [Epub ahead of print].

Microbes have the unique ability to acquire immunological memories from mobile genetic invaders to protect themselves from predation. To confer CRISPR resistance, new spacers need to be compatible with a targeting requirement in the invader's DNA called the protospacer adjacent motif (PAM). Many CRISPR systems encode Cas4 proteins to ensure new spacers are integrated that meet this targeting prerequisite. Here we report that a gene fusion between cas4 and cas1 from the Geobacter sulfurreducens I-U CRISPR-Cas system is capable of introducing functional spacers carrying interference proficient TTN PAM sequences at much higher frequencies than unfused Cas4 adaptation modules. Mutations of Cas4-domain catalytic residues resulted in dramatically decreased naïve and primed spacer acquisition, and a loss of PAM selectivity showing that the Cas4 domain controls Cas1 activity. We propose the fusion gene evolved to drive the acquisition of only PAM-compatible spacers to optimize CRISPR interference.

RevDate: 2019-04-04

Dong L, Guan X, Li N, et al (2019)

An anti-CRISPR protein disables type V Cas12a by acetylation.

Nature structural & molecular biology, 26(4):308-314.

Phages use anti-CRISPR proteins to deactivate the CRISPR-Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained unknown how the type V CRISPR-Cas12a (Cpf1) system is inhibited by anti-CRISPRs. Here we identify the anti-CRISPR protein AcrVA5 and report the mechanisms by which it inhibits CRISPR-Cas12a. Our structural and biochemical data show that AcrVA5 functions as an acetyltransferase to modify Moraxella bovoculi (Mb) Cas12a at Lys635, a residue that is required for recognition of the protospacer-adjacent motif. The AcrVA5-mediated modification of MbCas12a results in complete loss of double-stranded DNA (dsDNA)-cleavage activity. In contrast, the Lys635Arg mutation renders MbCas12a completely insensitive to inhibition by AcrVA5. A cryo-EM structure of the AcrVA5-acetylated MbCas12a reveals that Lys635 acetylation provides sufficient steric hindrance to prevent dsDNA substrates from binding to the Cas protein. Our study reveals an unprecedented mechanism of CRISPR-Cas inhibition and suggests an evolutionary arms race between phages and bacteria.

RevDate: 2019-04-02

Zhang Z, Pan S, Liu T, et al (2019)

Cas4 nucleases can effect specific integration of CRISPR spacers.

Journal of bacteriology pii:JB.00747-18 [Epub ahead of print].

CRISPR-Cas systems incorporate short DNA fragments from invasive genetic elements into host CRISPR arrays in order to generate host immunity. Recently, we demonstrated that the Csa3a regulator protein triggers CCN PAM-dependent CRISPR spacer acquisition in the subtype I-A CRISPR-Cas system of Sulfolobus islandicus However, the mechanisms underlying specific protospacer selection and spacer insertion remained unclear. Here, we demonstrate that two Cas4 family proteins (Cas4 and Csa1) have essential roles (a) in recognizing the 5' PAM and 3' nucleotide motif of protospacers and (b) in determining both the spacer length and its orientation. Furthermore, we identify amino acid residues of the Cas4 proteins that facilitate these functions. Overexpression of the Cas4 and Csa1 proteins, and also of an archaeal virus-encoded Cas4 protein, resulted in strongly reduced adaptation efficiency and the former proteins yielded a high incidence of PAM-dependent atypical spacer integration, or of PAM-independent spacer integration. We further demonstrated that, in the plasmid challenging experiments, overexpressed Cas4-mediated defective spacer acquisition, in turn, potentially enabled targeted DNA to escape subtype I-A CRISPR-Cas interference. In summary, these results define the specific involvement of diverse Cas4 proteins in in vivo CRISPR spacer acquisition. Furthermore, we provide support for an anti-CRISPR role for virus-encoded Cas4 proteins that involves compromising CRISPR-Cas interference activity by hindering spacer acquisition.Importance The Cas4 family endonuclease is an essential component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems. The Crenarchaeota Sulfolobus islandicus REY15A encodes two cas4 genes (cas4 and csa1) linked to the CRISPR arrays. Here, we demonstrate that Cas4 and Csa1 are essential to CRISPR spacer acquisition in this organism. Both proteins specify the upstream and downstream conserved nucleotide motifs of the protospacers and define the spacer length and orientation in the acquisition process. Conserved amino acid residues, in addition to the recently reported, were identified to be important for above functions. More importantly, overexpression of the Sulfolobus viral Cas4 abolished spacer acquisition, providing support for an anti-CRISPR role for virus-encoded Cas4 proteins that inhibit spacer acquisition.

RevDate: 2019-04-03

Koonin EV (2019)

CRISPR: a new principle of genome engineering linked to conceptual shifts in evolutionary biology.

Biology & philosophy, 34(1):9.

