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Current Topics in Genome Analysis 2014 Course Syllabus, Handouts and Videos

Current Topics in Genome Analysis 2014 Course Syllabus, Handouts and Videos | Awesome Science That  I Like | Scoop.it

"A lecture series covering contemporary areas in genomics and bioinformatics"

Handouts and videos are online, from the National Human Genome Research Institute


Via Mary Williams
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Really updated, complete and... free!

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In vivo genome editing using Staphylococcus aureus Cas9

In vivo genome editing using Staphylococcus aureus Cas9 | Awesome Science That  I Like | Scoop.it

The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

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New Cas9 from Staphylococcus aureus: PAM identification and in vivo efficiency. This shorter Cas9 expands the CRISPR/Cas9 toolbox.

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The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations

The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations | Awesome Science That  I Like | Scoop.it

An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype while individuals homozygous for two copies of the allele will display a mutant phenotype. Here, we develop a method that we refer to as the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome editing system for generating autocatalytic mutations to generate homozygous loss-of-function mutations. We demonstrate in Drosophila that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms.

Jorge Lozano-Juste's insight:

Gene drive by CRISPR in Drosophila. A really exciting approach that calls for changes in legislation.

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A split-Cas9 architecture for inducible genome editing and transcription modulation

A split-Cas9 architecture for inducible genome editing and transcription modulation | Awesome Science That  I Like | Scoop.it

The RNA-guided CRISPR-associated (Cas) endonuclease Cas9 has been harnessed as a tool for genome editing in mammalian cells1, 2. In addition, strategies employing catalytically inactive Cas9 can direct effector proteins to genomic targets3, 4, 5 to modulate transcription. Here, we demonstrate that Cas9 can be split into two fragments and rendered chemically inducible by rapamycin-binding dimerization domains for controlled reassembly to mediate genome editing and transcription modulation.

Jorge Lozano-Juste's insight:

Another step forward. Chemically induced CRISPR/Cas9.

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Maternal temperature history activates Flowering Locus T in fruits to control progeny dormancy according to time of year

Maternal temperature history activates Flowering Locus T in fruits to control progeny dormancy according to time of year | Awesome Science That  I Like | Scoop.it

Seasonal behavior is important for fitness in temperate environments but it is unclear how progeny gain their initial seasonal entrainment. Plants use temperature signals to measure time of year, and changes to life histories are therefore an important consequence of climate change. Here we show that in Arabidopsis the current and prior temperature experience of the mother plant is used to control germination of progeny seeds, via the activation of the florigen Flowering Locus T (FT) in fruit tissues. We demonstrate that maternal past and current temperature experience are transduced to the FT locus in silique phloem. In turn, FT controls seed dormancy through inhibition of proanthocyanidin synthesis in fruits, resulting in altered seed coat tannin content. Our data reveal that maternal temperature history is integrated through FT in the fruit to generate a metabolic signal that entrains the behavior of progeny seeds according to time of year.

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Plants surprise me more and more... Mother plants use the flowering time protein FT to reduce the germination of the offspring under adverse conditions

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Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo

Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo | Awesome Science That  I Like | Scoop.it

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

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Best of Plants 2014: Plant-based antibodies used to treat Ebola Virus Disease

Best of Plants 2014: Plant-based antibodies used to treat Ebola Virus Disease | Awesome Science That  I Like | Scoop.it

 

One of the biggest stories of 2014 was the spread of Ebola Virus Disease, and antibodies produced in plants are one of the few therapies shown to be effective. Plant-derived antibodies produced by MAPP pharmaceuticals in tobacco had previously been tested successfully on monkeys (http://www.pnas.org/content/109/44/18030), but this summer were used to save the lives of two American missionaries (http://edition.cnn.com/2014/10/03/health/ebola-tobacco-plant/). Plant-production of phamaceuticals (aka molecular pharming) can be cheaper than production in animals or cell lines, and this year has demonstrated this technology to be fast, versatile and effective.


