Lei Qi (Stanley)

All Publications

• CRISPhieRmix: a hierarchical mixture model for CRISPR pooled screens. Genome biology Daley, T. P., Lin, Z., Lin, X., Liu, Y., Wong, W. H., Qi, L. S. 2018; 19 (1): 159

  Abstract

  Pooled CRISPR screens allow researchers to interrogate genetic causes of complex phenotypes at the genome-wide scale and promise higher specificity and sensitivity compared to competing technologies. Unfortunately, two problems exist, particularly for CRISPRi/a screens: variability in guide efficiency and large rare off-target effects. We present a method, CRISPhieRmix, that resolves these issues by using a hierarchical mixture model with a broad-tailed null distribution. We show that CRISPhieRmix allows for more accurate and powerful inferences in large-scale pooled CRISPRi/a screens. We discuss key issues in the analysis and design of screens, particularly the number of guides needed for faithful full discovery.

  View details for DOI 10.1186/s13059-018-1538-6

  View details for PubMedID 30296940

• CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization. Cell Wang, H., Xu, X., Nguyen, C. M., Liu, Y., Gao, Y., Lin, X., Daley, T., Kipniss, N. H., La Russa, M., Qi, L. S. 2018

  Abstract

  Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30-600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.

  View details for DOI 10.1016/j.cell.2018.09.013

  View details for PubMedID 30318144

• CRISPR Activation Screens Systematically Identify Factors that Drive Neuronal Fate and Reprogramming. Cell stem cell Liu, Y., Yu, C., Daley, T. P., Wang, F., Cao, W. S., Bhate, S., Lin, X., Still, C. 2., Liu, H., Zhao, D., Wang, H., Xie, X. S., Ding, S., Wong, W. H., Wernig, M., Qi, L. S. 2018

  Abstract

  Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming, but systematic interrogation of transcription factors, and their interactions with each other, has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells, revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation, with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons, which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition.

  View details for DOI 10.1016/j.stem.2018.09.003

  View details for PubMedID 30318302

• Genetic interaction mapping in mammalian cells using CRISPR interference. Nature methods Du, D., Roguev, A., Gordon, D. E., Chen, M., Chen, S., Shales, M., Shen, J. P., Ideker, T., Mali, P., Qi, L. S., Krogan, N. J. 2017; 14 (6): 577-580

  Abstract

  We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein-protein-interaction data revealed a functional map of chromatin regulation.

  View details for DOI 10.1038/nmeth.4286

  View details for PubMedID 28481362

• Engineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system. Nature communications Kipniss, N. H., Dingal, P. C., Abbott, T. R., Gao, Y., Wang, H., Dominguez, A. A., Labanieh, L., Qi, L. S. 2017; 8 (1): 2212

  Abstract

  G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and detect a wide array of cues in the human body. Here we describe a molecular device that couples CRISPR-dCas9 genome regulation to diverse natural and synthetic extracellular signals via GPCRs. We generate alternative architectures for fusing CRISPR to GPCRs utilizing the previously reported design, Tango, and our design, ChaCha. Mathematical modeling suggests that for the CRISPR ChaCha design, multiple dCas9 molecules can be released across the lifetime of a GPCR. The CRISPR ChaCha is dose-dependent, reversible, and can activate multiple endogenous genes simultaneously in response to extracellular ligands. We adopt the design to diverse GPCRs that sense a broad spectrum of ligands, including synthetic compounds, chemokines, mitogens, fatty acids, and hormones. This toolkit of CRISPR-coupled GPCRs provides a modular platform for rewiring diverse ligand sensing to targeted genome regulation for engineering cellular functions.

  View details for DOI 10.1038/s41467-017-02075-1

  View details for PubMedID 29263378

  View details for PubMedCentralID PMC5738360

• Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nature methods Gao, Y., Xiong, X., Wong, S., Charles, E. J., Lim, W. A., Qi, L. S. 2016

  Abstract

  The ability to dynamically manipulate the transcriptome is important for studying how gene networks direct cellular functions and how network perturbations cause disease. Nuclease-dead CRISPR-dCas9 transcriptional regulators, while offering an approach for controlling individual gene expression, remain incapable of dynamically coordinating complex transcriptional events. Here, we describe a flexible dCas9-based platform for chemical-inducible complex gene regulation. From a screen of chemical- and light-inducible dimerization systems, we identified two potent chemical inducers that mediate efficient gene activation and repression in mammalian cells. We combined these inducers with orthogonal dCas9 regulators to independently control expression of different genes within the same cell. Using this platform, we further devised AND, OR, NAND, and NOR dCas9 logic operators and a diametric regulator that activates gene expression with one inducer and represses with another. This work provides a robust CRISPR-dCas9-based platform for enacting complex transcription programs that is suitable for large-scale transcriptome engineering.

