Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where David R. Liu is active.

Publication


Featured researches published by David R. Liu.


Science | 2009

Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1

Mamta Tahiliani; Kian Peng Koh; Yinghua Shen; William A. Pastor; Hozefa S. Bandukwala; Yevgeny Brudno; Suneet Agarwal; Lakshminarayan M. Iyer; David R. Liu; L. Aravind; Anjana Rao

Methylation Mediation Methylation of cytosine bases, 5-methylcytosine (5mC), in DNA plays an important regulatory role in mammalian genomes. Methylation patterns are often inherited across generations, but they can also be dynamic, suggesting that active DNA demethylation pathways exist. One such pathway, best characterized in plants, involves the removal of the 5mC base, and its replacement by C, via a DNA repair mechanism. Kriaucionis and Heintz (p. 929, published online 16 April) now show that, as well as 5mC in mammalian genomes, there are also significant amounts of 5-hydroxymethylcytosine (5hmC) in DNA of Purkinje neurons, which have large nuclei with apparently very little heterochromatin. Tahiliani et al. (p. 930, published online 16 April) find that the protein TET1 is capable of converting 5mC into 5hmC both in vitro and in vivo. 5-Hydroxymethylcytosine is also present in embryonic stem cells, and levels of 5hmC and TET1 show correlated variation during cell differentiation. Methylated C bases, an important epigenetic mark in genomic DNA, can be enzymically converted to 5-hydroxymethylcytosine. DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference–mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.


Nature Biotechnology | 2013

High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity

Vikram Pattanayak; Steven Lin; John Paul Guilinger; Enbo Ma; Jennifer A. Doudna; David R. Liu

The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 1012 potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.


Cell Stem Cell | 2009

A Small-Molecule Inhibitor of Tgf-β Signaling Replaces Sox2 in Reprogramming by Inducing Nanog

Justin K. Ichida; Joel Blanchard; Kelvin Lam; Esther Y. Son; Julia E. Chung; Dieter Egli; Kyle M. Loh; Ava C. Carter; Francesco Paolo Di Giorgio; Kathryn Koszka; Danwei Huangfu; Hidenori Akutsu; David R. Liu; Lee L. Rubin; Kevin Eggan

The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.


PLOS ONE | 2010

The Behaviour of 5-Hydroxymethylcytosine in Bisulfite Sequencing

Yun Huang; William A. Pastor; Yinghua Shen; Mamta Tahiliani; David R. Liu; Anjana Rao

Background We recently showed that enzymes of the TET family convert 5-mC to 5-hydroxymethylcytosine (5-hmC) in DNA. 5-hmC is present at high levels in embryonic stem cells and Purkinje neurons. The methylation status of cytosines is typically assessed by reaction with sodium bisulfite followed by PCR amplification. Reaction with sodium bisulfite promotes cytosine deamination, whereas 5-methylcytosine (5-mC) reacts poorly with bisulfite and is resistant to deamination. Since 5-hmC reacts with bisulfite to yield cytosine 5-methylenesulfonate (CMS), we asked how DNA containing 5-hmC behaves in bisulfite sequencing. Methodology/Principal Findings We used synthetic oligonucleotides with different distributions of cytosine as templates for generation of DNAs containing C, 5-mC and 5-hmC. The resulting DNAs were subjected in parallel to bisulfite treatment, followed by exposure to conditions promoting cytosine deamination. The extent of conversion of 5-hmC to CMS was estimated to be 99.7%. Sequencing of PCR products showed that neither 5-mC nor 5-hmC undergo C-to-T transitions after bisulfite treatment, confirming that these two modified cytosine species are indistinguishable by the bisulfite technique. DNA in which CMS constituted a large fraction of all bases (28/201) was much less efficiently amplified than DNA in which those bases were 5-mC or uracil (the latter produced by cytosine deamination). Using a series of primer extension experiments, we traced the inefficient amplification of CMS-containing DNA to stalling of Taq polymerase at sites of CMS modification, especially when two CMS bases were either adjacent to one another or separated by 1–2 nucleotides. Conclusions We have confirmed that the widely used bisulfite sequencing technique does not distinguish between 5-mC and 5-hmC. Moreover, we show that CMS, the product of bisulfite conversion of 5-hmC, tends to stall DNA polymerases during PCR, suggesting that densely hydroxymethylated regions of DNA may be underrepresented in quantitative methylation analyses.


Nature | 2016

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

Alexis C. Komor; Yongjoo Kim; Michael S. Packer; John A Zuris; David R. Liu

Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of ‘base editing’, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting ‘base editors’ convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15–75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.