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Best of Plants 2014: A few research highlights, summaries and educational resources, and some fun

Best of Plants 2014: A few research highlights, summaries and educational resources, and some fun | Awesome Science That  I Like | Scoop.it

A very few research highlights

Biosensors: Plant biologists FRET over stress. Two independent research labs have developed fluorescent biosensors to report the levels of the stress hormone, abscisic acid, within cells in living plants in real-time. http://elifesciences.org/content/3/e02763

PLETHORA gradient formation mechanism separates auxin responses. http://www.nature.com/nature/journal/v515/n7525/full/nature13663.html

Multiscale digital Arabidopsis predicts individual organ and whole-organism growth. http://www.pnas.org/content/111/39/E4127.abstract

The Structure of the Catalytic Domain of a

Plant cellulose synthase and its assembly into dimers. http://www.plantcell.org/content/26/7/2996.abstract

Increasing CO2 threatens human nutrition. http://www.nature.com/nature/journal/v510/n7503/full/nature13179.html

A reference genome for common bean and genome-wide analysis of dual domestications. http://www.nature.com/ng/journal/v46/n7/full/ng.3008.html

 

Research syntheses and other educational resources

The Art of Being Flexible: How to Escape from Shade, Salt, and Drought http://www.plantphysiol.org/content/166/1/5

Epigenetic Memory for Stress Response and Adaptation in Plants http://pcp.oxfordjournals.org/content/55/11/1859.abstract

Traversing organizational scales in plant salt-stress responses http://www.sciencedirect.com/science/article/pii/S1369526614001435

Rice special issue in Nature http://www.nature.com/nature/outlook/rice/

Plant Physiology published Focus Issues on

Water (http://www.plantphysiol.org/content/164/4.toc),  

Roots (http://www.plantphysiol.org/content/166/2.toc),

Weed Control (http://www.plantphysiol.org/content/166/3.toc),  and

The Plant Cell started the year with an excellent set of review articles on Photobiology (http://www.plantcell.org/content/26/1.toc).

CourseSource. CourseSource is an open-access journal of peer-reviewed teaching resources for undergraduate biological sciences; the development of these resources, including plant-based resources, was supported by ASPB and BSA. http://www.coursesource.org/

The Plant Detectives Manual. A research-led approach for teaching plant science, by Gonzalo M. Estavillo, Ulrike Mathesius, Michael Djordjevic and Adrienne B. Nicotra. http://press.anu.edu.au/titles/anu-etext/the-plant-detectives-manual/

Campus Flora Oz App. Explore campus flora on your phone! https://campusflora.wordpress.com/

Countdown to 400: Oxford Herbarium’s ongoing weekly series of plants, counting downt to its 400th anniversary!  http://herbaria.plants.ox.ac.uk/bol/plants400

In 2014, Teaching Tools in Plant Biology started a series of topics on plant physiology, including water relations and plant nutrition! http://www.plantcell.org/site/teachingtools/teaching.xhtml

 

Finally, some end of year fun

Fifi the Oomycete, a holiday song, by Kamoun Lab. https://www.youtube.com/watch?v=l9ikfDWZaT8&feature=youtu.be

#AdventBotany. By Alastair Culham and Dr. M. (AKA Jonathan Mitchley) (@BotanyRNG and @drmgoeswild) http://blogs.reading.ac.uk/crg/2014/12/page/4/ & http://drmgoeswild.com/advent-botany/


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Carla Pinheiro's curator insight, December 31, 2014 5:23 AM

A great tool to explore subcellular compartmentalization

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Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains | Awesome Science That  I Like | Scoop.it

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo–electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions (“CAT tails”).

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High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis

High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis | Awesome Science That  I Like | Scoop.it

With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.

Jorge Lozano-Juste's insight:

High Resolution Melting (HRM) seems to be a good strategy to screen for mutants by pools

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RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors | Awesome Science That  I Like | Scoop.it

argeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3::uidA targets in plant cells. Further, the dCas9:SRDX-mediated transcriptional repression of an endogenous gene. Thus, our results suggest that the synthetic transcriptional repressor (dCas9:SRDX) and activators (dCas9:EDLL and dCas9:TAD) can be used as endogenous transcription factors to repress or activate transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9 DNA-targeting platform can be used in plants as a functional genomics tool and for biotechnological applications.

Jorge Lozano-Juste's insight:

Moderate regulation of gene expression by dCas9-TF in plant cells

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Data access for the 1,000 Plants (1KP) project

The 1,000 plants (1KP) project is an international multi-disciplinary consortium that has generated transcriptome data from over 1,000 plant species, with exemplars for all of the major lineages across the Viridiplantae (green plants) clade. Here, we describe how to access the data used in a phylogenomics analysis of the first 85 species, and how to visualize our gene and species trees. Users can develop computational pipelines to analyse these data, in conjunction with data of their own that they can upload. Computationally estimated protein-protein interactions and biochemical pathways can be visualized at another site. Finally, we comment on our future plans and how they fit within this scalable system for the dissemination, visualization, and analysis of large multi-species data sets.

