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Introduction DNA-based transposable elements constitute a part of the genome of a wide range of organisms as dispersed repetitive sequences. Elements that retain their transposition activity in their host species [...], The majority of DNA-based transposable elements comprise autonomous and nonautonomous copies, or only nonautonomous copies, where the autonomous copy contains an intact gene for a transposase protein and the nonautonomous copy does not. Even if autonomous copies coexist, they are generally less frequent. The Tol2 element of medaka fish is one of the few elements for which a nonautonomous copy has not yet been found. Here, we report the presence of a nonautonomous Tol2 copy that was identified by surveying the medaka genome sequence database. This copy contained three local sequence alterations that affected the deduced amino acid sequence of the transposase: a deletion of 15 nucleotides resulting in a deletion of 5 amino acids, a base substitution causing a single amino acid change, and another base substitution giving rise to a stop codon. Transposition assays using cultured human cells revealed that transposase activity was reduced by the 15-nucleotide deletion and abolished by the nonsense mutation. This is the first example of a nonautonomous Tol2 copy. Thus, Tol2 is in an early stage of decay in the medaka genome, and is therefore a unique element to observe an almost complete decay process that progresses in natural populations. Key words: transposition, transposase, autonomy, horizontal transfer, vertebrate. La majorité des transposons à ADN comprennent soit des copies autonomes et non-autonomes, ou encore seulement des copies non-autonomes, où la copie autonome contient un gène intact codant pour la trans-posase tandis que la copie non-autonome n'en contient pas. Même lorsque des copies autonomes co-existent avec des copies non-autonmes, ces premières sont généralement moins nombreuses. L'élément Tol2 chez le médaka est un des rares éléments pour lesquels aucune copie non-autonome n'a été trouvée à ce jour. Dans ce travail, les auteurs rapportent la présence d'une copie non-autonome de Tol2, laquelle a été identifiée en inspectant la séquence du génome de ce poisson. Cette copie contient trois altérations locales de séquence qui affectent la séquence prédite des acides aminés de la transposase : une délétion de 15 nucléotides entraîne la perte de 5 acides aminés, une substitution nucléotidique cause le changement d'un seul acide aminé et une autre substitution donne naissance à un codon stop. L'analyse de l'activité transpositionnelle chez des cellules humaines en culture a révélé qu'elle était réduite par la délétion de 15 nucléotides et abolie par la mutation non-sens. Il s'agit de la première description d'une copie non-autonome de Tol2. Ainsi, Tol2 est à un stade précoce de dégradation au sein du génome du médaka et constitue un élément unique pour observer la presque totalité du processus de dégradation au fur et à mesure qu'il se produit au sein de populations naturelles. [Traduit par la Rédaction] Mots-clés : transposition, transposase, autonomie, transfert horizontal, vertébrés.

