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The last 50 bibliographies

Baboon Envelope Pseudotyped "Nanoblades" Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34(+) Cells and Knock-in of AAV6-Encoded Donor DNA in CD34(+) Cells.

Author(s) : Gutierrez-Guerrero A, Abrey Recalde M, Mangeot P, Costa C, Bernadin O, Périan S, Fusil F, Froment G, Martinez-Turtos A, Krug A, Martin F, Benabdellah K, Ricci E, Giovannozzi S, Gijsbers R, Ayuso E, Cosset F, Verhoeyen E,
Journal : Front Genome Ed
Programmable nucleases have enabled rapid and accessible genome engineering ineukaryotic cells and living organisms. However, their delivery into human bloodcells can be challenging. Here, we have utilized "nanoblades," a new technology thatdelivers a genomic cleaving agent into cells. These are modified murine leukemiavirus (MLV) or HIV-derived virus-like particle (VLP), in which the viral structuralprotein Gag has been fused to Cas9. These VLPs are thus loaded with Cas9 proteincomplexed with the guide RNAs. Highly efficient gene editing was obtained in celllines, IPS and primary mouse and human cells. Here, we showed that nanoblades wereremarkably efficient for entry into human T, B, and hematopoietic stem andprogenitor cells (HSPCs) thanks to their surface co-pseudotyping with baboonretroviral and VSV-G envelope glycoproteins. A brief incubation of human T and Bcells with nanoblades incorporating two gRNAs resulted in 40 and 15% edited deletionin the Wiskott-Aldrich syndrome (WAS) gene locus, respectively. CD34(+) cells(HSPCs) treated with the same nanoblades allowed 30-40% exon 1 drop-out in the WASgene locus. Importantly, no toxicity was detected upon nanoblade-mediated geneediting of these blood cells. Finally, we also treated HSPCs with nanoblades incombination with a donor-encoding rAAV6 vector resulting in up to 40% of stableexpression cassette knock-in into the WAS gene locus. Summarizing, this newtechnology is simple to implement, shows high flexibility for different targetsincluding primary immune cells of human and murine origin, is relatively inexpensiveand therefore gives important prospects for basic and clinical translation in thearea of gene therapy.

Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease.

Author(s) : Eshraghi M, Karunadharma P, Blin J, Shahani N, Ricci E, Michel A, Urban N, Galli N, Sharma M, Ramírez-Jarquín U, Florescu K, Hernandez J, Subramaniam S,
Journal : Nat Commun
The polyglutamine expansion of huntingtin (mHTT) causes Huntington disease (HD) andneurodegeneration, but the mechanisms remain unclear. Here, we found that mHttpromotes ribosome stalling and suppresses protein synthesis in mouse HD striatalneuronal cells. Depletion of mHtt enhances protein synthesis and increases the speedof ribosomal translocation, while mHtt directly inhibits protein synthesis in vitro.Fmrp, a known regulator of ribosome stalling, is upregulated in HD, but itsdepletion has no discernible effect on protein synthesis or ribosome stalling in HDcells. We found interactions of ribosomal proteins and translating ribosomes withmHtt. High-resolution global ribosome footprint profiling (Ribo-Seq) and mRNA-Seqindicates a widespread shift in ribosome occupancy toward the 5' and 3' end andunique single-codon pauses on selected mRNA targets in HD cells, compared tocontrols. Thus, mHtt impedes ribosomal translocation during translation elongation,a mechanistic defect that can be exploited for HD therapeutics.

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades").

Author(s) : Mangeot P, Guiguettaz L, Sohier T, Ricci E,
Journal : J Vis Exp
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systemhas democratized genome-editing in eukaryotic cells and led to the development ofnumerous innovative applications. However, delivery of the Cas9 protein andsingle-guide RNA (sgRNA) into target cells can be technically challenge. Classicalviral vectors, such as those derived from lentiviruses (LVs) or adeno-associatedviruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9protein and its associated sgRNA in many primary cells and in vivo. Nevertheless,these vectors can suffer from drawbacks such as integration of the transgene in thetarget cell genome, a limited cargo capacity, and long-term expression of the Cas9protein and guide RNA in target cells. To overcome some of these problems, adelivery vector based on the murine Leukemia virus (MLV) was developed to packagethe Cas9 protein and its associated guide RNA in the absence of any codingtransgene. By fusing the Cas9 protein to the C-terminus of the structural proteinGag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA(named "Nanoblades") were formed. Nanoblades can be collected from the culturemedium of producer cells, purified, quantified, and used to transduce target cellsand deliver the active Cas9/sgRNA complex. Nanoblades deliver theirribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary andimmortalized cells and can be programmed for other applications, such as transienttranscriptional activation of targeted genes, using modified Cas9 proteins.Nanoblades are capable of in vivo genome-editing in the liver of injected adult miceand in oocytes to generate transgenic animals. Finally, they can be complexed withdonor DNA for "transfection-free" homology-directed repair. Nanoblade preparation issimple, relatively low-cost, and can be easily carried out in any cell biologylaboratory.