Via Biswapriya Biswavas Misra
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Biswapriya Biswavas Misra's curator insight, November 1, 2014 12:44 AM

The 1,000 plants (1KP) project is an international multi-disciplinary consortium that has generated transcriptome data from over 1,000 plant species, with exemplars for all of the major lineages across the Viridiplantae (green plants) clade. Here, we describe how to access the data used in a phylogenomics analysis of the first 85 species, and how to visualize our gene and species trees. Users can develop computational pipelines to analyse these data, in conjunction with data of their own that they can upload. Computationally estimated protein-protein interactions and biochemical pathways can be visualized at another site. Finally, we comment on our future plans and how they fit within this scalable system for the dissemination, visualization, and analysis of large multi-species data sets.

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A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging: Cell

A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging: Cell | Awesome Science That  I Like | Scoop.it

Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs

Jorge Lozano-Juste's insight:

Smart trick to increase gene activation by CRISPR/Cas9

 

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Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress

Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress | Awesome Science That  I Like | Scoop.it

Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.

Jorge Lozano-Juste's insight:

A detailed structural analysis of CIPK23 and CIPK24

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Primary transcripts of microRNAs encode regulatory peptides

Primary transcripts of microRNAs encode regulatory peptides | Awesome Science That  I Like | Scoop.it

MicroRNAs (miRNAs) are small regulatory RNA molecules that inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs or otherwise inhibiting their translation into proteins1. miRNAs are transcribed as much larger primary transcripts (pri-miRNAs), the function of which is not fully understood. Here we show that plant pri-miRNAs contain short open reading frame sequences that encode regulatory peptides. The pri-miR171b of Medicago truncatula and the pri-miR165a of Arabidopsis thaliana produce peptides, which we term miPEP171b and miPEP165a, respectively, that enhance the accumulation of their corresponding mature miRNAs, resulting in downregulation of target genes involved in root development. The mechanism of miRNA-encoded peptide (miPEP) action involves increasing transcription of the pri-miRNA. Five other pri-miRNAs of A. thaliana and M. truncatula encode active miPEPs, suggesting that miPEPs are widespread throughout the plant kingdom. Synthetic miPEP171b and miPEP165a peptides applied to plants specifically trigger the accumulation of miR171b and miR165a, leading to reduction of lateral root development and stimulation of main root growth, respectively, suggesting that miPEPs might have agronomical applications.

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Surprising!!

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Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells

Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells | Awesome Science That  I Like | Scoop.it

The bacterial CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ), but it remains inefficient for precise editing of genome sequences. Here we develop a reporter-based screening approach for high-throughput identification of chemical compounds that can modulate precise genome editing through homology-directed repair (HDR). Using our screening method, we have identified small molecules that can enhance CRISPR-mediated HDR efficiency, 3-fold for large fragment insertions and 9-fold for point mutations. Interestingly, we have also observed that a small molecule that inhibits HDR can enhance frame shift insertion and deletion (indel) mutations mediated by NHEJ. The identified small molecules function robustly in diverse cell types with minimal toxicity. The use of small molecules provides a simple and effective strategy to enhance precise genome engineering applications and facilitates the study of DNA repair mechanisms in mammalian cells.

Jorge Lozano-Juste's insight:

A chemical screening identifies new drugs able to increase CRISPR/Cas9 genome editing success rates. Although the reduced number  of small molecules screened (4k) suggests that new and better chemical leads are yet to come.

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Agrochemical control of plant water use using engineered abscisic acid receptors

Agrochemical control of plant water use using engineered abscisic acid receptors | Awesome Science That  I Like | Scoop.it

Rising temperatures and lessening fresh water supplies are threatening agricultural productivity and have motivated efforts to improve plant water use and drought tolerance. During water deficit, plants produce elevated levels of abscisic acid (ABA), which improves water consumption and stress tolerance by controlling guard cell aperture and other protective responses1, 2. One attractive strategy for controlling water use is to develop compounds that activate ABA receptors, but agonists approved for use have yet to be developed. In principle, an engineered ABA receptor that can be activated by an existing agrochemical could achieve this goal. Here we describe a variant of the ABA receptor PYRABACTIN RESISTANCE 1 (PYR1) that possesses nanomolar sensitivity to the agrochemical mandipropamid and demonstrate its efficacy for controlling ABA responses and drought tolerance in transgenic plants. Furthermore, crystallographic studies provide a mechanistic basis for its activity and demonstrate the relative ease with which the PYR1 ligand-binding pocket can be altered to accommodate new ligands. Thus, we have successfully repurposed an agrochemical for a new application using receptor engineering. We anticipate that this strategy will be applied to other plant receptors and represents a new avenue for crop improvement.