  View details for DOI 10.1038/nmeth.4042

  View details for PubMedID 27776111

  View details for PubMedCentralID PMC5436902

• A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria CELL Peters, J. M., Colavin, A., Shi, H., Czarny, T. L., Larson, M. H., Wong, S., Hawkins, J. S., Lu, C. H., Koo, B., Marta, E., Shiver, A. L., Whitehead, E. H., Weissman, J. S., Brown, E. D., Qi, L. S., Huang, K. C., Gross, C. A. 2016; 165 (6): 1493-1506

  Abstract

  Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.

  View details for DOI 10.1016/j.cell.2016.05.003

  View details for Web of Science ID 000377045400021

  View details for PubMedID 27238023

  View details for PubMedCentralID PMC4894308

• Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells. Cell stem cell Yu, C., Liu, Y., Ma, T., Liu, K., Xu, S., Zhang, Y., Liu, H., La Russa, M., Xie, M., Ding, S., Qi, L. S. 2015; 16 (2): 142-147

  Abstract

  The bacterial CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ), but it remains inefficient for precise editing of genome sequences. Here we develop a reporter-based screening approach for high-throughput identification of chemical compounds that can modulate precise genome editing through homology-directed repair (HDR). Using our screening method, we have identified small molecules that can enhance CRISPR-mediated HDR efficiency, 3-fold for large fragment insertions and 9-fold for point mutations. Interestingly, we have also observed that a small molecule that inhibits HDR can enhance frame shift insertion and deletion (indel) mutations mediated by NHEJ. The identified small molecules function robustly in diverse cell types with minimal toxicity. The use of small molecules provides a simple and effective strategy to enhance precise genome engineering applications and facilitates the study of DNA repair mechanisms in mammalian cells.

  View details for DOI 10.1016/j.stem.2015.01.003

  View details for PubMedID 25658371

  View details for PubMedCentralID PMC4461869

• Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds CELL Zalatan, J. G., Lee, M. E., Almeida, R., Gilbert, L. A., Whitehead, E. H., La Russa, M., Tsai, J. C., Weissman, J. S., Dueber, J. E., Qi, L. S., Lim, W. A. 2015; 160 (1-2): 339-350

  Abstract

  Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.

  View details for DOI 10.1016/j.cell.2014.11.052

  View details for Web of Science ID 000347923200029

  View details for PubMedID 25533786

• Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System CELL Chen, B., Gilbert, L. A., Cimini, B. A., Schnitzbauer, J., Zhang, W., Li, G., Park, J., Blackburn, E. H., Weissman, J. S., Qi, L. S., Huang, B. 2013; 155 (7): 1479-1491

  Abstract

  The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.

  View details for DOI 10.1016/j.cell.2013.12.001

  View details for Web of Science ID 000328693300006

  View details for PubMedID 24360272

• CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes CELL Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., Stern-Ginossar, N., Brandman, O., Whitehead, E. H., Doudna, J. A., Lim, W. A., Weissman, J. S., Qi, L. S. 2013; 154 (2): 442-451

  Abstract

  The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our 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.

  View details for DOI 10.1016/j.cell.2013.06.044

  View details for Web of Science ID 000321950700019

  View details for PubMedID 23849981

• Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression CELL Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., Lim, W. A. 2013; 152 (5): 1173-1183

  Abstract

  Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.

  View details for DOI 10.1016/j.cell.2013.02.022

  View details for Web of Science ID 000315710300022

  View details for PubMedID 23452860

• Evolution at the Cutting Edge: CRISPR-Mediated Directed Evolution. Molecular cell Abbott, T. R., Qi, L. S. 2018; 72 (3): 402–3

  Abstract

  In a recent issue of Nature, Halperin etal. (2018) develop a new technology to continuously diversify specific genomic loci by combining CRISPR-Cas9 with error-prone DNA polymerases.