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity have become a household name among biologists and even the general public thanks to the unprecedented success of the new generation of genome editing tools utilizing Cas proteins. However, the fundamental biological features of CRISPR-Cas are of no lesser interest and have major impacts on our understanding of the evolution of antivirus defense, host-parasite coevolution, self versus non-self discrimination and mechanisms of adaptation. CRISPR-Cas systems present the best known case in point for Lamarckian evolution, i.e. generation of heritable, adaptive genomic changes in response to encounters with external factors, in this case, foreign nucleic acids. CRISPR-Cas systems employ multiple mechanisms of self versus non-self discrimination but, as is the case with immune systems in general, are nevertheless costly because autoimmunity cannot be eliminated completely. In addition to the autoimmunity, the fitness cost of CRISPR-Cas systems appears to be determined by their inhibitory effect on horizontal gene transfer, curtailing evolutionary innovation. Hence the dynamic evolution of CRISPR-Cas loci that are frequently lost and (re)acquired by archaea and bacteria. Another fundamental biological feature of CRISPR-Cas is its intimate connection with programmed cell death and dormancy induction in microbes. In this and, possibly, other immune systems, active immune response appears to be coupled to a different form of defense, namely, "altruistic" shutdown of cellular functions resulting in protection of neighboring cells. Finally, analysis of the evolutionary connections of Cas proteins reveals multiple contributions of mobile genetic elements (MGE) to the origin of various components of CRISPR-Cas systems, furthermore, different biological systems that function by genome manipulation appear to have evolved convergently from unrelated MGE. The shared features of adaptive defense systems and MGE, namely the ability to recognize and cleave unique sites in genomes, make them ideal candidates for genome editing and engineering tools.

RevDate: 2019-04-06

Wang F, Wang L, Zou X, et al (2019)

Advances in CRISPR-Cas systems for RNA targeting, tracking and editing.

Biotechnology advances pii:S0734-9750(19)30052-7 [Epub ahead of print].

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely used in gene/genome targeting. Modifications of Cas9 enable these systems to become platforms for precise DNA manipulations. However, the utilization of CRISPR-Cas systems in RNA targeting remains preliminary. The discovery of type VI CRISPR-Cas systems (Cas13) shed light on RNA-guided RNA targeting. Cas13d, the smallest Cas13 protein, with a length of only ~930 amino acids, is a promising platform for RNA targeting compatible with viral delivery systems. Much effort has also been made to develop Cas9, Cas13a and Cas13b applications for RNA-guided RNA targeting. The discovery of new RNA-targeting CRISPR-Cas systems as well as the development of RNA-targeting platforms with Cas9 and Cas13 will promote RNA-targeting technology substantially. Here, we review new advances in RNA-targeting CRISPR-Cas systems as well as advances in applications of these systems in RNA targeting, tracking and editing. We also compare these Cas protein-based technologies with traditional technologies for RNA targeting, tracking and editing. Finally, we discuss remaining questions and prospects for the future.

RevDate: 2019-03-27

Slaymaker IM, Mesa P, Kellner MJ, et al (2019)

High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage.

Cell reports, 26(13):3741-3751.e5.

Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells.

RevDate: 2019-04-09

Babu K, Amrani N, Jiang W, et al (2019)

Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance.

Biochemistry, 58(14):1905-1917.

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. The programmable nature of CRISPR-targeting mechanisms has enabled their adaptation as powerful genome engineering tools. Cas9, a type II CRISPR effector protein, has been widely used for gene-editing applications owing to the fact that a single-guide RNA can direct Cas9 to cleave desired genomic targets. An understanding of the role of different domains of the protein and guide RNA-induced conformational changes of Cas9 in selecting target DNA has been and continues to enable development of Cas9 variants with reduced off-targeting effects. It has been previously established that an arginine-rich bridge helix (BH) present in Cas9 is critical for its activity. In the present study, we show that two proline substitutions within a loop region of the BH of Streptococcus pyogenes Cas9 impair the DNA cleavage activity by accumulating nicked products and reducing target DNA linearization. This in turn imparts a higher selectivity in DNA targeting. We discuss the probable mechanisms by which the BH-loop contributes to target DNA recognition.

RevDate: 2019-03-26

Fu J, Yang F, Xie H, et al (2019)

[Application and optimization of CRISPR/Cas system in bacteria].

Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 35(3):341-350.

Clustered regular interspaced short palindromic repeats (CRISPR) system has been widely used in recent years. Compared with traditional genome editing technology, CRISPR/Cas system has notable advantages, including high editing efficiency, high specificity, low cost and the convenience for manipulation. Type Ⅱ and Ⅴ CRISPR/Cas system only requires a single Cas9 protein or a single Cpf1 protein as effector nucleases for cutting double-stranded DNA, developed as genome editing tools. At present, CRISPR/Cas9 technology has been successfully applied to the genome editing of eukaryotes such as zebrafish, mice and human cells, whereas limited progress has been made in the genome editing of bacteria. In our review, we describe CRISPR/Cas system, its mechanism and summarize the optimization and progress of genome editing in bacteria.

RevDate: 2019-03-26

Wadhwa R, Aggarwal T, Malyla V, et al (2019)

Identification of biomarkers and genetic approaches toward chronic obstructive pulmonary disease.

Journal of cellular physiology [Epub ahead of print].

Chronic obstructive pulmonary disease accounts as the leading cause of mortality worldwide prominently affected by genetic and environmental factors. The disease is characterized by persistent coughing, breathlessness airways inflammation followed by a decrease in forced expiratory volume1 and exacerbations, which affect the quality of life. Determination of genetic, epigenetic, and oxidant biomarkers to evaluate the progression of disease has proved complicated and challenging. Approaches including exome sequencing, genome-wide association studies, linkage studies, and inheritance and segregation studies played a crucial role in the identification of genes, their pathways and variation in genes. This review highlights multiple approaches for biomarker and gene identification, which can be used for differential diagnosis along with the genome editing tools to study genes associated with the development of disease and models their function. Further, we have discussed the approaches to rectify the abnormal gene functioning of respiratory tissues and various novel gene editing techniques like Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9).