Coupled protein synthesis and ribosome-guided piRNA processing on mRNAs.

Author(s) : Sun Y, Wang R, Du K, Zhu J, Zheng J, Xie L, Pereira A, Zhang C, Ricci E, Li X,
Journal : Nat Commun
PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essentialfor fertility. piRNAs originate from transposable element (TE) RNAs, long non-codingRNAs, or 3´ untranslated regions (3´UTRs) of protein-coding messenger genes, withthe last being the least characterized of the three piRNA classes. Here, wedemonstrate that the precursors of 3´UTR piRNAs are full-length mRNAs and thatpost-termination 80S ribosomes guide piRNA production on 3´UTRs in mice andchickens. At the pachytene stage, when other co-translational RNA surveillancepathways are sequestered, piRNA biogenesis degrades mRNAs right after pioneer roundsof translation and fine-tunes protein production from mRNAs. Although 3´UTR piRNAprecursor mRNAs code for distinct proteins in mice and chickens, they all harborembedded TEs and produce piRNAs that cleave TEs. Altogether, we discover a functionof the piRNA pathway in fine-tuning protein production and reveal a conserved piRNAbiogenesis mechanism that recognizes translating RNAs in amniotes.

Ribosome dynamics and mRNA turnover, a complex relationship under constant cellular scrutiny.

Author(s) : Morris C, Cluet D, Ricci E,
Journal : Wiley Interdiscip Rev RNA
Eukaryotic gene expression is closely regulated by translation and turnover ofmRNAs. Recent advances highlight the importance of translation in the control ofmRNA degradation, both for aberrant and apparently normal mRNAs. During translation,the information contained in mRNAs is decoded by ribosomes, one codon at a time, andtRNAs, by specifically recognizing codons, translate the nucleotide code into aminoacids. Such a decoding step does not process regularly, with various obstacles thatcan hinder ribosome progression, then leading to ribosome stalling or collisions.The progression of ribosomes is constantly monitored by the cell which has evolvedseveral translation-dependent mRNA surveillance pathways, includingnonsense-mediated decay (NMD), no-go decay (NGD), and non-stop decay (NSD), todegrade certain problematic mRNAs and the incomplete protein products. Recentprogress in sequencing and ribosome profiling has made it possible to discover newmechanisms controlling ribosome dynamics, with numerous crosstalks betweentranslation and mRNA decay. We discuss here various translation features criticalfor mRNA decay, with particular focus on current insights from the complexity of thegenetic code and also the emerging role for the ribosome as a regulatory huborchestrating mRNA decay, quality control, and stress signaling. Even if theinterplay between mRNA translation and degradation is no longer to be demonstrated,a better understanding of their precise coordination is worthy of furtherinvestigation. This article is categorized under: RNA Turnover and Surveillance >Regulation of RNA Stability Translation > Translation Regulation RNA Interactionswith Proteins and Other Molecules > RNA-Protein Complexes.

Best practices for the visualization, mapping, and manipulation of R-loops.

Author(s) : Chédin F, Hartono S, Sanz L, Vanoosthuyse V,
Journal : EMBO J
R-loops represent an abundant class of large non-B DNA structures in genomes. Eventhough they form transiently and at modest frequencies, interfering with R-loopformation or dissolution has significant impacts on genome stability. Addressing themechanism(s) of R-loop-mediated genome destabilization requires a precisecharacterization of their distribution in genomes. A number of independent methodshave been developed to visualize and map R-loops, but their results are at timesdiscordant, leading to confusion. Here, we review the main existing methodologiesfor R-loop mapping and assess their limitations as well as the robustness ofexisting datasets. We offer a set of best practices to improve the reproducibilityof maps, hoping that such guidelines could be useful for authors and referees alike.Finally, we propose a possible resolution for the apparent contradictions in R-loopmapping outcomes between antibody-based and RNase H1-based mapping approaches.