  View details for DOI 10.1016/j.molcel.2018.10.027

  View details for PubMedID 30388408

• DNMT3A and TET1 cooperate to regulate promoter epigenetic landscapes in mouse embryonic stem cells GENOME BIOLOGY Gu, T., Lin, X., Cullen, S. M., Luo, M., Jeong, M., Estecio, M., Shen, J., Hardikar, S., Sun, D., Su, J., Rux, D., Guzman, A., Lee, M., Qi, L., Chen, J., Kyba, M., Huang, Y., Chen, T., Li, W., Goodell, M. A. 2018; 19: 88

  Abstract

  DNA methylation is a heritable epigenetic mark, enabling stable but reversible gene repression. In mammalian cells, DNA methyltransferases (DNMTs) are responsible for modifying cytosine to 5-methylcytosine (5mC), which can be further oxidized by the TET dioxygenases to ultimately cause DNA demethylation. However, the genome-wide cooperation and functions of these two families of proteins, especially at large under-methylated regions, called canyons, remain largely unknown.Here we demonstrate that DNMT3A and TET1 function in a complementary and competitive manner in mouse embryonic stem cells to mediate proper epigenetic landscapes and gene expression. The longer isoform of DNMT3A, DNMT3A1, exhibits significant enrichment at distal promoters and canyon edges, but is excluded from proximal promoters and canyons where TET1 shows prominent binding. Deletion of Tet1 increases DNMT3A1 binding capacity at and around genes with wild-type TET1 binding. However, deletion of Dnmt3a has a minor effect on TET1 binding on chromatin, indicating that TET1 may limit DNA methylation partially by protecting its targets from DNMT3A and establishing boundaries for DNA methylation. Local CpG density may determine their complementary binding patterns and therefore that the methylation landscape is encoded in the DNA sequence. Furthermore, DNMT3A and TET1 impact histone modifications which in turn regulate gene expression. In particular, they regulate Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 enrichment to constrain gene expression from bivalent promoters.We conclude that DNMT3A and TET1 regulate the epigenome and gene expression at specific targets via their functional interplay.

  View details for DOI 10.1186/s13059-018-1464-7

  View details for Web of Science ID 000438536700001

  View details for PubMedID 30001199

  View details for PubMedCentralID PMC6042404

• A CRISPR-dCas Toolbox for Genetic Engineering and Synthetic Biology. Journal of molecular biology Xu, X., Qi, L. S. 2018

  Abstract

  Programmable control of gene expression is essential to understanding gene function, engineering cellular behaviors, and developing therapeutics. Beyond the gene editing applications enabled by the nuclease CRISPR-Cas9 and CRISPR-Cas12a, the invention of the nuclease-dead Cas molecules (dCas9 and dCas12a) offers a platform for the precise control of genome function without gene editing. Diverse dCas tools have been developed, which constitute a comprehensive toolbox that allows interrogation of gene function and modulation of the cellular behaviors. This review summarizes current applications of the dCas tools for transcription regulation, epigenetic engineering, genome imaging, genetic screens, and chromatin immunoprecipitation. We also highlight the advantages and existing challenges of the current dCas tools in genetic engineering and synthetic biology, and provide perspectives on future directions and applications.

  View details for DOI 10.1016/j.jmb.2018.06.037

  View details for PubMedID 29958882

• Low-intensity extracorporeal shock wave therapy promotes myogenesis through PERK/ATF4 pathway NEUROUROLOGY AND URODYNAMICS Wang, B., Zhou, J., Banie, L., Reed-Maldonado, A. B., Ning, H., Lu, Z., Ruan, Y., Zhou, T., Wang, H., Oh, B., Wang, G., Qi, S., Lin, G., Lue, T. F. 2018; 37 (2): 699–707

  Abstract

  Stress urinary incontinence (SUI) is a significant health problem for women. Treatments employing muscle derived stem cells (MDSCs) may be a promising approach to this prevalent, bothersome condition, but these treatments are invasive and require collection of cells from one site for injection into another. It is also unknown whether or not these cells establish themselves and function as muscle cells in the target tissues. Alternatively, low-intensity extracorporeal shock wave therapy (Li-ESWT) is non-invasive and has shown positive outcomes in the treatment of multiple musculoskeletal disorders, but the biological effects responsible for clinical success are not yet well understood. The aim of this study is to explore the possibility of employing Li-ESWT for activation of MDSCs in situ and to further elucidate the underlying biological effects and mechanisms of action in urethral muscle.Urethral muscle derived stem cells (uMDSCs) were harvest from Zucker Lean (ZUC-LEAN) (ZUC-Leprfa 186) rats and characterized with flow cytometry. Li-ESWT (0.02 mJ/mm2 , 3 Hz, 200 pulses) and GSK2656157, an inhibitor of PERK pathway, were applied to L6 rat myoblast cells. To assess for myotube formation, we used immunofluorescence staining and western blot analysis in uMDSCs and L6 cells.The results indicate that uMDSCs could form myotubes. Myotube formation was significantly increased by the Li-ESWT as was the expression of muscle heavy chain (MHC) and myogenic factor 5 (Myf5) in L6 cells in vitro. Li-ESWT activated protein kinase RNA-like ER kinase (PERK) pathway by increasing the phosphorylation levels of PERK and eukaryotic initiation factor 2a (eIF2α) and by increasing activating transcription factor 4 (ATF4). In addition, GSK2656157, an inhibitor of PERK, effectively inhibited the myotube formation in L6 rat myoblast cells. Furthermore, GSK2656157 also attenuated myotube formation induced by Li-ESWT.In conclusion, this experiment reveals that rat uMDSCs can be isolated successfully and can form myotubes in vitro. PERK/ATF4 pathway was involved in myotube formation, and L6 rat myoblast cells were activated by Li-ESWT to form myotubes. These findings suggest that PERK/ATF4 pathway is activated by Li-ESWT. This study elucidates one of the biochemical pathways responsible for the clinical improvements seen after Li-ESWT. It is possible that this information will help to establish Li-ESWT as an acceptable treatment modality and may help to further refine the use of Li-ESWT in the clinical practice of medicine.