Science | 2013

Sequence-Controlled Polymers

Jean-François Lutz; Makoto Ouchi; David R. Liu; Mitsuo Sawamoto

Background During the last few decades, progress has been made in manipulating the architecture of synthetic polymer materials. However, the primary structure—that is, the sequential arrangement of monomer units in a polymer chain—is generally poorly controlled in synthetic macromolecules. Common synthetic polymers are usually homopolymers, made of the same monomer unit, or copolymers with simple chain microstructures, such as random or block copolymers. These polymers are used in many areas but do not have the structural and functional complexity of sequence-defined biopolymers, such as nucleic acids or proteins. Indeed, monomer sequence regulation plays a key role in biology and is a prerequisite for crucial features of life, such as heredity, self-replication, complex self-assembly, and molecular recognition. In this context, developing synthetic polymers containing controlled monomer sequences is an important area for research. Precise molecular encoding of synthetic polymer chains. In most synthetic copolymers, monomer units (represented here as colored square boxes A, B, C, and D) are distributed randomly along the polymer chains (left). In sequence-controlled polymers, they are arranged in a specific order in all of the chains (right). Monomer sequence regularity strongly influences the molecular, supramolecular, andmacroscopic properties of polymer materials. Advances Various synthetic methods for controlling monomer sequences in polymers have been identified, and two major trends in the field of sequence-controlled polymers have emerged. Some approaches use biological concepts that have been optimized by nature for sequence regulation. For instance, DNA templates, enzymes, or even living organisms can be used to prepare sequence-defined polymers. These natural mechanisms can be adapted to tolerate nonnatural monomers. The other trend is the preparation of sequence-controlled polymers by synthetic chemistry. In the most popular approach, monomer units are attached one by one to a support, which is an efficient method but demanding in practice. Recently, some strategies have been proposed for controlling sequences in chain-growth and step-growth polymerizations. These mechanisms usually allow fast and large-scale synthesis of polymers. Specific kinetics and particular catalytic or template conditions allow sequence regulation in these processes. Outlook The possibility of controlling monomer sequences in synthetic macromolecules has many scientific and technological implications. Information can be controlled at the molecular level in synthetic polymer chains. This opens up interesting perspectives for the field of data storage. In addition, having power over monomer sequences could mean structural control of the resulting polymer, as it strongly influences macromolecular folding and self-assembly. For instance, functional synthetic assemblies that mimic the properties of globular proteins, such as enzymes and transporters, can be foreseen. Moreover, monomer sequence control influences some macroscopic properties. For example, bulk properties such as conductivity, rigidity, elasticity, or biodegradability can be finely tuned in sequence-controlled polymers. The behavior of polymers in solution, particularly in water, is also strongly dependent on monomer sequences. Thus, sequence regulation may enable a more effective control of structure-property relations in tomorrow’s polymer materials. Controlled Polymers Nature has achieved exquisite sequence control in the synthesis of polymers like DNA. In contrast, synthetic polymers rarely have the same fidelity in their chemistry or uniformity in chain-length distribution, especially when more than one monomer is involved. Lutz et al. (1238149) review the progress that has been made in making sequence-controlled polymers of increasing length and complexity. These developments have come from both advances in synthetic chemistry methods and the exploitation of biological machinery. Sequence-controlled polymers are macromolecules in which monomer units of different chemical nature are arranged in an ordered fashion. The most prominent examples are biological and have been studied and used primarily by molecular biologists and biochemists. However, recent progress in protein- and DNA-based nanotechnologies has shown the relevance of sequence-controlled polymers to nonbiological applications, including data storage, nanoelectronics, and catalysis. In addition, synthetic polymer chemistry has provided interesting routes for preparing nonnatural sequence-controlled polymers. Although these synthetic macromolecules do not yet compare in functional scope with their natural counterparts, they open up opportunities for controlling the structure, self-assembly, and macroscopic properties of polymer materials.


Nature Biotechnology | 2015

Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo

John A Zuris; David B. Thompson; Yilai Shu; John Paul Guilinger; Jeffrey L. Bessen; Johnny H. Hu; Morgan L. Maeder; J. Keith Joung; Zheng-Yi Chen; David R. Liu

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape, and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcriptional activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.


Nature Methods | 2011

Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection

Vikram Pattanayak; Cherie L. Ramirez; J. Keith Joung; David R. Liu

Engineered zinc-finger nucleases (ZFNs) are promising tools for genome manipulation, and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 1011 DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGFA genes, respectively. Analysis of identified sites in one cultured human cell line revealed CCR5-224–induced changes at nine off-target loci, though this remains to be tested in other relevant cell types. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future ZFN design.


