Bio

Academic Appointments


Honors & Awards


  • Donald and Delia Baxter Foundation Faculty Scholar, Baxter Foundation (2019)
  • CRI Irvington Fellow, Cancer Research Institute
  • HHMI International Fellow, Howard Hughes Medical Institute

Professional Education


  • PhD, Harvard University, Harvard Medical School., Biological and Biomedical Sciences (2014)
  • LHB, Harvard Medical School., Certificate in Leder Human Biology and Translational Medicine
  • B.S., Tsinghua University, Biological Sciences, Electronic Engineering (2009)

Community and International Work


  • Neuro-engineering and Gene-editing., Cold Spring Harbor Laboratory

    Topic

    Advanced Techniques in Molecular Neuroscience

    Partnering Organization(s)

    Cold Spring Harbor Laboratory

    Location

    International

    Ongoing Project

    Yes

    Opportunities for Student Involvement

    Yes

Patents


  • Feng Zhang, Le Cong, Patrick Hsu, Fei Ann Ran. "United States Patent 8,906,616 Engineering of systems, methods and optimized guide compositions for sequence manipulation"
  • Le Cong, Feng Zhang. "United States Patent 8,932,814 CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes."
  • Feng Zhang, LeCong, Randall Platt, Neville Sanjana, Fei Ann Ran. "United States Patent 8,993,233 Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains"
  • Cong, Egloff, Garraway, Grandis, Lander, Stransky, Tward, Zhang.. "United States Patent 9,370,551. Compositions and methods of treating head and neck cancer."
  • Le Cong, Feng Zhang, Patrick Hsu, Fei Ann Ran. "United StatesEngineering of systems, methods and optimized guide compositions for sequence manipulation."

Teaching

2018-19 Courses


Stanford Advisees


Graduate and Fellowship Programs


Publications

All Publications


  • Take Risks and Constantly Challenge the Status Quo STEM CELLS AND DEVELOPMENT Cong, L. 2019
  • Combined Computational-Experimental Approach to Explore the Molecular Mechanism of SaCas9 with a Broadened DNA Targeting Range JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Luan, B., Xu, G., Feng, M., Cong, L., Zhou, R. 2019; 141 (16): 6545–52
  • Efficient Generation of Transcriptomic Profiles by Random Composite Measurements. Cell Cleary, B., Cong, L., Cheung, A., Lander, E. S., Regev, A. 2017; 171 (6): 1424–36.e18

    Abstract

    RNA profiles are an informative phenotype of cellular and tissue states but can be costly to generate at massive scale. Here, we describe how gene expression levels can be efficiently acquired with random composite measurements-in which abundances are combined in a random weighted sum. We show (1) that the similarity between pairs of expression profiles can be approximated with very few composite measurements; (2) that by leveraging sparse, modular representations of gene expression, we can use random composite measurements to recover high-dimensional gene expression levels (with 100 times fewer measurements than genes); and (3) that it is possible to blindly recover gene expression from composite measurements, even without access to training data. Our results suggest new compressive modalities as a foundation for massive scaling in high-throughput measurements and new insights into the interpretation of high-dimensional data.

    View details for PubMedID 29153835

    View details for PubMedCentralID PMC5726792

  • A Distinct Gene Module for Dysfunction Uncoupled from Activation in Tumor-Infiltrating T Cells CELL Singer, M., Wang, C., Cong, L., Marjanovic, N. D., Kowalczyk, M. S., Zhang, H., Nyman, J., Sakuishi, K., Kurtulus, S., Gennert, D., Xia, J., Kwon, J. Y., Nevin, J., Herbst, R. H., Yanai, I., Rozenblatt-Rosen, O., Kuchroo, V. K., Regev, A., Anderson, A. C. 2016; 166 (6): 1500-?

    Abstract

    Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8(+) tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8(+) TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact.

    View details for DOI 10.1016/j.cell.2016.08.052

    View details for Web of Science ID 000386339900021

    View details for PubMedID 27610572

    View details for PubMedCentralID PMC5019125

  • In vivo gene editing in dystrophic mouse muscle and muscle stem cells SCIENCE Tabebordbar, M., Zhu, K., Cheng, J. K., Chew, W. L., Widrick, J. J., Yan, W. X., Maesner, C., Wu, E. Y., Xiao, R., Ran, F. A., Cong, L., Zhang, F., Vandenberghe, L. H., Church, G. M., Wagers, A. J. 2016; 351 (6271): 407-411

