Mechanisms and design of Tc1/mariner transposons for genome engineering

Transposons are DNA segments that autonomously move within and between genomes across the tree of life. Tc1/mariners in particular have frequently crossed species boundaries in nature and provide powerful broad-host-range genetic vectors. Among them, the Sleeping Beauty (SB) transposon inserts DNA in vertebrate genomes with extraordinarily high efficiency, making it a prime genetic tool with applications expanding to gene therapy clinical trials. Nevertheless, the molecular principles of SB’s distinctive activity remain elusive, greatly hampering its further development. In the first part of this thesis, I investigated the molecular mechanisms of the SB transposon in comparison to Human mariner 1 (Hsmar1), a representative transposon of the same superfamily. Using biochemical and biophysical techniques together with fluorescence-based assays, I have characterized the initial steps of SB and Hsmar1 transposition and shown that the two transposons assemble their molecular machineries (or transpososomes) differently. By combining crystallographic data and SAXS-based modelling, I visualized the structural basis of these differences and explained how transpososome assembly is coupled to catalysis in the Hsmar1 transposon. Moreover, the data demonstrated that the unique assembly pathway of SB largely contributes to its exceptional efficiency and that it can be chemically modulated to control insertion rates in living cells. I have further reconstituted in vitro the ordered series of events comprising SB transposition, including transposon end binding, cleavage, and integration, and dissected previously unrevealed molecular features of the process. In the second part of my work, building on these mechanistic insights, I developed a novel SB transposase variant (hsSB) by employing a structure-based protein design approach. Using hsSB allowed for establishing a new genome engineering method based on the direct delivery of recombinant SB protein to cells. We showed that this new method, named SBprotAct, provides safer and more controlled genome modification of several cell types (including stem cells and human T cells), as compared to the state-of-art technology. This work sheds first light on the molecular determinants of SB transposition and its hyper-activity, providing a unique resource for the rational design of improved genome engineering platforms for research and medicine

R-loop formation and conformational activation mechanisms of Cas9

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage$^{1-3}$. The programmable activity of Cas9 has been widely utilized for genome editing applications$^{4-6}$, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity

DNA on the move: mechanisms, functions and applications of transposable elements

Transposons are mobile genetic elements that have invaded all domains of life by moving between and within their host genomes. Due to their mobility (or transposition), transposons facilitate horizontal gene transfer in bacteria and foster the evolution of new molecular functions in prokaryotes and eukaryotes. As transposition can lead to detrimental genomic rearrangements, organisms have evolved a multitude of molecular strategies to control transposons, including genome defense mechanisms provided by CRISPR‐Cas systems. Apart from their biological impacts on genomes, DNA transposons have been leveraged as efficient gene insertion vectors in basic research, transgenesis and gene therapy. However, the close to random insertion profile of transposon‐based tools limits their programmability and safety. Despite recent advances brought by the development of CRISPR‐associated genome editing nucleases, a strategy for efficient insertion of large, multi‐kilobase transgenes at user‐defined genomic sites is currently challenging. The discovery and experimental characterization of bacterial CRISPR‐associated transposons (CASTs) led to the attractive hypothesis that these systems could be repurposed as programmable, site‐specific gene integration technologies. Here, we provide a broad overview of the molecular mechanisms underpinning DNA transposition and of its biological and technological impact. The second focus of the article is to describe recent mechanistic and functional analyses of CAST transposition. Finally, current challenges and desired future advances of CAST‐based genome engineering applications are briefly discussed

Structural basis for the assembly of the type V CRISPR-associated transposon complex

CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads

Target site selection and remodelling by type V CRISPR-transposon systems

Canonical CRISPR–Cas systems provide adaptive immunity against mobile genetic elements1. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion2,3,4,5,6,7. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ7, 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

Engineered Sleeping Beauty transposase redirects transposon integration away from genes

The Sleeping Beauty (SB) transposon system is a popular tool for genome engineering, but random integration into the genome carries a certain genotoxic risk in therapeutic applications. Here we investigate the role of amino acids H187, P247 and K248 in target site selection of the SB transposase. Structural modeling implicates these three amino acids located in positions analogous to amino acids with established functions in target site selection in retroviral integrases and transposases. Saturation mutagenesis of these residues in the SB transposase yielded variants with altered target site selection properties. Transposon integration profiling of several mutants reveals increased specificity of integrations into palindromic AT repeat target sequences in genomic regions characterized by high DNA bendability. The H187V and K248R mutants redirect integrations away from exons, transcriptional regulatory elements and nucleosomal DNA in the human genome, suggesting enhanced safety and thus utility of these SB variants in gene therapy applications

Sleeping Beauty transposase structure allows rational design of hyperactive variants for genetic engineering

Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB’s applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases

A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming

The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc+/int− transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc+/int− transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change