  View details for DOI 10.1002/nau.23380

  View details for Web of Science ID 000428455000020

  View details for PubMedID 28763567

  View details for PubMedCentralID PMC5794657

• CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency CELL STEM CELL Liu, P., Chen, M., Liu, Y., Qi, L. S., Ding, S. 2018; 22 (2): 252-+

  View details for DOI 10.1016/j.stem.2017.12.001

  View details for Web of Science ID 000423840400015

• Multiplexed Dynamic Imaging of Genomic Loci by Combined CRISPR Imaging and DNA Sequential FISH BIOPHYSICAL JOURNAL Takei, Y., Shah, S., Harvey, S., Qi, L. S., Cai, L. 2017; 112 (9): 1773-1776

  Abstract

  Visualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labeled and uniquely identified. Here we describe an approach we call "track first and identify later" for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR imaging and DNA sequential fluorescence in situ hybridization. Our approach first labels and tracks chromosomal loci in live cells with the CRISPR-Cas9 system, then barcodes those loci by DNA sequential fluorescence in situ hybridization in fixed cells and resolves their identities. We demonstrate our approach by tracking telomere dynamics, identifying 12 unique subtelomeric regions with variable detection efficiencies, and tracking back the telomere dynamics of respective chromosomes in mouse embryonic stem cells.

  View details for DOI 10.1016/j.bpj.2017.03.024

  View details for Web of Science ID 000401301600006

  View details for PubMedID 28427715

• A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription Zhou, X. X., Zou, X., Chung, H. K., Gao, Y., Liu, Y., QI, L. S., Lin, M. Z. 2017: 443–48

  Abstract

  Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

  View details for DOI 10.1021/acschembio.7b00603

  View details for PubMedCentralID PMC5820652

• Genetic and epigenetic control of gene expression by CRISPR-Cas systems. F1000Research Lo, A., Qi, L. 2017; 6

  Abstract

  The discovery and adaption of bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems has revolutionized the way researchers edit genomes. Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state. These CRISPR-Cas-based technologies have contributed to the rapid development of disease models and functional genomics screening approaches, which can facilitate genetic target identification and drug discovery. In this short review, we will cover recent advances of CRISPR-dCas9 systems and their use for transcriptional repression and activation, epigenome editing, and engineered synthetic circuits for complex control of the mammalian genome.

  View details for DOI 10.12688/f1000research.11113.1

  View details for PubMedID 28649363

  View details for PubMedCentralID PMC5464239

• Applications of CRISPR Genome Engineering in Cell Biology. Trends in cell biology Wang, F., Qi, L. S. 2016; 26 (11): 875-888

  Abstract

  Recent advances in genome engineering are starting a revolution in biological research and translational applications. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided endonuclease CRISPR associated protein 9 (Cas9) and its variants enable diverse manipulations of genome function. In this review, we describe the development of Cas9 tools for a variety of applications in cell biology research, including the study of functional genomics, the creation of transgenic animal models, and genomic imaging. Novel genome engineering methods offer a new avenue to understand the causality between the genome and phenotype, thus promising a fuller understanding of cell biology.

  View details for DOI 10.1016/j.tcb.2016.08.004

  View details for PubMedID 27599850

• CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs CELL STEM CELL Mandegar, M. A., Huebsch, N., Frolov, E. B., Shin, E., Truong, A., Olvera, M. P., Chan, A. H., Miyaoka, Y., Holmes, K., Spencer, C. I., Judge, L. M., Gordon, D. E., Eskildsen, T. V., Villalta, J. E., Horlbeck, M. A., Gilbert, L. A., Krogan, N. J., Sheikh, S. P., Weissman, J. S., Qi, L. S., So, P., Conklin, B. R. 2016; 18 (4): 541-553

  Abstract

  Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.