    Abstract

    Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

    View details for DOI 10.1126/science.aad5177

    View details for Web of Science ID 000368440500046

    View details for PubMedID 26721686

    View details for PubMedCentralID PMC4924477

  • RBPJ Controls Development of Pathogenic Th17 Cells by Regulating IL-23 Receptor Expression. Cell reports Meyer Zu Horste, G., Wu, C., Wang, C., Cong, L., Pawlak, M., Lee, Y., Elyaman, W., Xiao, S., Regev, A., Kuchroo, V. K. 2016; 16 (2): 392–404

    Abstract

    Interleukin-17 (IL-17)-producing helper T cells (Th17 cells) play an important role in autoimmune diseases. However, not all Th17 cells induce tissue inflammation or autoimmunity. Th17 cells require IL-23 receptor (IL-23R) signaling to become pathogenic. The transcriptional mechanisms controlling the pathogenicity of Th17 cells and IL-23R expression are unknown. Here, we demonstrate that the canonical Notch signaling mediator RBPJ is a key driver of IL-23R expression. In the absence of RBPJ, Th17 cells fail to upregulate IL-23R, lack stability, and do not induce autoimmune tissue inflammation in vivo, whereas overexpression of IL-23R rescues this defect and promotes pathogenicity of RBPJ-deficient Th17 cells. RBPJ binds and trans-activates the Il23r promoter and induces IL-23R expression and represses anti-inflammatory IL-10 production in Th17 cells. We thus find that Notch signaling influences the development of pathogenic and non-pathogenic Th17 cells by reciprocally regulating IL-23R and IL-10 expression.

    View details for PubMedID 27346359

    View details for PubMedCentralID PMC4984261

  • Definitive localization of intracellular proteins: Novel approach using CRISPR-Cas9 genome editing, with glucose 6-phosphate dehydrogenase as a model. Analytical biochemistry Spencer, N. Y., Yan, Z., Cong, L., Zhang, Y., Engelhardt, J. F., Stanton, R. C. 2016; 494: 55–67

    Abstract

    Studies to determine subcellular localization and translocation of proteins are important because subcellular localization of proteins affects every aspect of cellular function. Such studies frequently utilize mutagenesis to alter amino acid sequences hypothesized to constitute subcellular localization signals. These studies often utilize fluorescent protein tags to facilitate live cell imaging. These methods are excellent for studies of monomeric proteins, but for multimeric proteins, they are unable to rule out artifacts from native protein subunits already present in the cells. That is, native monomers might direct the localization of fluorescent proteins with their localization signals obliterated. We have developed a method for ruling out such artifacts, and we use glucose 6-phosphate dehydrogenase (G6PD) as a model to demonstrate the method's utility. Because G6PD is capable of homodimerization, we employed a novel approach to remove interference from native G6PD. We produced a G6PD knockout somatic (hepatic) cell line using CRISPR-Cas9 mediated genome engineering. Transfection of G6PD knockout cells with G6PD fluorescent mutant proteins demonstrated that the major subcellular localization sequences of G6PD are within the N-terminal portion of the protein. This approach sets a new gold standard for similar studies of subcellular localization signals in all homodimerization-capable proteins.

    View details for PubMedID 26576833

    View details for PubMedCentralID PMC4695245

  • Crystal Structure of Staphylococcus aureus Cas9 CELL Nishimasu, H., Cong, L., Yan, W. X., Ran, F. A., Zetsche, B., Li, Y., Kurabayashi, A., Ishitani, R., Zhang, F., Nureki, O. 2015; 162 (5): 1113-1126

    Abstract

    The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets with a protospacer adjacent motif (PAM) and complementarity to the guide RNA. Recently, we harnessed Staphylococcus aureus Cas9 (SaCas9), which is significantly smaller than Streptococcus pyogenes Cas9 (SpCas9), to facilitate efficient in vivo genome editing. Here, we report the crystal structures of SaCas9 in complex with a single guide RNA (sgRNA) and its double-stranded DNA targets, containing the 5'-TTGAAT-3' PAM and the 5'-TTGGGT-3' PAM, at 2.6 and 2.7 Å resolutions, respectively. The structures revealed the mechanism of the relaxed recognition of the 5'-NNGRRT-3' PAM by SaCas9. A structural comparison of SaCas9 with SpCas9 highlighted both structural conservation and divergence, explaining their distinct PAM specificities and orthologous sgRNA recognition. Finally, we applied the structural information about this minimal Cas9 to rationally design compact transcriptional activators and inducible nucleases, to further expand the CRISPR-Cas9 genome editing toolbox.

    View details for DOI 10.1016/j.cell.2015.08.007

    View details for Web of Science ID 000360589900020

    View details for PubMedID 26317473

    View details for PubMedCentralID PMC4670267

  • In vivo genome editing using Staphylococcus aureus Cas9 NATURE Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., Koonin, E. V., Sharp, P. A., Zhang, F. 2015; 520 (7546): 186-U98

    Abstract

    The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

    View details for DOI 10.1038/nature14299

    View details for Web of Science ID 000352454600031

    View details for PubMedID 25830891

    View details for PubMedCentralID PMC4393360

  • Genome engineering using CRISPR-Cas9 system. Methods in molecular biology (Clifton, N.J.) Cong, L., Zhang, F. 2015; 1239: 197-217

    Abstract

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts.