RevDate: 2019-04-10

Vochozkova P, Simmet K, Jemiller EM, et al (2019)

Gene Editing in Primary Cells of Cattle and Pig.

Methods in molecular biology (Clifton, N.J.), 1961:271-289.

Gene Editing by CRISPR/Cas has revolutionized many aspects of biotechnology within a short period of time. This is also true for the genetic manipulation of livestock species, but their specific challenges such as the lack of stem cells, the limited proliferative capacity of primary cells, and the genetic diversity of the pig and cattle populations need consideration when CRISPR/Cas is applied. Here we present guidelines for CRISPRing primary cells in pig and cattle, with a specific focus on testing gRNA in vitro, on generating single cell clones, and on identifying modifications in single cell clones.

RevDate: 2019-04-10

Brinkman EK, B van Steensel (2019)

Rapid Quantitative Evaluation of CRISPR Genome Editing by TIDE and TIDER.

Methods in molecular biology (Clifton, N.J.), 1961:29-44.

Current genome editing tools enable targeted mutagenesis of selected DNA sequences in many species. However, the efficiency and the type of introduced mutations by the genome editing method are largely dependent on the target site. As a consequence, the outcome of the editing operation is difficult to predict. Therefore, a quick assay to quantify the frequency of mutations is vital for a proper assessment of genome editing actions. We developed two methods that are rapid, cost-effective, and readily applicable: (1) TIDE, which can accurately identify and quantify insertions and deletions (indels) that arise after introduction of double strand breaks (DSBs); (2) TIDER, which is suited for template-mediated editing events including point mutations. Both methods only require a set of PCR reactions and standard Sanger sequencing runs. The sequence traces are analyzed by the TIDE or TIDER algorithm (available at https://tide.nki.nl or https://deskgen.com). The routine is easy, fast, and provides much more detailed information than current enzyme-based assays. TIDE and TIDER accelerate testing and designing of DSB-based genome editing strategies.

RevDate: 2019-03-25

Brandt K, R Barrangou (2019)

Applications of CRISPR Technologies Across the Food Supply Chain.

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

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

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

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

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

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

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

RevDate: 2019-03-29

Kim JH (2018)

Genetics of Alzheimer's Disease.

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

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

RevDate: 2019-03-25

Zhang F (2019)

Exploration of Microbial Diversity to Discover Novel Molecular Technologies.

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

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

RevDate: 2019-03-25

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

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

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

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

RevDate: 2019-03-25

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

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

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

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

RevDate: 2019-03-25

Ramachandran G, D Bikard (2019)

Editing the microbiome the CRISPR way.

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

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

RevDate: 2019-03-25

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

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

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

RevDate: 2019-03-25

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

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

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

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

RevDate: 2019-03-25

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

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

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

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

RevDate: 2019-03-25

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

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

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

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

RevDate: 2019-03-25

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

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

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

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

RevDate: 2019-03-25

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

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

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

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

RevDate: 2019-04-18

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

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

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

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

RevDate: 2019-03-25

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

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

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

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

RevDate: 2019-03-25

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

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

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

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

RevDate: 2019-03-25

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

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

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

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

RevDate: 2019-03-25

Koonin EV, KS Makarova (2019)

Origins and evolution of CRISPR-Cas systems.

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

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

RevDate: 2019-03-25

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

Variability in the durability of CRISPR-Cas immunity.

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

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

RevDate: 2019-03-25

Gurney J, Pleška M, BR Levin (2019)

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

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

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

RevDate: 2019-03-25

Bradde S, Mora T, AM Walczak (2019)

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

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

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

RevDate: 2019-03-22

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

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

Proteomics [Epub ahead of print].

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

RevDate: 2019-04-16

Schmidt C, Pacher M, H Puchta (2019)

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

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

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

RevDate: 2019-03-22

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

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

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

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

RevDate: 2019-04-03
CmpDate: 2019-04-03

Paul A, Bharati J, Punetha M, et al (2019)

Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 52(3):532-552.

BACKGROUND/AIMS: Thrombospondins (TSPs) are large multi-modular proteins, identified as natural angiogenesis inhibitors that exert their activity by binding to CD36 and CD47 receptors. The anti-angiogenic effect of TSPs in luteal regression of water buffalo has not been addressed. The present study characterized the expression pattern and localization of TSPs and their receptors in ovarian corpus luteum during different stages of development in buffalo. This study also elucidated the effect of exogenous Thrombospondin1 (TSP1) or the knocking out of the endogenous protein on luteal cell viability and function. Further, the in vitro transcriptional interaction of TSP1 with hormones, LH, PGF2α and angiogenic growth factors, VEGF and FGF2 were also evaluated.