Jorge Lozano-Juste's insight:

Proof of concept for the use of engineered receptors to control plant phenotypes.

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Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development

Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development | Awesome Science That  I Like | Scoop.it

The plant hormone auxin is a key regulator of plant growth. It has been hypothesized that some auxin responses are mediated by a candidate auxin receptor called auxin binding protein 1 (ABP1). Support for this hypothesis mainly comes from the analyses of Arabidopsis ABP1knockdown lines generated by cellular immunization or antisense approaches. However, these approaches are subject to off-target effects. As an alternative, we have recovered two new null alleles of abp1. Surprisingly, neither of the mutants exhibits defects in growth and development, or auxin response, indicating that ABP1 does not have a major role in these responses under normal growth conditions. These results require that the role of ABP1 in plant growth and auxin response be reexamined.

Jorge Lozano-Juste's insight:

apb1 has no phenotype. Really!??

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Insights into the origin and evolution of plant hormone signaling machinery

Insights into the origin and evolution of plant hormone signaling machinery | Awesome Science That  I Like | Scoop.it

"Our multi-species genome-wide analysis reveals: i) AUX, CK and SL signaling pathways originated in charophyte lineages; ii) ABA, JA, and SA signaling pathways arose in the last common ancestor of land plants; iii) the GA signaling evolved after the divergence of bryophytes from land plants; iv) the canonical BR signaling originated before the emergence of angiosperms but likely after the split of gymnosperms and angiosperms; v) the origin of the canonical ETH signaling pathway postdates shortly the emergence of angiosperms. Our findings might have important implications in understanding the molecular mechanisms underlying the emergence of land plants"


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Jorge Lozano-Juste's insight:

Nice!

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Mary Williams's curator insight, January 8, 5:36 AM

This is a pretty fabulous paper - I'm sure it'll find many uses, not the least being the one-page figure that summarizes all of the hormone signaling pathways. The figure shown here examines the presence or absence of signaling gene homologs in green algal species.

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Best of Plants 2014: From Phenotype to Genotype and Back Again, great strides in plant breeding tools

Best of Plants 2014: From Phenotype to Genotype and Back Again, great strides in plant breeding tools | Awesome Science That  I Like | Scoop.it

 

2014 saw numerous advances in plant breeding technologies. Genetic diversity can be sourced from the broader gene pool of crop relatives, induced, or engineered. The rapidly growing body of genomic information and tools facilitate the molecular identification of genetic variation, high-throughput phenotyping platforms allow precise characterization of its effects, and modeling approaches are helping to predict optimal genotypes for success in different environments.  Through these rapid advances we are moving toward two important outcomes; the development of plants whose genomes are optimized for their environment (in terms of water and nutrient availability, presence of pathogens or herbivores etc), and their development by way of methods that don’t cause them get stuck in an endless regulatory process.

 Just a few of many hundreds of articles published in 2014 highlighting some of these advances:

 

Reinventing the Green Revolution by Harnessing Crop Mutant Resources (http://www.plantphysiol.org/content/166/4/1682.full)

MSH1-Induced Non-Genetic Variation Provides a Source of Phenotypic Diversity in Sorghum bicolor  (http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0108407)

Combining high-throughput phenotyping and genome-wide association studies to reveal natural genetic variation in rice (http://www.nature.com/ncomms/2014/141008/ncomms6087/full/ncomms6087.html)

Integrated Analysis Platform: An Open-Source Information System for High-Throughput Plant Phenotyping (http://www.plantphysiol.org/content/165/2/506)

Field high-throughput phenotyping: the new crop breeding frontier (http://www.cell.com/trends/plant-science/abstract/S1360-1385%2813%2900199-4)

The Sol Genomics Network (SGN)—from genotype to phenotype to breeding (http://nar.oxfordjournals.org/content/early/2014/11/26/nar.gku1195.full)

Precise plant breeding using new genome editing techniques: opportunities, safety and regulation in the EU (http://onlinelibrary.wiley.com/doi/10.1111/tpj.12413/abstract)

Evolution of physiological responses to salt stress in hexaploid wheat (http://www.pnas.org/content/111/32/11882.abstract)

Microbial genome-enabled insights into plant–microorganism interactions  (http://www.nature.com/nrg/journal/v15/n12/abs/nrg3748.html)

Are we ready for back-to-nature crop breeding? (www.cell.com/trends/plant-science/abstract/S1360-1385(14)00290-8)

TraitCapture: genomic and environment modelling of plant phenomic data (http://www.sciencedirect.com/science/article/pii/S1369526614000181)


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Science Graphic of the Week: Scientists Discover the First Protein That Can Edit Other Proteins | WIRED

Science Graphic of the Week: Scientists Discover the First Protein That Can Edit Other Proteins | WIRED | Awesome Science That  I Like | Scoop.it
A new graphic shows how that DNA isn't the only thing in charge of handing out code for proteins.