  View details for DOI 10.1016/j.stem.2016.01.022

  View details for Web of Science ID 000373722100017

  View details for PubMedID 26971820

• YAP Induces Human Naive Pluripotency. Cell reports Qin, H., Hejna, M., Liu, Y., Percharde, M., Wossidlo, M., Blouin, L., Durruthy-Durruthy, J., Wong, P., Qi, Z., Yu, J., Qi, L. S., Sebastiano, V., Song, J. S., Ramalho-Santos, M. 2016; 14 (10): 2301-2312

  Abstract

  The human naive pluripotent stem cell (PSC) state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP(-/-) cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology.

  View details for DOI 10.1016/j.celrep.2016.02.036

  View details for PubMedID 26947063

  View details for PubMedCentralID PMC4807727

• CRISPR Technology for Genome Activation and Repression in Mammalian Cells. Cold Spring Harbor protocols Du, D., Qi, L. S. 2016; 2016 (1): pdb prot090175-?

  Abstract

  Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful system for targeted genome activation and repression, in addition to its use in genome editing. This protocol describes how to design, construct, and experimentally validate the function of sequence-specific single guide RNAs (sgRNAs) for sequence-specific repression (CRISPRi) or activation (CRISPRa) of transcription in mammalian cells. In this technology, the CRISPR-associated protein Cas9 is catalytically deactivated (dCas9) to provide a general platform for RNA-guided DNA targeting of any locus in the genome. Fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in mammalian cells. Delivery of multiple sgRNAs further enables activation or repression of multiple genes. By using scaffold RNAs (scRNAs), different effectors can be recruited to different genes for simultaneous activation of some and repression of others. The CRISPRi and CRISPRa methods provide powerful tools for sequence-specific control of gene expression on a genome-wide scale to aid understanding gene functions and for engineering genetic regulatory systems.

  View details for DOI 10.1101/pdb.prot090175

  View details for PubMedID 26729910

• An Introduction to CRISPR Technology for Genome Activation and Repression in Mammalian Cells. Cold Spring Harbor protocols Du, D., Qi, L. S. 2016; 2016 (1): pdb top086835-?

  Abstract

  CRISPR interference/activation (CRISPRi/a) technology provides a simple and efficient approach for targeted repression or activation of gene expression in the mammalian genome. It is highly flexible and programmable, using an RNA-guided nuclease-deficient Cas9 (dCas9) protein fused with transcriptional regulators for targeting specific genes to effect their regulation. Multiple studies have shown how this method is an effective way to achieve efficient and specific transcriptional repression or activation of single or multiple genes. Sustained transcriptional modulation can be obtained by stable expression of CRISPR components, which enables directed reprogramming of cell fate. Here, we introduce the basics of CRISPRi/a technology for genome repression or activation.

  View details for DOI 10.1101/pdb.top086835

  View details for PubMedID 26729914

• Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation NATURE REVIEWS MOLECULAR CELL BIOLOGY Dominguez, A. A., Lim, W. A., Qi, L. S. 2016; 17 (1)

  Abstract

  The bacterial CRISPR-Cas9 system has emerged as a multifunctional platform for sequence-specific regulation of gene expression. This Review describes the development of technologies based on nuclease-deactivated Cas9, termed dCas9, for RNA-guided genomic transcription regulation, both by repression through CRISPR interference (CRISPRi) and by activation through CRISPR activation (CRISPRa). We highlight different uses in diverse organisms, including bacterial and eukaryotic cells, and summarize current applications of harnessing CRISPR-dCas9 for multiplexed, inducible gene regulation, genome-wide screens and cell fate engineering. We also provide a perspective on future developments of the technology and its applications in biomedical research and clinical studies.

  View details for DOI 10.1038/nrm.2015.2

  View details for Web of Science ID 000366920600007

  View details for PubMedID 26670017

• CRISPR/Cas9 for Human Genome Engineering and Disease Research ANNUAL REVIEW OF GENOMICS AND HUMAN GENETICS, VOL 17 Xiong, X., Chen, M., Lim, W. A., Zhao, D., Qi, L. S. 2016; 17: 131-154

  Abstract

  The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, a versatile RNA-guided DNA targeting platform, has been revolutionizing our ability to modify, manipulate, and visualize the human genome, which greatly advances both biological research and therapeutics development. Here, we review the current development of CRISPR/Cas9 technologies for gene editing, transcription regulation, genome imaging, and epigenetic modification. We discuss the broad application of this system to the study of functional genomics, especially genome-wide genetic screening, and to therapeutics development, including establishing disease models, correcting defective genetic mutations, and treating diseases.