METHODS: First, the CLs were classified into four groups based on macroscopic observation and progesterone concentration. mRNA expression of examined factors was measured by qPCR, localization by immunoblotting and immunohistochemistry. TSP1 was knocked out (KO) in cultured luteal cells isolated from late luteal stage CLs (day 1116) by CRISPR/Cas9 mediated gene editing technology in order to functionally validate the TSP1 gene. Isolated cells from late stage CLs were also stimulated with different doses of TSP1, LH, PGF2α, VEGF and FGF2 for various time intervals to determine transcriptional regulation of thrombospondins.

RESULTS: mRNA expression of TSPs and their receptors were found to be significantly higher in late and regressed stage of CL as compared to other groups which was consistent with the findings of immunoblotting and immunolocalization experiments. It was observed that TSP1 induced apoptosis, down regulated angiogenic growth factors, VEGF and FGF2 and attenuated progesterone production in cultured luteal cells. However, knocking out of endogenous TSP1 with CRISPR/Cas9 system improved the viability of luteal cells, progesterone synthesis and upregulated the expression of VEGF and FGF2 in the KO luteal cells. PGF2α induced the upregulation of TSPs and Caspase 3 transcripts, whereas treatment with LH and angiogenic growth factors (VEGF and FGF2) down regulated the TSP system in luteal cells.

CONCLUSION: Collectively, these data provide evidence that thrombospondins along with their receptors are expressed at varying levels in different stages of CL progression with maximum expression during the late and regressing stages. These results are consistent with the hypothesis that thrombospondins stimulated by PGF2α plays an essential modulatory role in bringing about structural and functional luteolysis in buffalo.

RevDate: 2019-03-23

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

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

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

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

RevDate: 2019-04-02

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

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells.

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

In spite of the progresses in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine-aminopterin-thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019.

RevDate: 2019-03-29

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

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

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

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

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

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

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

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

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

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

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

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


RevDate: 2019-04-03
CmpDate: 2019-04-03

Sandoz J, Nagy Z, Catez P, et al (2019)

Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression.

Nature communications, 10(1):1288 pii:10.1038/s41467-019-09270-2.

The TFIIH subunit XPB is involved in combined Xeroderma Pigmentosum and Cockayne syndrome (XP-B/CS). Our analyses reveal that XPB interacts functionally with KAT2A, a histone acetyltransferase (HAT) that belongs to the hSAGA and hATAC complexes. XPB interacts with KAT2A-containing complexes on chromatin and an XP-B/CS mutation specifically elicits KAT2A-mediated large-scale chromatin decondensation. In XP-B/CS cells, the abnormal recruitment of TFIIH and KAT2A to chromatin causes inappropriate acetylation of histone H3K9, leading to aberrant formation of transcription initiation complexes on the promoters of several hundred genes and their subsequent overexpression. Significantly, this cascade of events is similarly sensitive to KAT2A HAT inhibition or to the rescue with wild-type XPB. In agreement, the XP-B/CS mutation increases KAT2A HAT activity in vitro. Our results unveil a tight connection between TFIIH and KAT2A that controls higher-order chromatin structure and gene expression and provide new insights into transcriptional misregulation in a cancer-prone DNA repair-deficient disorder.

RevDate: 2019-04-10

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

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

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

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

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

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

RevDate: 2019-03-29

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

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

Frontiers in bioengineering and biotechnology, 7:31.

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

RevDate: 2019-04-05
CmpDate: 2019-04-05

Harutyunyan AS, Krug B, Chen H, et al (2019)

H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3 and is essential for glioma tumorigenesis.

Nature communications, 10(1):1262 pii:10.1038/s41467-019-09140-x.

Lys-27-Met mutations in histone 3 genes (H3K27M) characterize a subgroup of deadly gliomas and decrease genome-wide H3K27 trimethylation. Here we use primary H3K27M tumor lines and isogenic CRISPR-edited controls to assess H3K27M effects in vitro and in vivo. We find that whereas H3K27me3 and H3K27me2 are normally deposited by PRC2 across broad regions, their deposition is severely reduced in H3.3K27M cells. H3K27me3 is unable to spread from large unmethylated CpG islands, while H3K27me2 can be deposited outside these PRC2 high-affinity sites but to levels corresponding to H3K27me3 deposition in wild-type cells. Our findings indicate that PRC2 recruitment and propagation on chromatin are seemingly unaffected by K27M, which mostly impairs spread of the repressive marks it catalyzes, especially H3K27me3. Genome-wide loss of H3K27me3 and me2 deposition has limited transcriptomic consequences, preferentially affecting lowly-expressed genes regulating neurogenesis. Removal of H3K27M restores H3K27me2/me3 spread, impairs cell proliferation, and completely abolishes their capacity to form tumors in mice.

RevDate: 2019-04-04

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

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

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

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

RevDate: 2019-03-19

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

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

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

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

RevDate: 2019-03-19

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

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

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

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

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

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

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

RevDate: 2019-03-18

Moses C, P Kaur (2019)

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

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

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

RevDate: 2019-03-29

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

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

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

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

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

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

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

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

RevDate: 2019-04-05
CmpDate: 2019-04-05

Chen Z, Cai X, Li M, et al (2019)

CRISPR/Cas9-based liver-derived reporter cells for screening of mPGES-1 inhibitors.

