Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost

Benoit, Roger; Ostermeier, Christian; Geiser, Martin; Li, Julia Su Zhou; Widmer, Hans; Auer, Manfred

doi:10.1371/journal.pone.0153158

Cited by 33 publications

(39 citation statements)

References 30 publications

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“…S4) to fulfill seamless assembly of DNA fragment(s) derived from Cas9 RNP cleavage. In accordance with previous reports [43][44][45] , we found that the efficiency of EI method drops sharply when the number of gene fragments is over three. So, the T5-assisted cloning is preferable when assembling three or more gene fragments.…”

Section: Single Cas9 Rnp-based Seamless Dna Assemblysupporting

confidence: 93%

“…Though GA is fast and versatile, it is very expensive because it requires three enzymes including T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase. Meanwhile, enzyme-independent cloning (EI) methods 43,44 have been developed capable of assembling DNA fragments without any enzyme at low cost. In a typical EI cloning (Fig.…”

Section: Single Cas9 Rnp-based Seamless Dna Assemblymentioning

confidence: 99%

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Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

Qiao³

et al. 2018

Preprint

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CRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to produce Cas9 RNP and supply a convenient and cost-effective DNA assembly method which is an invaluable addition to the synthetic biological toolboxes.Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated-protein 9 (Cas9) genome-editing system is a kind of the adaptive immune systems in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids 1-3 . The widely used Streptococcus pyogenes Cas9 (SpCas9) can be targeted to a 20 bp specific DNA sequence by an associated complementary guide RNA (gRNA), provided that a protospacer adjacent motif (PAM) of the form NRG (where R = G or A) is present 4,5 . The CRISPR-Cas9-based genetic technologies, in particular as genome-editing tools, have been extensively applied to biomedical area with great promises to revolutionize the treatment of genetic diseases [6][7][8][9] . Compared to other protein-based genome-editing technologies, including zinc-finger nucleases (ZFNs) 10 and transcription activator-like effector nucleases (TALENs) 11 , the newly developed RNA-guided endonucleases CRISPR-Cas9 system has attracted wide attentions due to its simplicity, high efficacy, and ease of use 8,12 . CRISPR-Cas9 has been mostly used for in vivo genome excising and editing 1,3,6 , otherwise, it and its variants such as catalytically inactivated Cas9 (dCas9) have been successfully applied in high-throughput interrogation of gene functions in health and diseases 13,14 . In spite of numerous in vivo applications, recently, CRISPR-Cas9 has been employed as in vitro molecular cloning tools as well. For instance, a technique named CATCH utilized Cas9 nuclease to excise the target genome segment up to 100 kilobases and ligated it to cloning vectors in a single step 15 . Besides, another technique called DASH harnessed Cas9 to effectively deplete unwanted sequence from DNA libraries prior to sequencing to reduce sequencing cost 16 . In these techniques, Cas9 functioned as a ribonucleprotein complex (RNP), in which the specific negative single guide RNA molecules (sgRNAs) bound to positively charged Cas9 enzymes. Compared to regular methods that deliver plasm...

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Section: Single Cas9 Rnp-based Seamless Dna Assemblysupporting

confidence: 93%

Section: Single Cas9 Rnp-based Seamless Dna Assemblymentioning

confidence: 99%

Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

Qiao³

et al. 2018

Preprint

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“…Therefore, any improvement in protein yield and purity makes the research more cost-effective and supports approaches to investigate protein function, applying for example alanine scan methodology [41, 42]. The availability of restriction enzyme free cloning strategies, such as the seamless cloning technique [43], allows a broad application of our expression cassette using the strong paf promoter/terminator. Penicillium sp.…”

Section: Resultsmentioning

confidence: 99%

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses

et al. 2016

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BackgroundSmall, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure–function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses.ResultsThe APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI–MS, ECD and NMR spectroscopy and growth inhibition assays.ConclusionThis study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure–function analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0586-4) contains supplementary material, which is available to authorized users.

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“…Furthermore, the Hot Fusion method 15, 23 can be used for assembling two fragments instead of the Gibson reaction, which further reduces the cost of mutagenesis by omitting a DNA ligase.…”

Section: Resultsmentioning

confidence: 99%

High-throughput mutagenesis using a two-fragment PCR approach

Heydenreich

Miljuš

Jaussi

et al. 2017

Sci Rep

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Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efficiency of the mutagenesis step is the key for fast and economical generation of such libraries. PCR artefacts such as misannealing and tandem primer repeats are often observed in mutagenesis cloning and reduce the efficiency of mutagenesis. Here we present a high-throughput mutagenesis pipeline based on established methods that significantly reduces PCR artefacts. We combined a two-fragment PCR approach, in which mutagenesis primers are used in two separate PCR reactions, with an in vitro assembly of resulting fragments. We show that this approach, despite being more laborious, is a very efficient pipeline for the creation of large libraries of mutants.

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Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost

Cited by 33 publications

References 30 publications

Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses

High-throughput mutagenesis using a two-fragment PCR approach

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