  View details for DOI 10.1146/annurev-genom-083115-022258

  View details for Web of Science ID 000382615800007

  View details for PubMedID 27216776

• CRISPR/Cas9 in Genome Editing and Beyond ANNUAL REVIEW OF BIOCHEMISTRY, VOL 85 Wang, H., La Russa, M., Qi, L. S. 2016; 85: 227-264

  Abstract

  The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

  View details for DOI 10.1146/annurev-biochem-060815-014607

  View details for Web of Science ID 000379324700011

  View details for PubMedID 27145843

• CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation BIOINFORMATICS Liu, H., Wei, Z., Dominguez, A., Li, Y., Wang, X., Qi, L. S. 2015; 31 (22): 3676-3678

  View details for DOI 10.1093/bioinformatics/btv423

  View details for Web of Science ID 000365134700017

  View details for PubMedID 26209430

• The New State of the Art: Cas9 for Gene Activation and Repression MOLECULAR AND CELLULAR BIOLOGY La Russa, M. F., Qi, L. S. 2015; 35 (22): 3800-3809

  Abstract

  CRISPR-Cas9 technology has rapidly changed the landscape for how biologists and bioengineers study and manipulate the genome. Derived from the bacterial adaptive immune system, CRISPR-Cas9 has been coopted and repurposed for a variety of new functions, including the activation or repression of gene expression (termed CRISPRa or CRISPRi, respectively). This represents an exciting alternative to previously used repression or activation technologies such as RNA interference (RNAi) or the use of gene overexpression vectors. We have only just begun exploring the possibilities that CRISPR technology offers for gene regulation and the control of cell identity and behavior. In this review, we describe the recent advances of CRISPR-Cas9 technology for gene regulation and outline advantages and disadvantages of CRISPRa and CRISPRi (CRISPRa/i) relative to alternative technologies.

  View details for DOI 10.1128/MCB.00512-15

  View details for Web of Science ID 000365714400001

  View details for PubMedID 26370509

  View details for PubMedCentralID PMC4609748

• Bacterial CRISPR: accomplishments and prospects CURRENT OPINION IN MICROBIOLOGY Peters, J. M., Silvis, M. R., Zhao, D., Hawkins, J. S., Gross, C. A., Qi, L. S. 2015; 27: 121-126

  View details for DOI 10.1016/j.mib.2015.08.007

  View details for Web of Science ID 000365065400019

  View details for PubMedID 26363124

• Specific Gene Repression by CRISPRi System Transferred through Bacterial Conjugation ACS SYNTHETIC BIOLOGY Ji, W., Lee, D., Wong, E., Dadlani, P., Dinh, D., Huang, V., Kearns, K., Teng, S., Chen, S., Haliburton, J., Heimberg, G., Heineike, B., Ramasubramanian, A., Stevens, T., Helmke, K. J., Zepeda, V., Qi, L. S., Lim, W. A. 2014; 3 (12): 929-931

  Abstract

  In microbial communities, bacterial populations are commonly controlled using indiscriminate, broad range antibiotics. There are few ways to target specific strains effectively without disrupting the entire microbiome and local environment. Here, we use conjugation, a natural DNA horizontal transfer process among bacterial species, to deliver an engineered CRISPR interference (CRISPRi) system for targeting specific genes in recipient Escherichia coli cells. We show that delivery of the CRISPRi system is successful and can specifically repress a reporter gene in recipient cells, thereby establishing a new tool for gene regulation across bacterial cells and potentially for bacterial population control.

  View details for DOI 10.1021/sb500036q

  View details for Web of Science ID 000347140300010

  View details for PubMedID 25409531

  View details for PubMedCentralID PMC4277763

• A versatile framework for microbial engineering using synthetic non-coding RNAs NATURE REVIEWS MICROBIOLOGY Qi, L. S., Arkin, A. P. 2014; 12 (5): 341-354

  Abstract

  Synthetic non-coding RNAs have emerged as a versatile class of molecular devices that have a diverse range of programmable functions, including signal sensing, gene regulation and the modulation of molecular interactions. Owing to their small size and the central role of Watson-Crick base pairing in determining their structure, function and interactions, several distinct types of synthetic non-coding RNA regulators that are functional at the DNA, mRNA and protein levels have been experimentally characterized and computationally modelled. These engineered devices can be incorporated into genetic circuits, enabling the more efficient creation of complex synthetic biological systems. In this Review, we summarize recent progress in engineering synthetic non-coding RNA devices and their application to genetic and cellular engineering in a broad range of microorganisms.