RNA polymerase backtracking results in the accumulation of fission yeast condensin at active genes.

Author(s) : Rivosecchi J, Jost D, Vachez L, Gautier F, Bernard P, Vanoosthuyse V,
Journal : Life Sci Alliance
The mechanisms leading to the accumulation of the SMC complexes condensins aroundspecific transcription units remain unclear. Observations made in bacteria suggestedthat RNA polymerases (RNAPs) constitute an obstacle to SMC translocation,particularly when RNAP and SMC travel in opposite directions. Here we show infission yeast that gene termini harbour intrinsic condensin-accumulating featureswhatever the orientation of transcription, which we attribute to the frequentbacktracking of RNAP at gene ends. Consistent with this, to relocate backtrackedRNAP2 from gene termini to gene bodies was sufficient to cancel the accumulation ofcondensin at gene ends and to redistribute it evenly within transcription units,indicating that RNAP backtracking may play a key role in positioning condensin.Formalization of this hypothesis in a mathematical model suggests that the inclusionof a sub-population of RNAP with longer dwell-times is essential to fullyrecapitulate the distribution profiles of condensin around active genes. Takentogether, our data strengthen the idea that dense arrays of proteins tightly boundto DNA alter the distribution of condensin on chromosomes.

Mutational sources of trans-regulatory variation affecting gene expression in Saccharomyces cerevisiae.

Author(s) : Duveau F, Vande Zande P, Metzger B, Diaz C, Walker E, Tryban S, Siddiq M, Yang B, Wittkopp P,
Journal : Elife
Heritable variation in a gene's expression arises from mutations impacting cis- andtrans-acting components of its regulatory network. Here, we investigate howtrans-regulatory mutations are distributed within the genome and within a generegulatory network by identifying and characterizing 69 mutations withtrans-regulatory effects on expression of the same focal gene in Saccharomycescerevisiae. Relative to 1766 mutations without effects on expression of this focalgene, we found that these trans-regulatory mutations were enriched in codingsequences of transcription factors previously predicted to regulate expression ofthe focal gene. However, over 90% of the trans-regulatory mutations identifiedmapped to other types of genes involved in diverse biological processes includingchromatin state, metabolism, and signal transduction. These data show how geneticchanges in diverse types of genes can impact a gene's expression in trans, revealingproperties of trans-regulatory mutations that provide the raw material fortrans-regulatory variation segregating within natural populations.

Polymer modelling unveils the roles of heterochromatin and nucleolar organizing regions in shaping 3D genome organization in Arabidopsis thaliana.

Author(s) : Di Stefano M, Nützmann H, Marti-Renom M, Jost D,
Journal : Nucleic Acids Res
The 3D genome is characterized by a complex organization made of genomic andepigenomic layers with profound implications on gene regulation and cell function.However, the understanding of the fundamental mechanisms driving the crosstalkbetween nuclear architecture and (epi)genomic information is still lacking. Theplant Arabidopsis thaliana is a powerful model organism to address these questionsowing to its compact genome for which we have a rich collection of microscopy,chromosome conformation capture (Hi-C) and ChIP-seq experiments. Using polymermodelling, we investigate the roles of nucleolus formation and epigenomics-driveninteractions in shaping the 3D genome of A. thaliana. By validation of severalpredictions with published data, we demonstrate that self-attracting nucleolarorganizing regions and repulsive constitutive heterochromatin are major mechanismsto regulate the organization of chromosomes. Simulations also suggest thatinterphase chromosomes maintain a partial structural memory of the V-shapes, typicalof (sub)metacentric chromosomes in anaphase. Additionally, self-attraction betweenfacultative heterochromatin regions facilitates the formation of Polycomb bodieshosting H3K27me3-enriched gene-clusters. Since nucleolus and heterochromatin arehighly-conserved in eukaryotic cells, our findings pave the way for a comprehensivecharacterization of the generic principles that are likely to shape and regulate the3D genome in many species.