ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

Nie, Yan; Chaillet, Maxime; Becke, Christian; Haffke, Matthias; Pelosse, Martin; Fitzgerald, Daniel J.; Collinson, Ian; Schaffitzel, Christiane; Berger, Imre

doi:10.1007/978-3-319-27216-0_3

Cited by 20 publications

(17 citation statements)

References 73 publications

(102 reference statements)

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“…We had previously developed methods to rapidly assemble functional DNA elements into multicomponent circuitry in baculoviral vectors [24][25][26] . Here, we optimized and fine-tuned our approach by incorporating time-tested MultiSite Gateway recombination modalities to assemble with ease currently up to 25 distinct DNA elements of various sizes (Fig.1a, Extended Data Fig.1a-e, Supplementary Methods), importantly aiming to significantly reduce prokaryotic elements carried over into the baculovirus which can compromise vector integrity during manufacturing 27 (Extended Data Fig.1f).…”

Section: Resultsmentioning

confidence: 99%

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Baculovirus-vectored precision delivery of large DNA cargoes in human genomes

Aulicino¹,

Pelosse²,

Toelzer³

et al. 2020

Preprint

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Precise gene editing and genome engineering by CRISPR technology requires simultaneous delivery of multiple DNA-encoded components into living cells rapidly exceeding the cargo capacity of currently utilized viral vector systems. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck. We implement hybrid DNA techniques (MultiMate) for rapid and error-free assembly of currently up to 25 functional DNA modules in a single baculoviral vector enabling CRISPR-based genome engineering. Utilizing homology-independent targeted integration (HITI), we achieve up to 30% correct genome interventions in human cells, including precision docking of large DNA payloads in the ACTB locus. We demonstrate baculovirus-vectored delivery of prime-editing toolkits for seamless DNA search-and-replace interventions achieving, with a single vector, highly efficient cleavage-free trinucleotide insertion in the HEK3 locus without any detectable indels. Our approach thus unlocks a wide range of editing and engineering applications in human cell genomes.

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Section: Resultsmentioning

confidence: 99%

“…Cre-mediated recombination of DNA elements One acceptor and one or multiple donor vectors were assembled using Cre-mediated recombination as previously described 26,32,47 . One acceptor and one or more donors were mixed with a ratio of 1:1.1 in distilled H2O with 0.5 ul (7.…”

Section: Lr Recombinationmentioning

confidence: 99%

Baculovirus-vectored precision delivery of large DNA cargoes in human genomes

Aulicino¹,

Pelosse²,

Toelzer³

et al. 2020

Preprint

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“…After overnight incubation, prepare Acceptor-Donor fusion plasmid using standard kits (Qiagen). Predict Acceptor-Donor fusion plasmid sequence by using web-based Cre-ACEMBLER software (21). Check plasmids by restriction digestion using appropriate enzymes identified by restriction pattern prediction (see Note 2).…”

Section: Methodsmentioning

confidence: 99%

Multiprotein Complex Production in E. coli: The SecYEG-SecDFYajC-YidC Holotranslocon

Berger

Jiang

Schulze

et al. 2017

Methods in Molecular Biology

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General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms SummaryA modular approach for balanced overexpression of recombinant multiprotein complexes in E. coli is described, with the prokaryotic protein secretase/insertase complex, the SecYEGSecDFYajC-YidC holotranslocon (HTL) used as an example. This procedure has been implemented here in the ACEMBL system. The protocol details the design principles of the mono-or polycistronic DNA constructs, the expression and purification of functional HTL and its association with translating ribosome nascent chain (RNC) complexes into a RNC-HTL supercomplex.1.

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“…To facilitate the assembly of multi-gene expression cassettes, we developed recombineering-based, parallelized, automatable approaches relying on synthetic DNA plasmid modules that could be conjoined into elaborate transfer plasmids for integration into the Tn7 and LoxP sites, respectively [29,30,31,32]. We followed a ‘reduce to the max’ approach in designing these reagents, eliminating from our plasmid modules DNA elements with unclear or, for our purposes unnecessary, functions present in common plasmids, keeping only the bare minimum of DNA elements conferring defined functions (origin, resistance marker, promoters and terminators), adding the specific integration sequences we needed (LoxP, Tn7) and short DNA sequences for multiplying the expression cassettes.…”

Section: The Multibac Bics: Enabling Multiprotein Complex Structurmentioning

confidence: 99%

MultiBac: Baculovirus-Mediated Multigene DNA Cargo Delivery in Insect and Mammalian Cells

Gupta

Tölzer

Sari-Ak

et al. 2019

Viruses

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The baculovirus/insect cell system (BICS) is widely used in academia and industry to produce eukaryotic proteins for many applications, ranging from structure analysis to drug screening and the provision of protein biologics and therapeutics. Multi-protein complexes have emerged as vital catalysts of cellular function. In order to unlock the structure and mechanism of these essential molecular machines and decipher their function, we developed MultiBac, a BICS particularly tailored for heterologous multigene transfer and multi-protein complex production. Baculovirus is unique among common viral vectors in its capacity to accommodate very large quantities of heterologous DNA and to faithfully deliver this cargo to a host cell of choice. We exploited this beneficial feature to outfit insect cells with synthetic DNA circuitry conferring new functionality during heterologous protein expression, and developing customized MultiBac baculovirus variants in the process. By altering its tropism, recombinant baculovirions can be used for the highly efficient delivery of a customized DNA cargo in mammalian cells and tissues. Current advances in synthetic biology greatly facilitate the construction or recombinant baculoviral genomes for gene editing and genome engineering, mediated by a MultiBac baculovirus tailored to this purpose. Here, recent developments and exploits of the MultiBac system are presented and discussed.

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ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

Cited by 20 publications

References 73 publications

Baculovirus-vectored precision delivery of large DNA cargoes in human genomes

Baculovirus-vectored precision delivery of large DNA cargoes in human genomes

Multiprotein Complex Production in E. coli: The SecYEG-SecDFYajC-YidC Holotranslocon

MultiBac: Baculovirus-Mediated Multigene DNA Cargo Delivery in Insect and Mammalian Cells

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