  View details for DOI 10.1038/nrmicro3244

  View details for Web of Science ID 000334846500011

  View details for PubMedID 24736794

• Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System (vol 155, pg 1479, 2013) CELL Chen, B., Gilbert, L. A., Cimini, B. A., Schnitzbauer, J., Zhang, W., Li, G., Park, J., Blackburn, E. H., Weissman, J. S., Qi, L. S., Huang, B. 2014; 156 (1-2): 373-373

  View details for DOI 10.1016/j.cell.2013.12.033

  View details for Web of Science ID 000329912200034

• CRISPR interference (CRISPRi) for sequence-specific control of gene expression NATURE PROTOCOLS Larson, M. H., Gilbert, L. A., Wang, X., Lim, W. A., Weissman, J. S., Qi, L. S. 2013; 8 (11): 2180-2196

  Abstract

  Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1-2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.

  View details for DOI 10.1038/nprot.2013.132

  View details for Web of Science ID 000326164100008

  View details for PubMedID 24136345

• An adaptor from translational to transcriptional control enables predictable assembly of complex regulation NATURE METHODS Liu, C. C., Qi, L., Lucks, J. B., Segall-Shapiro, T. H., Wang, D., Mutalik, V. K., Arkin, A. P. 2012; 9 (11): 1088-?

  Abstract

  Bacterial regulators of transcriptional elongation are versatile units for building custom genetic switches, as they control the expression of both coding and noncoding RNAs, act on multigene operons and can be predictably tethered into higher-order regulatory functions (a property called composability). Yet the less versatile bacterial regulators of translational initiation are substantially easier to engineer. To bypass this tradeoff, we have developed an adaptor that converts regulators of translational initiation into regulators of transcriptional elongation in Escherichia coli. We applied this adaptor to the construction of several transcriptional attenuators and activators, including a small molecule-triggered attenuator and a group of five mutually orthogonal riboregulators that we assembled into NOR gates of two, three or four RNA inputs. Continued application of our adaptor should produce large collections of transcriptional regulators whose inherent composability can facilitate the predictable engineering of complex synthetic circuits.

  View details for DOI 10.1038/NMETH.2184

  View details for Web of Science ID 000310848700022

  View details for PubMedID 23023598

• RNA processing enables predictable programming of gene expression NATURE BIOTECHNOLOGY Qi, L., Haurwitz, R. E., Shao, W., Doudna, J. A., Arkin, A. P. 2012; 30 (10): 1002-?

  Abstract

  Complex interactions among genetic components often result in variable systemic performance in designed multigene systems. Using the bacterial clustered regularly interspaced short palindromic repeat (CRISPR) pathway we develop a synthetic RNA-processing platform, and show that efficient and specific cleavage of precursor mRNA enables reliable and predictable regulation of multigene operons. Physical separation of linked genetic elements by CRISPR-mediated cleavage is an effective strategy to achieve assembly of promoters, ribosome binding sites, cis-regulatory elements, and riboregulators into single- and multigene operons with predictable functions in bacteria. We also demonstrate that CRISPR-based RNA cleavage is effective for regulation in bacteria, archaea and eukaryotes. Programmable RNA processing using CRISPR offers a general approach for creating context-free genetic elements and can be readily used in the bottom-up construction of increasingly complex biological systems in a plug-and-play manner.

  View details for DOI 10.1038/nbt.2355

  View details for Web of Science ID 000309965500028

  View details for PubMedID 22983090

• Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals NUCLEIC ACIDS RESEARCH Qi, L., Lucks, J. B., Liu, C. C., Mutalik, V. K., Arkin, A. P. 2012; 40 (12): 5775-5786

  Abstract

  Non-coding RNAs (ncRNAs) are versatile regulators in cellular networks. While most trans-acting ncRNAs possess well-defined mechanisms that can regulate transcription or translation, they generally lack the ability to directly sense cellular signals. In this work, we describe a set of design principles for fusing ncRNAs to RNA aptamers to engineer allosteric RNA fusion molecules that modulate the activity of ncRNAs in a ligand-inducible way in Escherichia coli. We apply these principles to ncRNA regulators that can regulate translation (IS10 ncRNA) and transcription (pT181 ncRNA), and demonstrate that our design strategy exhibits high modularity between the aptamer ligand-sensing motif and the ncRNA target-recognition motif, which allows us to reconfigure these two motifs to engineer orthogonally acting fusion molecules that respond to different ligands and regulate different targets in the same cell. Finally, we show that the same ncRNA fused with different sensing domains results in a sensory-level NOR gate that integrates multiple input signals to perform genetic logic. These ligand-sensing ncRNA regulators provide useful tools to modulate the activity of structurally related families of ncRNAs, and building upon the growing body of RNA synthetic biology, our ability to design aptamer-ncRNA fusion molecules offers new ways to engineer ligand-sensing regulatory circuits.

