Rapid 40 kb genome construction from 52 parts

Pryor, John M.; Potapov, Vladimir; Pokhrel, Nilisha; Lohman, Gregory J. S.

doi:10.1101/2020.12.22.424019

Cited by 11 publications

(12 citation statements)

References 44 publications

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“…By Golden Gate assembly, 40 kb genome of T7 phage was constructed from 52 DNA fragments. 38 Combining iPac with Golden Gate assembly may improve the efficiency of multifragment construction. Here, I demonstrated the construction of the phage genomes and the large plasmids using portions of phage genomes as DNA fragments.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Rapid and Accurate Assembly of Large DNA Assisted by In Vitro Packaging of Bacteriophage

Nozaki

2022

ACS Synth. Biol.

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Development of DNA assembly methods made it possible to construct large DNA. However, achieving a large DNA assembly easily, accurately, and at a low cost remains a challenge. This study shows that DNA assembled only by annealing of overlapping single-stranded DNA ends, which are generated by exonuclease treatment, without ligation can be packaged in phage particles and can also be transduced into bacterial cells. Based on this, I developed a simple method to construct long DNA of about 40–50 kb from five to ten PCR fragments using the bacteriophage in vitro packaging system. This method, namely, iPac ( in vitro Packaging-assisted DNA assembly), allowed accurate and rapid construction of large plasmids and phage genomes. This simple method will accelerate research in molecular and synthetic biology, including the construction of gene circuits or the engineering of metabolic pathways.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Rapid and Accurate Assembly of Large DNA Assisted by In Vitro Packaging of Bacteriophage

Nozaki

2022

ACS Synth. Biol.

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“…It is probable that this factor outweighs the costs of reduced reaction volume. Pryor et al recently demonstrated the use of Golden Gate assembly to efficiently assemble a 40 kbp bacteriophage genome from 52 parts . These data suggest that the EMMA pipeline is likely to be able to handle far larger constructs.…”

Section: Discussionmentioning

confidence: 99%

Automation and Expansion of EMMA Assembly for Fast-Tracking Mammalian System Engineering

et al. 2022

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With applications from functional genomics to the production of therapeutic biologics, libraries of mammalian expression vectors have become a cornerstone of modern biological investigation and engineering. Multiple modular vector platforms facilitate the rapid design and assembly of vectors. However, such systems approach a technical bottleneck when a library of bespoke vectors is required. Utilizing the flexibility and robustness of the Extensible Mammalian Modular Assembly (EMMA) toolkit, we present an automated workflow for the library-scale design, assembly, and verification of mammalian expression vectors. Vector design is simplified using our EMMA computer-aided design tool (EMMA-CAD), while the precision and speed of acoustic droplet ejection technology are applied in vector assembly. Our pipeline facilitates significant reductions in both reagent usage and researcher hands-on time compared with manual assembly, as shown by system Q-metrics. To demonstrate automated EMMA performance, we compiled a library of 48 distinct plasmid vectors encoding either CRISPR interference or activation modalities. Characterization of the workflow parameters shows that high assembly efficiency is maintained across vectors of various sizes and design complexities. Our system also performs strongly compared with manual assembly efficiency benchmarks. Alongside our automated pipeline, we present a straightforward strategy for integrating gRNA and Cas modules into the EMMA platform, enabling the design and manufacture of valuable genome editing resources.

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“…The PCR amplicons were gel purified and mixed with the entry vector. They were assembled together using Golden Gate assembly with Bsm BI 44 . The final plasmids were confirmed by Sanger sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…They were assembled together using Golden Gate assembly with BsmBI 44 . The final plasmids were confirmed by Sanger sequencing.…”

Section: Creepy Plasmids and Grna Assemblymentioning

confidence: 99%

Episomal editing of synthetic constructs in yeast using CRISPR

Zhao

Coelho

Lauer

et al. 2022

Preprint

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Use of synthetic genomics to design and build ″big″ DNA has revolutionized our ability to answer fundamental biological questions by employing a bottom-up approach. S. cerevisiae, or budding yeast, has become the major platform to assemble large synthetic constructs thanks to its powerful homologous recombination machinery and the availability of well-established molecular biology techniques. However, efficiently and precisely introducing designer variations to episomal assemblies remains challenging. Here, we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, for rapid engineering of mammalian DNA constructs larger than 100 kb. We demonstrate that editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes with CRISPR. After optimizing CREEPY for episomal editing, we achieve efficient simplex and multiplex editing as demonstrated by engineering a mouse Sox2-harboring episome.

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Rapid 40 kb genome construction from 52 parts

Cited by 11 publications

References 44 publications

Rapid and Accurate Assembly of Large DNA Assisted by In Vitro Packaging of Bacteriophage

Rapid and Accurate Assembly of Large DNA Assisted by In Vitro Packaging of Bacteriophage

Automation and Expansion of EMMA Assembly for Fast-Tracking Mammalian System Engineering

Episomal editing of synthetic constructs in yeast using CRISPR

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