Journal of enzyme inhibition and medicinal chemistry, 34(1):799-807.

mPGES-1 is a terminal rate-limiting enzyme responsible for inflammation-induced PGE2 production. The inhibition of mPGES-1 has been considered as a safe and effective target for the treatment of inflammation and cancer. However, a specific, efficient, and simple method for high-throughput screening of mPGES-1 inhibitors is still lacking. In this study, we developed a fluorescence imaging strategy to monitor the expression of mPGES-1 via CRISPR/Cas9 knock-in system. Immunofluorescence colocalisation, Sanger sequencing, RNAi, and IL-1β treatment all confirmed the successful construction of mPGES-1 reporter cells. The fluorescence signal intensity of the reporter cells treated with four conventional mPGES-1 inhibitors was considerably attenuated via flow cytometry and fluorescent microplate reader, demonstrating that the reporter cells can be used as an efficient and convenient means for screening and optimising mPGES-1 inhibitors. Moreover, it provides a new technical support for the development of targeted small molecule compounds for anti-inflammatory and tumour therapy.

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

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

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

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

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

RevDate: 2019-04-05

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

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

Applied microbiology and biotechnology, 103(8):3239-3248.

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

RevDate: 2019-04-06

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

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

Molecular microbiology [Epub ahead of print].

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

RevDate: 2019-03-15

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

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

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

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

RevDate: 2019-03-15

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

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

Viruses, 11(3): pii:v11030246.

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

RevDate: 2019-04-02
CmpDate: 2019-03-29

Thomas M, Burgio G, Adams DJ, et al (2019)

Collateral damage and CRISPR genome editing.

PLoS genetics, 15(3):e1007994 pii:PGENETICS-D-18-01868.

The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic modification of virtually every organism and offer immense therapeutic potential for the treatment of human disease. Although these systems may function efficiently within eukaryotic cells, there remain concerns about the accuracy of Cas endonuclease effectors and their use for precise gene editing. Recently, two independent reports investigating the editing accuracy of the CRISPR-Cas9 system were published by separate groups at the Wellcome Sanger Institute; our study-Iyer and colleagues [1]-defined the landscape of off-target mutations, whereas the other by Kosicki and colleagues [2] detailed the existence of on-target, potentially deleterious deletions. Although both studies found evidence of large on-target CRISPR-induced deletions, they reached seemingly very different conclusions.

RevDate: 2019-03-29

Odamaki T, Bottacini F, Mitsuyama E, et al (2019)

Impact of a bathing tradition on shared gut microbe among Japanese families.

Scientific reports, 9(1):4380 pii:10.1038/s41598-019-40938-3.

Sharing of Bifidobacterium longum strains had recently been shown to occur among Japanese family members, a phenomenon that is not confined to mother-infant pairs. In the current study, we investigated if bathtub water is a possible vehicle for the exchange of strains as a consequence of a Japanese custom to share bathtub water by family members during bathing practices. A total of twenty-one subjects from five Japanese families, each consisting of parents with either 2 or 3 children, were enrolled in this study and the fecal microbiota of all participants was determined. Viable bifidobacterial strains were isolated from all bathtub water samples. A subsequent comparative genome analysis using ninety-eight strains indicated that certain strain-sets, which were isolated from feces and bathtub water, share near identical genome sequences, including CRISPR/Cas protospacers. By means of unweighted UniFrac distance analysis based on 16S rRNA gene analysis of 59 subjects from sixteen Japanese families, we showed that the fecal microbiota composition among family members that share bathtub water is significantly closer than that between family members that do not engage in this practice. Our results indicate that bathtub water represents a vehicle for the transmission of gut bacteria, and that the Japanese custom of sharing bathtub water contributes to the exchange of gut microbes, in particular bifidobacteria, among family members.

RevDate: 2019-03-29

Thormann V, Glaser LV, Rothkegel MC, et al (2019)

Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements.

Life science alliance, 2(2): pii:2/2/e201800283.

The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used CRISPR/Cas-mediated homology-directed repair to add a single GR-binding site directly upstream of the transcriptional start site of each of four genes. To our surprise, we found that the addition of a single GR-binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Furthermore, by introducing GR-binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR-binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.

RevDate: 2019-03-29

Veigl SJ (2019)

A use/disuse paradigm for CRISPR-Cas systems.

Biology & philosophy, 34(1):13.

RevDate: 2019-04-18
CmpDate: 2019-04-18

Savell KE, Bach SV, Zipperly ME, et al (2019)

A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation.

eNeuro, 6(1): pii:eN-MNT-0495-18.

CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.

RevDate: 2019-03-12

Le Rhun A, Escalera-Maurer A, Bratovič M, et al (2019)

CRISPR-Cas in Streptococcus pyogenes.

RNA biology [Epub ahead of print].

The discovery and characterization of the prokaryotic CRISPR-Cas immune system has led to a revolution in genome editing and engineering technologies. Despite the fact that most applications emerged after the discovery of the type II-A CRISPR-Cas9 system of Streptococcus pyogenes, its biological importance in this organism has received little attention. Here, we provide a comprehensive overview of the current knowledge about CRISPR-Cas systems from S. pyogenes. We discuss how the interplay between CRISPR-mediated immunity and horizontal gene transfer might have modeled the evolution of this pathogen. We review the current literature about the CRISPR-Cas systems present in S. pyogenes (types I-C and II-A), and describe their distinctive biochemical and functional features. Finally, we summarize the main biotechnological applications that have arisen from the discovery of the CRISPR-Cas9 system in S. pyogenes.