  View details for DOI 10.1093/nar/gks168

  View details for Web of Science ID 000305829000057

  View details for PubMedID 22383579

• Rationally designed families of orthogonal RNA regulators of translation NATURE CHEMICAL BIOLOGY Mutalik, V. K., Qi, L., Guimaraes, J. C., Lucks, J. B., Arkin, A. P. 2012; 8 (5): 447-454

  Abstract

  Our ability to routinely engineer genetic networks for applications is limited by the scarcity of highly specific and non-cross-reacting (orthogonal) gene regulators with predictable behavior. Though antisense RNAs are attractive contenders for this purpose, quantitative understanding of their specificity and sequence-function relationship sufficient for their design has been limited. Here, we use rationally designed variants of the RNA-IN-RNA-OUT antisense RNA-mediated translation system from the insertion sequence IS10 to quantify >500 RNA-RNA interactions in Escherichia coli and integrate the data set with sequence-activity modeling to identify the thermodynamic stability of the duplex and the seed region as the key determinants of specificity. Applying this model, we predict the performance of an additional ~2,600 antisense-regulator pairs, forecast the possibility of large families of orthogonal mutants, and forward engineer and experimentally validate two RNA pairs orthogonal to an existing group of five from the training data set. We discuss the potential use of these regulators in next-generation synthetic biology applications.

  View details for DOI 10.1038/NCHEMBIO.919

  View details for Web of Science ID 000302962500011

  View details for PubMedID 22446835

• Versatile RNA-sensing transcriptional regulators for engineering genetic networks PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lucks, J. B., Qi, L., Mutalik, V. K., Wang, D., Arkin, A. P. 2011; 108 (21): 8617-8622

  Abstract

  The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.

  View details for DOI 10.1073/pnas.1015741108

  View details for Web of Science ID 000290908000025

  View details for PubMedID 21555549

• Regulation of transcription by unnatural amino acids NATURE BIOTECHNOLOGY Liu, C. C., Qi, L., Yanofsky, C., Arkin, A. P. 2011; 29 (2): 164-U111

  Abstract

  Small-molecule regulation of gene expression is intrinsic to cellular function and indispensable to the construction of new biological sensing, control and expression systems. However, there are currently only a handful of strategies for engineering such regulatory components and fewer still that can give rise to an arbitrarily large set of inducible systems whose members respond to different small molecules, display uniformity and modularity in their mechanisms of regulation, and combine to actuate universal logics. Here we present an approach for small-molecule regulation of transcription based on the combination of cis-regulatory leader-peptide elements with genetically encoded unnatural amino acids (amino acids that have been artificially added to the genetic code). In our system, any genetically encoded unnatural amino acid (UAA) can be used as a small-molecule attenuator or activator of gene transcription, and the logics intrinsic to the network defined by expanded genetic codes can be actuated.

  View details for DOI 10.1038/nbt.1741

  View details for Web of Science ID 000287023000025

  View details for PubMedID 21240267

• Toward scalable parts families for predictable design of biological circuits CURRENT OPINION IN MICROBIOLOGY Lucks, J. B., Qi, L., Whitaker, W. R., Arkin, A. P. 2008; 11 (6): 567-573

  Abstract

  Our current ability to engineer biological circuits is hindered by design cycles that are costly in terms of time and money, with constructs failing to operate as desired, or evolving away from the desired function once deployed. Synthetic biologists seek to understand biological design principles and use them to create technologies that increase the efficiency of the genetic engineering design cycle. Central to the approach is the creation of biological parts--encapsulated functions that can be composited together to create new pathways with predictable behaviors. We define five desirable characteristics of biological parts--independence, reliability, tunability, orthogonality and composability, and review studies of small natural and synthetic biological circuits that provide insights into each of these characteristics. We propose that the creation of appropriate sets of families of parts with these properties is a prerequisite for efficient, predictable engineering of new function in cells and will enable a large increase in the sophistication of genetic engineering applications.

  View details for DOI 10.1016/j.mib.2008.10.002

  View details for Web of Science ID 000261866200015

  View details for PubMedID 18983935