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GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases | Awesome Science That  I Like | Scoop.it

CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.

Jorge Lozano-Juste's insight:

What it seems the best approach for off-target analysis to date

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Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex | Awesome Science That  I Like | Scoop.it

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene signature based on the top screening hits correlated with a gene expression signature of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

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Engineered CRISPR/Cas9 (SAM) attracts several trasncriptional activators and increase the efficiency of CRISPR-TF. Finally! ...and then you can build many SAMs targeting  a whole genome and perform a screening... Feng Zhang's lab @ MIT

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Comparative assessments of CRISPR-Cas nucleases’ cleavage efficiency in planta - Online First - Springer

Comparative assessments of CRISPR-Cas nucleases’ cleavage efficiency in planta - Online First - Springer | Awesome Science That  I Like | Scoop.it

Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207–221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.

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Arabidopsis and Human codon optimized Cas9 are more efficient in plants

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Target-Selective Protein S-Nitrosylation by Sequence Motif Recognition: Cell

Target-Selective Protein S-Nitrosylation by Sequence Motif Recognition: Cell | Awesome Science That  I Like | Scoop.it

S-nitrosylation is a ubiquitous protein modification emerging as a principal mechanism of nitric oxide (NO)-mediated signal transduction and cell function. S-nitrosylases can use NO synthase (NOS)-derived NO to modify selected cysteines in target proteins. Despite proteomic identification of over a thousand S-nitrosylated proteins, few S-nitrosylases have been identified. Moreover, mechanisms underlying site-selective S-nitrosylation and the potential role of specific sequence motifs remain largely unknown. Here, we describe a stimulus-inducible, heterotrimeric S-nitrosylase complex consisting of inducible NOS (iNOS), S100A8, and S100A9. S100A9 exhibits transnitrosylase activity, shuttling NO from iNOS to the target protein, whereas S100A8 and S100A9 coordinately direct site selection. A family of proteins S-nitrosylated by iNOS-S100A8/A9 were revealed by proteomic analysis. A conserved I/L-X-C-X2-D/E motif was necessary and sufficient for iNOS-S100A8/A9-mediated S-nitrosylation. These results reveal an elusive parallel between protein S-nitrosylation and phosphorylation, namely, stimulus-dependent posttranslational modification of selected targets by primary sequence motif recognition.

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Breakthrough on S-nitrosylation!! S-nitrosylase complex revealed

 

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Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles - Springer

Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles - Springer | Awesome Science That  I Like | Scoop.it

Site-directed genome engineering in higher plants has great potential for basic research and molecular breeding. Here, we describe a method for site-directed mutagenesis of the Arabidopsis nuclear genome that efficiently generates heritable mutations using the RNA-guided endonuclease (RGEN) derived from bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated) protein system. To induce mutagenesis in proliferating tissues during embryogenesis and throughout the plant life cycle, the single guide RNA (sgRNA) and Cas9 DNA endonuclease were expressed from the U6 snRNA and INCURVATA2 promoters, respectively. After Agrobacterium-mediated introduction of T-DNAs encoding RGENs that targets FLOWERING LOCUS T (FT) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 4 genes, somatic mutagenesis at the targeted loci was observed in T1 transformants. In the results of FT-RGEN, T1 plants often showed late flowering indicative of the presence of large somatic sectors in which the FT gene is mutated on both chromosomes. DNA sequencing analysis estimated that about 90 % of independent chromosomal DNA fragments carried mutations in the analyzed tissue of a T1 plant showing late flowering. The most frequently detected somatic polymorphism showed a high rate of inheritance in T2 plants, and inheritance of less frequent polymorphisms was also observed. As a result, late-flowering plants homozygous for novel, heritable null alleles of FT including a 1 bp insertion or short deletions were recovered in the following T2 and T3 generations. Our results demonstrate that dividing tissue-targeted mutagenesis using RGEN provides an efficient heritable genome engineering method in A. thaliana.

Jorge Lozano-Juste's insight:

Using INCURVATA2 promoter to target Cas9 to cells in active division at the meristems, proliferating primordia, the vegetative shoot, inflorescences and flowers.

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