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

Porteus MH (2019)

A New Class of Medicines through DNA Editing.

The New England journal of medicine, 380(10):947-959.

RevDate: 2019-03-29

Parmeciano Di Noto G, Molina MC, C Quiroga (2019)

Insights Into Non-coding RNAs as Novel Antimicrobial Drugs.

Frontiers in genetics, 10:57.

Multidrug resistant bacteria are a serious worldwide problem, especially carbapenem-resistant Enterobacteriaceae (such as Klebsiella pneumoniae and Escherichia coli), Acinetobacter baumannii and Pseudomonas aeruginosa. Since the emergence of extensive and pan-drug resistant bacteria there are few antibiotics left to treat patients, thus novel RNA-based strategies are being considered. Here, we examine the current situation of different non-coding RNAs found in bacteria as well as their function and potential application as antimicrobial agents. Furthermore, we discuss the factors that may contribute in the efficient development of RNA-based drugs, the limitations for their implementation and the use of nanocarriers for delivery.

RevDate: 2019-03-29

Jaiswal S, Singh DK, P Shukla (2019)

Gene Editing and Systems Biology Tools for Pesticide Bioremediation: A Review.

Frontiers in microbiology, 10:87.

Bioremediation is the degradation potential of microorganisms to dissimilate the complex chemical compounds from the surrounding environment. The genetics and biochemistry of biodegradation processes in datasets opened the way of systems biology. Systemic biology aid the study of interacting parts involved in the system. The significant keys of system biology are biodegradation network, computational biology, and omics approaches. Biodegradation network consists of all the databases and datasets which aid in assisting the degradation and deterioration potential of microorganisms for bioremediation processes. This review deciphers the bio-degradation network, i.e., the databases and datasets (UM-BBD, PAN, PTID, etc.) aiding in assisting the degradation and deterioration potential of microorganisms for bioremediation processes, computational biology and multi omics approaches like metagenomics, genomics, transcriptomics, proteomics, and metabolomics for the efficient functional gene mining and their validation for bioremediation experiments. Besides, the present review also describes the gene editing tools like CRISPR Cas, TALEN, and ZFNs which can possibly make design microbe with functional gene of interest for degradation of particular recalcitrant for improved bioremediation.

RevDate: 2019-04-03
CmpDate: 2019-04-03

Cullot G, Boutin J, Toutain J, et al (2019)

CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.

Nature communications, 10(1):1136 pii:10.1038/s41467-019-09006-2.

CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on- and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.

RevDate: 2019-04-09
CmpDate: 2019-04-09

Park SJ, Kim B, Choi S, et al (2019)

Imaging inflammation using an activated macrophage probe with Slc18b1 as the activation-selective gating target.

Nature communications, 10(1):1111 pii:10.1038/s41467-019-08990-9.

Activated macrophages have the potential to be ideal targets for imaging inflammation. However, probe selectivity over non-activated macrophages and probe delivery to target tissue have been challenging. Here, we report a small molecule probe specific for activated macrophages, called CDg16, and demonstrate its application to visualizing inflammatory atherosclerotic plaques in vivo. Through a systematic transporter screen using a CRISPR activation library, we identify the orphan transporter Slc18b1/SLC18B1 as the gating target of CDg16.

RevDate: 2019-03-28
CmpDate: 2019-03-28

Kim HY, Kang SJ, Jeon Y, et al (2019)

Chimeric crRNAs with 19 DNA residues in the guide region show the retained DNA cleavage activity of Cas9 with potential to improve the specificity.

Chemical communications (Cambridge, England), 55(24):3552-3555.

We demonstrated that 19 out of 20 RNA residues in the guide region of crRNA can be replaced with DNA residues with high GC-contents. The cellular activity of the chimeric crRNAs to disrupt the target gene was comparable to that of the native crRNA.

RevDate: 2019-03-06

Mogila I, Kazlauskiene M, Valinskyte S, et al (2019)

Genetic Dissection of the Type III-A CRISPR-Cas System Csm Complex Reveals Roles of Individual Subunits.

Cell reports, 26(10):2753-2765.e4.

The type III-A Csm complex of Streptococcus thermophilus (StCsm) provides immunity against invading nucleic acids through the coordinated action of three catalytic domains: RNase (Csm3), ssDNase (Cas10-HD), and cyclic oligoadenylates synthase (Cas10-Palm). The matured StCsm complex is composed of Cas10:Csm2:Csm3:Csm4:Csm5 subunits and 40-nt CRISPR RNA (crRNA). We have carried out gene disruptions for each subunit and isolated deletion complexes to reveal the role of individual subunits in complex assembly and function. We show that the Cas10-Csm4 subcomplex binds the 5'-handle of crRNA and triggers Csm3 oligomerization to form a padlock for crRNA binding. We demonstrate that Csm5 plays a key role in target RNA binding while Csm2 ensures RNA cleavage at multiple sites by Csm3. Finally, guided by deletion analysis, we engineered a minimal Csm complex containing only the Csm3, Csm4, and Cas10 subunits and crRNA and demonstrated that it retains all three catalytic activities, thus paving the way for practical applications.

RevDate: 2019-03-29

Wegner M, Diehl V, Bittl V, et al (2019)

Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome.

eLife, 8: pii:42549.

Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.

RevDate: 2019-04-05
CmpDate: 2019-04-05

Zhang Y, Wang J, Wang Z, et al (2019)

A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae.

Nature communications, 10(1):1053 pii:10.1038/s41467-019-09005-3.

With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. Here, we report a gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering of Saccharomyces cerevisiae. Using reported gRNAs shown to be effective, this system enables simultaneous disruption of 8 genes with 87% efficiency. We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming the Golden Gate reaction mix to yeast. This approach enables disruption of 6 genes in 3 days with 60% efficiency using reported gRNAs and 23% using un-optimized gRNAs. Moreover, we applied the Lightning GTR-CRISPR to simplify yeast lipid networks, resulting in a 30-fold increase in free fatty acid production in 10 days using just two-round deletions of eight previously identified genes. The GTR-CRISPR should be an invaluable addition to the toolbox of synthetic biology and automation.

RevDate: 2019-04-04

Nasko DJ, Ferrell BD, Moore RM, et al (2019)

CRISPR Spacers Indicate Preferential Matching of Specific Virioplankton Genes.

mBio, 10(2): pii:mBio.02651-18.

Viral infection exerts selection pressure on marine microbes, as virus-induced cell lysis causes 20 to 50% of cell mortality, resulting in fluxes of biomass into oceanic dissolved organic matter. Archaeal and bacterial populations can defend against viral infection using the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system, which relies on specific matching between a spacer sequence and a viral gene. If a CRISPR spacer match to any gene within a viral genome is equally effective in preventing lysis, no viral genes should be preferentially matched by CRISPR spacers. However, if there are differences in effectiveness, certain viral genes may demonstrate a greater frequency of CRISPR spacer matches. Indeed, homology search analyses of bacterioplankton CRISPR spacer sequences against virioplankton sequences revealed preferential matching of replication proteins, nucleic acid binding proteins, and viral structural proteins. Positive selection pressure for effective viral defense is one parsimonious explanation for these observations. CRISPR spacers from virioplankton metagenomes preferentially matched methyltransferase and phage integrase genes within virioplankton sequences. These virioplankton CRISPR spacers may assist infected host cells in defending against competing phage. Analyses also revealed that half of the spacer-matched viral genes were unknown, some genes matched several spacers, and some spacers matched multiple genes, a many-to-many relationship. Thus, CRISPR spacer matching may be an evolutionary algorithm, agnostically identifying those genes under stringent selection pressure for sustaining viral infection and lysis. Investigating this subset of viral genes could reveal those genetic mechanisms essential to virus-host interactions and provide new technologies for optimizing CRISPR defense in beneficial microbes.IMPORTANCE The CRISPR-Cas system is one means by which bacterial and archaeal populations defend against viral infection which causes 20 to 50% of cell mortality in the ocean. We tested the hypothesis that certain viral genes are preferentially targeted for the initial attack of the CRISPR-Cas system on a viral genome. Using CASC, a pipeline for CRISPR spacer discovery, and metagenome data from oceanic microbes and viruses, we found a clear subset of viral genes with high match frequencies to CRISPR spacers. Moreover, we observed a many-to-many relationship of spacers and viral genes. These high-match viral genes were involved in nucleotide metabolism, DNA methylation, and viral structure. It is possible that CRISPR spacer matching is an evolutionary algorithm pointing to those viral genes most important to sustaining infection and lysis. Studying these genes may advance the understanding of virus-host interactions in nature and provide new technologies for leveraging CRISPR-Cas systems in beneficial microbes.

RevDate: 2019-03-05

Chen K, Wang Y, Zhang R, et al (2019)

CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture.

Annual review of plant biology [Epub ahead of print].

Enhanced agricultural production through innovative breeding technology is urgently needed to increase access to nutritious foods worldwide. Recent advances in CRISPR/Cas genome editing enable efficient targeted modification in most crops, thus promising to accelerate crop improvement. Here, we review advances in CRISPR/Cas9 and its variants and examine their applications in plant genome editing and related manipulations. We highlight base-editing tools that enable targeted nucleotide substitutions and describe the various delivery systems, particularly DNA-free methods, that have linked genome editing with crop breeding. We summarize the applications of genome editing for trait improvement, development of fine-tuning gene regulation, strategies for breeding virus resistance, and the use of high-throughput mutant libraries. We outline future perspectives for genome editing in plant synthetic biology and domestication, advances in delivery systems, editing specificity, homology-directed repair, and gene drives. Finally, we discuss the challenges and opportunities for precision plant breeding and its bright future in agriculture. Expected final online publication date for the Annual Review of Plant Biology Volume 70 is April 29, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

RevDate: 2019-03-05

van Sluijs L, van Houte S, van der Oost J, et al (2019)

Addiction systems antagonize bacterial adaptive immunity.

FEMS microbiology letters pii:5369624 [Epub ahead of print].

