Selective TnsC recruitment enhances the fidelity of RNA-guided transposition

Hoffmann, Florian T.; Kim, Minjoo; Beh, Leslie Y.; Wang, Jing; Vo, Phuc Leo H.; Gelsinger, Diego Rivera; George, Jerrin Thomas; Acree, Christopher; Mohabir, Jason; Fernandez, I.S.; Sternberg, Samuel H.

doi:10.1038/s41586-022-05059-4

Cited by 46 publications

(70 citation statements)

References 71 publications

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“…TnsC from prototypic Tn7 forms heptameric oligomers ( 20 ) and led to the original hypothesis that the ring would allow segregated interaction interfaces: the TnsD (a TniQ domain containing protein) interface maps to the N-terminal face, and the TnsA/B interface maps to the C-terminal face ( 20 ). Although I-F3 CAST TnsC also adopts a heptameric structure ( 12 ), it remains unknown whether TnsC in this system has similarly segregated interaction interfaces. However, ChIP-seq experiments suggest that TnsC plays a key role in associating with the Cascade complex, serving as a selectivity factor in subsequent recruitment of TnsA/B ( 12 ).…”

Section: Discussionmentioning

confidence: 99%

“…Although I-F3 CAST TnsC also adopts a heptameric structure ( 12 ), it remains unknown whether TnsC in this system has similarly segregated interaction interfaces. However, ChIP-seq experiments suggest that TnsC plays a key role in associating with the Cascade complex, serving as a selectivity factor in subsequent recruitment of TnsA/B ( 12 ). Therefore, despite the structural differences in ShCAST TnsC, we hypothesize that the segregated interactions we observe in the ShCAST transpososome are conserved across Tn7-like transposition systems.…”

Section: Discussionmentioning

confidence: 99%

“…The two classes were then subjected to homogenous refinement in cryoSPARC respectively. The particle stacks from both classes were subjected to 3D classification in RELION ( 11, 12 ), which removed junk particles with weak densities of Cas12k or TnsB. Each classification resulted in a particle stack that had well-defined configurations of ShCAST transpososome.…”

Section: Methodsmentioning

confidence: 99%

“…One class with better resolved TniQ (34%, 214,291 particles) was selected for downstream non-uniform refinement in cryoSPARC ( 10 ). The particle stack was subjected to 3D classification in RELION ( 11, 12 ) resulting in the intermediate particle stack with a stronger density of TniQ (70%, 150,358 particles). To improve the resolution of TniQ, this particle stack was subjected to two rounds of focused 3D classification (skipping alignments, tau fudge factor of 16), iterative CTF refinements ( 13 ) and bayesian polishing ( 14 ).…”

Section: Methodsmentioning

confidence: 99%

“…In CAST elements, TniQ protein associates with the target site via interactions with the CRISPR effector ( 9 ), and in some cases is shown to require a host factor(s) ( 10, 11 ). The AAA+ regulator TnsC serves as a ’matchmaker’ by interacting with both TniQ/TnsD and the transposase (TnsA/B) ( 12, 13 ). Very little structural information exists to explain how TnsC bridges the components in the hypothesized transpososome assembly.…”

Section: Introductionmentioning

confidence: 99%

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Structures of the holo CRISPR RNA-guided transposon integration complex

Park

Tsai

Rizo

et al. 2022

Preprint

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CRISPR-associated transposons (CAST) are programmable mobile elements that insert large DNA cargo by an RNA-guided mechanism. Multiple conserved components act in concert at the target site through formation of an integration complex (transpososome). We reconstituted the type V-K CAST transpososome fromScytonema hofmannii(ShCAST) and determined the structure using cryo-EM. Transpososome architecture ensures orientation-specific association: AAA+ regulator TnsC has defined polarity and length, with dedicated interaction interfaces for other CAST components. Interestingly, transposase (TnsB)-TnsC interactions we observe contribute to stimulating TnsC's ATP hydrolysis activity. TnsC deviates from previously observed helical configurations of TnsC, and target DNA does not track with TnsC protomers. Consequently, TnsC makes new, functionally important protein-DNA interactions throughout the transpososome. Finally, two distinct transpososome populations suggests that associations with the CRISPR effector are flexible. These ShCAST transpososome structures significantly enhances our understanding of CAST transposition systems and suggests avenues for improving CAST transposition for precision genome-editing applications.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structures of the holo CRISPR RNA-guided transposon integration complex

Park

Tsai

Rizo

et al. 2022

Preprint

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

Schmitz,

Querques

2023

FEBS Open Bio

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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.

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Precise Gene Knock‐In Tools with Minimized Risk of DSBs: A Trend for Gene Manipulation

Liu,

Kong,

Liu

et al. 2024

Advanced Science

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Gene knock‐in refers to the insertion of exogenous functional genes into a target genome to achieve continuous expression. Currently, most knock‐in tools are based on site‐directed nucleases, which can induce double‐strand breaks (DSBs) at the target, following which the designed donors carrying functional genes can be inserted via the endogenous gene repair pathway. The size of donor genes is limited by the characteristics of gene repair, and the DSBs induce risks like genotoxicity. New generation tools, such as prime editing, transposase, and integrase, can insert larger gene fragments while minimizing or eliminating the risk of DSBs, opening new avenues in the development of animal models and gene therapy. However, the elimination of off‐target events and the production of delivery carriers with precise requirements remain challenging, restricting the application of the current knock‐in treatments to mainly in vitro settings. Here, a comprehensive review of the knock‐in tools that do not/minimally rely on DSBs and use other mechanisms is provided. Moreover, the challenges and recent advances of in vivo knock‐in treatments in terms of the therapeutic process is discussed. Collectively, the new generation of DSBs‐minimizing and large‐fragment knock‐in tools has revolutionized the field of gene editing, from basic research to clinical treatment.

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Selective TnsC recruitment enhances the fidelity of RNA-guided transposition

Cited by 46 publications

References 71 publications

Structures of the holo CRISPR RNA-guided transposon integration complex

Structures of the holo CRISPR RNA-guided transposon integration complex

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

Precise Gene Knock‐In Tools with Minimized Risk of DSBs: A Trend for Gene Manipulation

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