Journal ArticleDOI
Enzymatic assembly of DNA molecules up to several hundred kilobases
Daniel G. Gibson,Lei Young,Ray-Yuan Chuang,J. Craig Venter,Clyde A. Hutchison,Hamilton O. Smith +5 more
TLDR
An isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase is described.Abstract:
We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.read more
Citations
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Journal ArticleDOI
Ultrasensitive fluorescent proteins for imaging neuronal activity.
Tsai Wen Chen,Trevor J. Wardill,Trevor J. Wardill,Yi Sun,Stefan R. Pulver,Sabine L. Renninger,Amy Baohan,Amy Baohan,Eric R. Schreiter,Rex Kerr,Michael B. Orger,Vivek Jayaraman,Loren L. Looger,Karel Svoboda,Douglas S. Kim +14 more
TL;DR: A family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo are developed and provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
Journal ArticleDOI
CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
Luke A. Gilbert,Matthew H. Larson,Leonardo Morsut,Zairan Liu,Gloria A. Brar,Sandra Elizabeth Torres,Noam Stern-Ginossar,Onn Brandman,Evan H. Whitehead,Jennifer A. Doudna,Wendell A. Lim,Jonathan S. Weissman,Lei S. Qi +12 more
TL;DR: The results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISpri as a general tool for the precise regulation of gene expression in eukaryotic cells.
Journal ArticleDOI
RNA-guided editing of bacterial genomes using CRISPR-Cas systems
Wenyan Jiang,David Bikard,David Daniel Cox,David Daniel Cox,Feng Zhang,Feng Zhang,Luciano A. Marraffini +6 more
TL;DR: The exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.
Journal ArticleDOI
Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome
Daniel G. Gibson,John I. Glass,Carole Lartigue,Vladimir N. Noskov,Ray-Yuan Chuang,Mikkel A. Algire,Gwynedd A. Benders,Michael G. Montague,Li Ma,Monzia Moodie,Chuck Merryman,Sanjay Vashee,Radha Krishnakumar,Nacyra Assad-Garcia,Cynthia Andrews-Pfannkoch,Evgeniya A. Denisova,Lei Young,Zhi-Qing Qi,Thomas H. Segall-Shapiro,Christopher H. Calvey,Prashanth P. Parmar,Clyde A. Hutchison,Hamilton O. Smith,J. Craig Venter +23 more
TL;DR: The design, synthesis, and assembly of the 1.08–mega–base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new cells that are controlled only by the synthetic chromosome are reported.
Journal ArticleDOI
Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers
Isaac B. Hilton,Anthony D'Ippolito,Christopher M. Vockley,Pratiksha I. Thakore,Gregory E. Crawford,Timothy E. Reddy,Charles A. Gersbach +6 more
TL;DR: A programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyl transferase p300 is described, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers.
References
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Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.
TL;DR: A method is described for the rapid generation and cloning of deletion derivatives well-suited for the sequencing of long stretches of DNA based on two useful features of exonuclease III: processive digestion at a very uniform rate and failure to initiate digestion at DNA ends with four-base 3'-protrusions.
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Foundations for engineering biology
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Gene Splicing by Overlap Extension: Tailor-Made Genes Using the Polymerase Chain Reaction
TL;DR: Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro.
Journal ArticleDOI
Ligation-independent cloning of PCR products (LIC-PCR)
C Aslanidis,P.J. de Jong +1 more
TL;DR: A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones, and the procedure is applied for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
Journal ArticleDOI
Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome.
Daniel G. Gibson,Gwynedd A. Benders,Cynthia Andrews-Pfannkoch,Evgeniya A. Denisova,Holly Baden-Tillson,Jayshree Zaveri,Timothy B. Stockwell,Anushka Brownley,David W. Thomas,Mikkel A. Algire,Chuck Merryman,Lei Young,Vladimir N. Noskov,John I. Glass,J. Craig Venter,Clyde A. Hutchison,Hamilton O. Smith +16 more
TL;DR: The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.