CRISPR-Cas systems provide adaptive immunity against mobile genetic elements, but employment of this resistance mechanism is often reported with a fitness cost for the host. Whether or not CRISPR-Cas systems are important barriers for the horizontal spread of conjugative plasmids, which play a crucial role in the spread of antibiotic resistance, will depend on the fitness costs of employing CRISPR-based defences and the benefits of resisting conjugative plasmids. To estimate these costs and benefits we measured bacterial fitness associated with plasmid immunity using Escherichia coli and the conjugative plasmid pOX38-Cm. We find that CRISPR-mediated immunity fails to confer a fitness benefit in the absence of antibiotics, despite the large fitness cost associated with carrying the plasmid in this context. Similar to many other conjugative plasmids, pOX38-Cm carries a CcdAB toxin-antitoxin (TA) addiction system. These addiction systems encode long-lived toxins and short-lived anti-toxins, resulting in toxic effects following the loss of the TA genes from the bacterial host. Our data suggest that the lack of a fitness benefit associated with CRISPR-mediated defence is due to expression of the TA system before plasmid detection and degradation. As most antibiotic resistance plasmids encode TA systems this could have important consequences for the role of CRISPR-Cas systems in limiting the spread of antibiotic resistance.

RevDate: 2019-04-16
CmpDate: 2019-04-03

Kelliher T, Starr D, Su X, et al (2019)

One-step genome editing of elite crop germplasm during haploid induction.

Nature biotechnology, 37(3):287-292.

Genome editing using CRISPR-Cas9 works efficiently in plant cells1, but delivery of genome-editing machinery into the vast majority of crop varieties is not possible using established methods2. We co-opted the aberrant reproductive process of haploid induction (HI)3-6 to induce edits in nascent seeds of diverse monocot and dicot species. Our method, named HI-Edit, enables direct genomic modification of commercial crop varieties. HI-Edit was tested in field and sweet corn using a native haploid-inducer line4 and extended to dicots using an engineered CENH3 HI system7. We also recovered edited wheat embryos using Cas9 delivered by maize pollen. Our data indicate that a transient hybrid state precedes uniparental chromosome elimination in maize HI. Edited haploid plants lack both the haploid-inducer parental DNA and the editing machinery. Therefore, edited plants could be used in trait testing and directly integrated into commercial variety development.

RevDate: 2019-04-03
CmpDate: 2019-04-03

Hodgson J (2019)

CRISPR target prediction remains blunt tool for clinical applications.

Nature biotechnology, 37(3):204-205.

RevDate: 2019-04-03
CmpDate: 2019-04-03

Anonymous (2019)

Vertex ramps up CRISPR repair.

Nature biotechnology, 37(3):205.

RevDate: 2019-03-05

Fu BXH, Smith JD, Fuchs RT, et al (2019)

Target-dependent nickase activities of the CRISPR-Cas nucleases Cpf1 and Cas9.

Nature microbiology pii:10.1038/s41564-019-0382-0 [Epub ahead of print].

Clustered regularly interspaced short palindromic repeats (CRISPR) machineries are prokaryotic immune systems that have been adapted as versatile gene editing and manipulation tools. We found that CRISPR nucleases from two families, Cpf1 (also known as Cas12a) and Cas9, exhibit differential guide RNA (gRNA) sequence requirements for cleavage of the two strands of target DNA in vitro. As a consequence of the differential gRNA requirements, both Cas9 and Cpf1 enzymes can exhibit potent nickase activities on an extensive class of mismatched double-stranded DNA (dsDNA) targets. These properties allow the production of efficient nickases for a chosen dsDNA target sequence, without modification of the nuclease protein, using gRNAs with a variety of patterns of mismatch to the intended DNA target. In parallel to the nicking activities observed with purified Cas9 in vitro, we observed sequence-dependent nicking for both perfectly matched and partially mismatched target sequences in a Saccharomyces cerevisiae system. Our findings have implications for CRISPR spacer acquisition, off-target potential of CRISPR gene editing/manipulation, and tool development using homology-directed nicking.

RevDate: 2019-03-06

Muñoz IV, Sarrocco S, Malfatti L, et al (2019)

CRISPR-Cas for Fungal Genome Editing: A New Tool for the Management of Plant Diseases.

Frontiers in plant science, 10:135.

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

Bowler M, Kong D, Sun S, et al (2019)

High-resolution characterization of centriole distal appendage morphology and dynamics by correlative STORM and electron microscopy.

Nature communications, 10(1):993 pii:10.1038/s41467-018-08216-4.

Centrioles are vital cellular structures that form centrosomes and cilia. The formation and function of cilia depends on a set of centriole's distal appendages. In this study, we use correlative super resolution and electron microscopy to precisely determine where distal appendage proteins localize in relation to the centriole microtubules and appendage electron densities. Here we characterize a novel distal appendage protein ANKRD26 and detail, in high resolution, the initial steps of distal appendage assembly. We further show that distal appendages undergo a dramatic ultra-structural reorganization before mitosis, during which they temporarily lose outer components, while inner components maintain a nine-fold organization. Finally, using electron tomography we reveal that mammalian distal appendages associate with two centriole microtubule triplets via an elaborate filamentous base and that they appear as almost radial finger-like protrusions. Our findings challenge the traditional portrayal of mammalian distal appendage as a pinwheel-like structure that is maintained throughout mitosis.


RJR Experience and Expertise


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


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


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


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


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


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


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


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

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

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E-mail: RJR8222@gmail.com

Collection of publications by R J Robbins

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

Research Gate page for R J Robbins

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

Curriculum Vitae for R J Robbins

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Curriculum Vitae for R J Robbins

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