Bio

Bio


Dr. Lei (Stanley) Qi is an Assistant Professor of Bioengineering and Chemical and Systems Biology at Stanford University, and a faculty fellow in Stanford ChEM-H. He is one of the major pioneers in the CRISPR technology for targeted genome engineering in mammalian cells. Different from most developers of the CRISPR tools, he has developed a series of gene regulation and imaging technologies, including CRISPR interference (CRISPRi), CRISPR imaging, and high-throughput CRISPR screening. He also worked in the fields of Synthetic Biology, and developed methods to generate synthetic noncoding RNA regulators of transcription, translation, and as molecular sensor for chemicals and intracellular proteins. He obtained his Ph.D. in Bioengineering from the University of California, Berkeley in 2012, and performed independent research as a Systems Biology Fellow in the University of California, San Francisco in 2012 to 2014. His lab is currently applying genome engineering and CRISPR technologies for the interrogation of genetic interaction networks related to cell differentiation, proliferation, epigenetic regulation and diseases.

Academic Appointments


Honors & Awards


  • Catalyst Award for the Development of Diagnostics, Devices, Therapeutics and Digital Health, UCSF (June 2014)
  • NIH Director's Independence Award, NIH (September 2013)
  • Chinese Government Award For Outstanding Self-Financed Students Abroad, Chinese government (June 2012)
  • Phi Beta Kappa Honor Society, Phi Beta Kappa (December 2011)

Professional Education


  • B.S., Tsinghua University, Mathematics and Physics (2005)
  • M.A., University of California, Berkeley, Physics (2007)
  • Ph.D., University of California, Berkeley, Bioengineering (2012)
  • Systems Biology Fellow, University of California, San Francisco, Systems and Synthetic Biology (2014)

Patents


  • Qi LS; Ding S; Chen Y. "United StatesSystems and methods for modulating CRISPR/Cas9 genome editing.", Leland Stanford Junior University
  • Jennifer A Doudna, Lei S Qi, Rachel E Haurwitz, Adam P Arkin. "United States Patent 14/248,980 Methods and compositions of controlling gene expression by RNA processing", University of California, Berkeley, Oct 9, 2014
  • Qi LS, Chen B, Huang B. "United States Patent US provisional patent application number 61/883,929. Optimized small guide RNAs and methods of use", University of California, San Francisco, Sep 1, 2013
  • Lei S Qi, Chang Liu, Adam P Arkin. "United States Patent WO Patent 2,013,049,330 Synthetic transcriptional control elements and methods of generating and using such elements", University of California, Berkeley, Apr 5, 2013
  • Lei S Qi, Jennifer A Doudna, Martin Jinek, Emmanuelle Charpentier,Krzysztof Chylinski, James HD Cate, Wendell A Lim. "United States Patent US Patent App. 13/842,859 Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription", University of California, Berkeley & University of California, San Francisco, Mar 15, 2013

Research & Scholarship

Current Research and Scholarly Interests


CRISPR technologies for genome engineering, stem cell engineering & cell therapy

Teaching

2016-17 Courses


Stanford Advisees


Publications

All Publications


  • 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 Web of Science ID 000349455000010

  • 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

  • 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

  • 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
  • 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 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

  • 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

  • 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