Introduction Transposable elements (TEs) are repetitive DNA sequences that can move from one chromosome to another within the host genome (Mackay 1989). They are present in the genome of most [...], Mariner-like elements (MLEs) are class II transposons belonging to the Tc1-mariner family that have successfully invaded many insect genomes. In the current study, the availability of the Hessian fly, Mayetiola destructor, genome has enabled us to perform in silico analysis of MLEs using a previously described mariner element (Desmar1) belonging to the mauritiana subfamily. Eighteen mauritiana-like elements were detected and clustered into three main groups: Desmar1-like, MauCons1, and MauCons2. Subsequently, in vitro analysis was carried out to investigate mauritiana-like elements in M. destructor as well as in Mayetiola hordei using primers designed from TIRs of previously identified MLEs. PCR amplifications were successful, and a total of 12 and 17 mauritiana-like elements were detected in M. destructor and M. hordei, respectively. Sequence analyses of mauritiana-like elements obtained in silico and in vitro have shown that MauCons1 and MauCons2 elements share low similarity with Desmar1 ranging from 50% to 55%, suggesting that different groups under the mauritiana subfamily have invaded the genomes of M. destructor and M. hordei. These groups were likely inherited by vertical transmission, which subsequently underwent different evolutionary histories. This work describes new mauritiana-like elements in M. destructor that are distinct from the previously discovered Desmar1 and provides the first evidence of MLEs belonging to the mauritiana subfamily in M. hordei. Key words: Mayetiola destructor, Mayetiola hordei, transposable elements, MLEs, mauritiana, diversity. Les éléments de type Mariner (MLE pour << mariner-like éléments >>) sont des transposons de classe II appartenant à la famille d&apos;éléments Tc1-mariner, lesquels ont envahi plusieurs génomes d&apos;insectes. Dans le présent travail, la disponibilité du génome du Mayetiola destructor (mouche de Hesse) a permis d&apos;effectuer une analyse in silico des MLE en recherchant un élément mariner déjà décrit (Desmar1) appartenant à la sous-famille mauritiana. Dix-huit éléments de type mauritiana ont été détectés et formaient trois groupes principaux nommés Desmar1-like, MauCons1 et MauCons2. Ensuite, des analyses in vitro ont été réalisées pour étudier les éléments mauritiana-like chez le M. destructor ainsi que chez le Mayetiola hordei à l&apos;aide d&apos;amorces conçues à partir des TIR des éléments MLE identifiés auparavant. Des amplications PCR ont permis d&apos;identifier un total de 12 et 17 éléments mauritiana-like chez le M. destructor et le M. hordei, respectivement. L&apos;analyse des séquences de ces éléments identifiés in silico et in vitro a révélé que les éléments MauCons1 et MauCons2 présentent une faible similarité avec Desmar1 (de 50 à 55%), ce qui suggère que différents groupes au sein de la sous-famille mauritiana auraient envahi les génomes du M. destructor et du M. hordei. Ces groupes ont vraisemblablement été transmis verticalement mais auraient ensuite connu une évolution différente. Ce travail décrit de nou-veaux éléments mauritiana-like chez le M. destructor qui sont distincts de l&apos;élément Desmar1 décrit précédemment et apporte les premières évidences de la présence de MLE de la sous-famille mauritiana chez le M. hordei. Mots-clés : Mayetiola destructor, Mayetiola hordei, éléments transposables, MLE, maurtiana, diversité.

Author(s): Tautvydas Karvelis [sup.1] , Gytis Druteika [sup.1] , Greta Bigelyte [sup.1] , Karolina Budre [sup.1] , Rimante Zedaveinyte [sup.1] , Arunas Silanskas [sup.1] , Darius Kazlauskas [sup.1] , Ceslovas [...], Transposition has a key role in reshaping genomes of all living organisms.sup.1. Insertion sequences of IS200/IS605 and IS607 families.sup.2 are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established.sup.3-5. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR-Cas9/Cas12 nucleases.sup.6-8. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5&apos;-TTGAT transposon-associated motif. We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR-Cas nucleases and establishes TnpB as a prototype of a new system for genome editing. The RNA-directed nuclease TnpB from Deinococcus radiodurans can be reprogrammed to cleave DNA target sites in human cells.

Author(s): Irma Querques [sup.1] , Michael Schmitz [sup.1] , Seraina Oberli [sup.1] , Christelle Chanez [sup.1] , Martin Jinek [sup.1] Author Affiliations: (1) Department of Biochemistry, University of Zurich, Zurich, [...], Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements.sup.1. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion.sup.2-7. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ.sup.7, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools. Structural studies on Scytonema hofmanni CRISPR-associated transposon protein complexes indicate a mechanism for RNA-guided DNA transposition involving Cas12k, TnsC and TnsB.

Introduction Substantial efforts over the past few years have led chimeric antigen receptor (CAR) T cell therapy to success in relapsed and refractory (r/r) B cell malignancies. The early concept [...], BACKGROUND. Chimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. METHODS. We report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells. RESULTS. The cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of [CD3.sup.+] lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion. CONCLUSION. SB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities. TRIAL REGISTRATION. ClinicalTrials.gov NCT03389035. FUNDING. This study was supported by grants from the Fondazione AIRC per la Ricerca sul Cancro (AIRC); Cancer Research UK (CRUK); the Fundacion Cientifica de la Asociacion Espanola Contra el Cancer (FC AECC); Ministero Della Salute; Fondazione Regionale per la Ricerca Biomedica (FRRB).