Journal of Clinical Oncology | 2013

Brivanib in Patients With Advanced Hepatocellular Carcinoma Who Were Intolerant to Sorafenib or for Whom Sorafenib Failed: Results From the Randomized Phase III BRISK-PS Study

Josep M. Llovet; Thomas Decaens; Jean-Luc Raoul; Eveline Boucher; Masatoshi Kudo; Charissa Y. Chang; Yoon-Koo Kang; Eric Assenat; H.Y. Lim; Valérie Boige; Philippe Mathurin; Laetitia Fartoux; Deng-Yn Lin; Jordi Bruix; Ronnie Tung-Ping Poon; Morris Sherman; Jean-Frédéric Blanc; Richard S. Finn; Won Young Tak; Yee Chao; Rana Ezzeddine; David R. Liu; Ian Walters; Joong-Won Park

PURPOSE Brivanib is a selective dual inhibitor of vascular endothelial growth factor and fibroblast growth factor receptors implicated in tumorigenesis and angiogenesis in hepatocellular carcinoma (HCC). An unmet medical need persists for patients with HCC whose tumors do not respond to sorafenib or who cannot tolerate it. This multicenter, double-blind, randomized, placebo-controlled trial assessed brivanib in patients with HCC who had been treated with sorafenib. PATIENTS AND METHODS In all, 395 patients with advanced HCC who progressed on/after or were intolerant to sorafenib were randomly assigned (2:1) to receive brivanib 800 mg orally once per day plus best supportive care (BSC) or placebo plus BSC. The primary end point was overall survival (OS). Secondary end points included time to progression (TTP), objective response rate (ORR), and disease control rate based on modified Response Evaluation Criteria in Solid Tumors (mRECIST) and safety. RESULTS Median OS was 9.4 months for brivanib and 8.2 months for placebo (hazard ratio [HR], 0.89; 95.8% CI, 0.69 to 1.15; P = .3307). Adjusting treatment effect for baseline prognostic factors yielded an OS HR of 0.81 (95% CI, 0.63 to 1.04; P = .1044). Exploratory analyses showed a median time to progression of 4.2 months for brivanib and 2.7 months for placebo (HR, 0.56; 95% CI, 0.42 to 0.76; P < .001), and an mRECIST ORR of 10% for brivanib and 2% for placebo (odds ratio, 5.72). Study discontinuation due to treatment-related adverse events (AEs) occurred in 61 brivanib patients (23%) and nine placebo patients (7%). The most frequent treatment-related grade 3 to 4 AEs for brivanib included hypertension (17%), fatigue (13%), hyponatremia (11%), and decreased appetite (10%). CONCLUSION In patients with HCC who had been treated with sorafenib, brivanib did not significantly improve OS. The observed benefit in the secondary outcomes of TTP and ORR warrants further investigation.


Nature | 2011

A system for the continuous directed evolution of biomolecules.

Kevin M. Esvelt; Jacob C. Carlson; David R. Liu

Laboratory evolution has generated many biomolecules with desired properties, but a single round of mutation, gene expression, screening or selection, and replication typically requires days or longer with frequent human intervention. Because evolutionary success is dependent on the total number of rounds performed, a means of performing laboratory evolution continuously and rapidly could dramatically enhance its effectiveness. Although researchers have accelerated individual steps in the evolutionary cycle, the only previous example of continuous directed evolution was the landmark study of Wright and Joyce, who continuously evolved RNA ligase ribozymes with an in vitro replication cycle that unfortunately cannot be easily adapted to other biomolecules. Here we describe a system that enables the continuous directed evolution of gene-encoded molecules that can be linked to protein production in Escherichia coli. During phage-assisted continuous evolution (PACE), evolving genes are transferred from host cell to host cell through a modified bacteriophage life cycle in a manner that is dependent on the activity of interest. Dozens of rounds of evolution can occur in a single day of PACE without human intervention. Using PACE, we evolved T7 RNA polymerase (RNAP) variants that recognize a distinct promoter, initiate transcripts with ATP instead of GTP, and initiate transcripts with CTP. In one example, PACE executed 200 rounds of protein evolution over the course of 8 days. Starting from undetectable activity levels in two of these cases, enzymes with each of the three target activities emerged in less than 1 week of PACE. In all three cases, PACE-evolved polymerase activities exceeded or were comparable to that of the wild-type T7 RNAP on its wild-type promoter, representing improvements of up to several hundred-fold. By greatly accelerating laboratory evolution, PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions about molecular evolution.

Collaboration


Dive into the David R. Liu's collaboration.

Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Zev J. Gartner

University of California

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Peter G. Schultz

Scripps Research Institute

View shared research outputs
Top Co-Authors

Avatar
Researchain Logo
Decentralizing Knowledge