Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system

Sakuma, Tetsushi; Nishikawa, Ayami; Kume, Satoshi; Chayama, Kazuaki; Yamamoto, Takashi

doi:10.1038/srep05400

Cited by 345 publications

(301 citation statements)

References 30 publications

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“…mMoClo enables the stable integration into defined sites in mammalian chromosomes of very large multigene constructs such as genetic switch programs that encode the regulated expression of counteracting transcription factors that establish hereditable memory in cells 34 . Golden Gate is best suited for applications where multiple genes must be expressed together, and consequently it is also used in kits for multiplex CRISPR-Cas9 genome editing 35 . The large number of plasmids required for Golden Gate is a drawback, but in return researchers can format their DNA to physical standards that facilitate part re-use and sharing.…”

Section: Endonuclease-mediated Assemblymentioning

confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

251

186

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PrefaceDNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade a plethora of powerful new DNA assembly methods including Gibson assembly, Golden Gate and LCR have been developed. In this Innovation article we discuss these methods and standards such as MoClo, GoldenBraid, MODAL and PaperClip, which have been developed to facilitate a streamlined assembly workflow, aid material exchange, and the creation of modular, reusable DNA parts. IntroductionOur capacity to cut and paste DNA from different sources and to assemble it into gene constructs has been one of the key drivers of biological research and biotechnology over the past four decades. However, despite countless advances in molecular biology, the assembly of DNA parts into new constructs remains a craft that is both time consuming and unpredictable. The decreasing costs of gene synthesis promises to alleviate these limitations by providing custom-made double-stranded DNA fragments typically between 200 and 2000 bp in length 1 . Nonetheless, gene synthesis does not eliminate the need for DNA assembly, which remains necessary for the production of constructs beyond one kilobase in size, both in research labs and at gene synthesis companies. DNA assembly also enables carrying out projects with more complex experimental needs and is especially valuable for building diverse plasmid libraries and creating multicomponent systems, and has even been used to construct synthetic cells 2 .Addressing the limitations of DNA assembly methods has been one of the key goals of synthetic biology, a scientific discipline focused on the construction and testing of new or redesigned versions of genes, gene networks, pathways and cells 3,4 . In order to tackle projects of increasing scale and complexity, researchers have invested significant effort into developing new tools for DNA assembly and into matching them with improved, lower-cost gene synthesis (for reviewes on gene synthesis see REFS. 1,5 1,5 ), as well as a suite of important new tools for genome editing (Box 1). With these combined advances the field is now at a point where gene synthesis and DNA assembly can empower even undergraduate students to construct entire eukaryotic chromosomes 6 . This acceleration in the scale of DNA assembly enables construction projects too complex to be drawn out on the back of an envelope, which instead require an engineering approach. In the past decade important 1 assembly methods such as Gibson assembly and Golden Gate have been developed 7,8 , which define new protocols for joining together DNA parts. Alongside these methods, researchers have also developed various physical standards such as MODAL (Modular Overlap-Directed Assembly with Linkers) 9 and MoClo (Modular Cloning system) 12 that define rules for the format of DNA parts that can be used with them. These physical standards facilitate the re-use of parts between experiments, exchange of parts between research groups and importantly provide modularity in construction. ...

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Section: Endonuclease-mediated Assemblymentioning

confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

251

186

View full text Add to dashboard Cite

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“…The techniques described here can be extended to use multiple guide RNAs to simultaneously target several loci 14,46,47 . Many protocols have been established to differentiate hPSCs into distinct cell types, allowing various genetic manipulations in cell types of interest 30 .…”

Section: Discussionmentioning

confidence: 99%

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Blair

Bateup

Hockemeyer

2016

JoVE

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Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell's endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent stem cells.

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“…Several editing agents are currently being developed, including TALENs and the CRISPR/Cas9 system. The latter has emerged as the genome editing system of choice due to its efficiency, ease of synthesis and ability to target multiple loci simultaneously [127,128]. In bacteria, the CRISPR system along with its associated CRISPR-associated (Cas) nuclease proteins provides a form of adaptive immunity by targeting nucleases against invading viral DNA [129,130].…”

Section: Other Gene-targeted Strategies In Rttmentioning

confidence: 99%

Gene Therapy for Rett Syndrome: Prospects and Challenges

et al. 2015

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Rett syndrome (RTT) is a neurological disorder that affects females and is caused by lossof-function mutations in the X-linked gene MECP2. Deletion of Mecp2 in mice results in a constellation of neurological features that resemble those seen in RTT patients. Experiments in mice have demonstrated that restoration of MeCP2, even at adult stages, reverses several aspects of the RTT-like pathology suggesting that the disorder may be inherently treatable. This has provided an impetus to explore several therapeutic approaches targeting RTT at the level of the gene, including gene therapy, activation of MECP2 on the inactive X chromosome and read-through and repair of RTT-causing mutations. Here, we review these different strategies and the challenges of gene-based approaches in RTT.

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Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system

Cited by 345 publications

References 30 publications

Bricks and blueprints: methods and standards for DNA assembly

Bricks and blueprints: methods and standards for DNA assembly

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Gene Therapy for Rett Syndrome: Prospects and Challenges

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