Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases

Tou, Connor J; Orr, Benno; Kleinstiver, Benjamin P.

doi:10.1038/s41587-022-01574-x

Cited by 57 publications

(52 citation statements)

References 66 publications

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“…Off-target insertions occur with both prokaryotic and synthetic CAST systems [29][30][31] , and we find that in our system these are the product of the active TE, and not off-target cleavage by the catalytically-active CRISPR/Cas9. Off-target insertions are reduced by the presence of Cas9, suggesting that the insertion of mPing is being directed to the ontarget site by the presence of the Cas9-induced double-strand DNA break.…”

Section: Discussionmentioning

confidence: 57%

“…Prokaryotic and synthetic CAST systems have been demonstrated in bacterial cultures, animal cell culture and animal tissue culture [29][30][31] . We have produced a functional synthetic CAST system to generate targeted insertions in whole individual plants of the model plant Arabidopsis, as well as translated this technology into soybean plants, which is a critically important crop for global oil and protein production.…”

Section: Discussionmentioning

confidence: 99%

“…The selection of the integration site is highly variable among different TE types, and can target accessible chromatin 23 , a favored chromatin state 24 , sites of double-strand breaks 25 or a short sequence such as TAA 26 . Bacterial genomes harbor natural CRISPR-associated transposases (CASTs) that have been engineered using different CRISPR gRNAs to transpose the TE to specific sites in bacterial genomes [27][28][29][30] . These CAST systems lack Cas cleavage activity and count on the transposase to perform the cut at the insertion site.…”

Section: Mainmentioning

confidence: 99%

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CRISPR-targeted transposable element insertion for efficient plant genome engineering

Slotkin

Liu

Panda

et al. 2023

Preprint

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The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome editing approaches to develop improved crops. Often considered genome ‘parasites’, transposable elements (TEs) evolved to insert their DNA seamlessly into genomes. TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type. We developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the TE’s natural ability to precisely insert into the genome. Inspired by CRISPR-associated transposases (CASTs) that target transposition in a programmable manner in bacteria, we generated a synthetic CAST by fusing the rice Pong TE transposase protein to the Cas9 or CPF1 programmable nucleases. We demonstrated sequence-specific targeted delivery (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and gene expression cassette into the genome of the model plant Arabidopsis. We additionally translated this system into soybean, a major global crop in need of targeted insertion technology. We have engineered a TE ‘parasite’ into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.

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Section: Discussionmentioning

confidence: 57%

Section: Discussionmentioning

confidence: 99%

Section: Mainmentioning

confidence: 99%

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CRISPR-targeted transposable element insertion for efficient plant genome engineering

Slotkin

Liu

Panda

et al. 2023

Preprint

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“…Most notably, Type V-K CAST systems lack TnsA altogether and exclusively generate cointegrate products, though these products can resolve to simple insertions through homologous recombination. 45,46,55,56 We and others have applied PCR-free long-read sequencing to unbiasedly characterize and quantify both types of integration products, demonstrating that Type I-F CAST systems show optimal product purity, but we also encourage interested readers to consider engineered Type V-K CAST systems (HELIX) that show improved properties for both transposition pathway and specificity. Lastly, both types of CAST systems can also generate tandem insertions (Fig.…”

Section: Boxes Box 1 Alternative Integration Byproductsmentioning

confidence: 98%

“…However, we point interested readers to recent studies that describe both mechanistic and technological advances in the study and application of Type V-K CAST systems 45,47,48 .…”

Section: Mechanism and Development Of Type I-f Castsmentioning

confidence: 99%

Bacterial genome engineering using CRISPR RNA-guided transposases

Gelsinger

Klompe

et al. 2023

Preprint

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CRISPR-associated transposons (CASTs) have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability, and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in E. coli at efficiencies approaching ~100%, generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CAST systems, including guidelines on the available homologs and vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational crRNA design algorithm to avoid potential off-targets and CRISPR array cloning pipeline for DNA insertion multiplexing. Starting from available plasmid constructs, the isolation of clonal strains containing a novel genomic integration event-of-interest can be achieved in 1 week using standard molecular biology techniques.

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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 cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases

Cited by 57 publications

References 66 publications

CRISPR-targeted transposable element insertion for efficient plant genome engineering

CRISPR-targeted transposable element insertion for efficient plant genome engineering

Bacterial genome engineering using CRISPR RNA-guided transposases

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

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