    View details for DOI 10.1007/978-1-4939-1862-1_10

    View details for PubMedID 25408407

  • Sequence determinants of improved CRISPR sgRNA design. Genome research Xu, H., Xiao, T., Chen, C. H., Li, W., Meyer, C. A., Wu, Q., Wu, D., Cong, L., Zhang, F., Liu, J. S., Brown, M., Liu, X. S. 2015; 25 (8): 1147–57

    Abstract

    The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation (CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies.

    View details for PubMedID 26063738

    View details for PubMedCentralID PMC4509999

  • Global microRNA depletion suppresses tumor angiogenesis. Genes & development Chen, S., Xue, Y., Wu, X., Le, C., Bhutkar, A., Bell, E. L., Zhang, F., Langer, R., Sharp, P. A. 2014; 28 (10): 1054–67

    Abstract

    MicroRNAs delicately regulate the balance of angiogenesis. Here we show that depletion of all microRNAs suppresses tumor angiogenesis. We generated microRNA-deficient tumors by knocking out Dicer1. These tumors are highly hypoxic but poorly vascularized, suggestive of deficient angiogenesis signaling. Expression profiling revealed that angiogenesis genes were significantly down-regulated as a result of the microRNA deficiency. Factor inhibiting hypoxia-inducible factor 1 (HIF-1), FIH1, is derepressed under these conditions and suppresses HIF transcription. Knocking out FIH1 using CRISPR/Cas9-mediated genome engineering reversed the phenotypes of microRNA-deficient cells in HIF transcriptional activity, VEGF production, tumor hypoxia, and tumor angiogenesis. Using multiplexed CRISPR/Cas9, we deleted regions in FIH1 3' untranslated regions (UTRs) that contain microRNA-binding sites, which derepresses FIH1 protein and represses hypoxia response. These data suggest that microRNAs promote tumor responses to hypoxia and angiogenesis by repressing FIH1.

    View details for PubMedID 24788094

    View details for PubMedCentralID PMC4035535

  • Multiplex Genome Engineering Using CRISPR/Cas Systems SCIENCE Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., Zhang, F. 2013; 339 (6121): 819-823

    Abstract

    Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

    View details for DOI 10.1126/science.1231143

    View details for Web of Science ID 000314874400049

    View details for PubMedID 23287718

  • Optical control of mammalian endogenous transcription and epigenetic states. Nature Konermann, S., Brigham, M. D., Trevino, A. E., Hsu, P. D., Heidenreich, M., Cong, L., Platt, R. J., Scott, D. A., Church, G. M., Zhang, F. 2013; 500 (7463): 472–76

    Abstract

    The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.

    View details for PubMedID 23877069

    View details for PubMedCentralID PMC3856241

  • Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains NATURE COMMUNICATIONS Cong, L., Zhou, R., Kuo, Y., Cunniff, M., Zhang, F. 2012; 3

    Abstract

    Transcription activator-like effectors are sequence-specific DNA-binding proteins that harbour modular, repetitive DNA-binding domains. Transcription activator-like effectors have enabled the creation of customizable designer transcriptional factors and sequence-specific nucleases for genome engineering. Here we report two improvements of the transcription activator-like effector toolbox for achieving efficient activation and repression of endogenous gene expression in mammalian cells. We show that the naturally occurring repeat-variable diresidue Asn-His (NH) has high biological activity and specificity for guanine, a highly prevalent base in mammalian genomes. We also report an effective transcription activator-like effector transcriptional repressor architecture for targeted inhibition of transcription in mammalian cells. These findings will improve the precision and effectiveness of genome engineering that can be achieved using transcription activator-like effectors.

    View details for DOI 10.1038/ncomms1962

    View details for Web of Science ID 000306995000040

    View details for PubMedID 22828628

    View details for PubMedCentralID PMC3556390

  • A transcription activator-like effector toolbox for genome engineering. Nature protocols Sanjana, N. E., Cong, L., Zhou, Y., Cunniff, M. M., Feng, G., Zhang, F. 2012; 7 (1): 171–92

    Abstract

    Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.

    View details for PubMedID 22222791

    View details for PubMedCentralID PMC3684555

  • Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription NATURE BIOTECHNOLOGY Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., Arlotta, P. 2011; 29 (2): 149-U90

    Abstract

    The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator-like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.

    View details for DOI 10.1038/nbt.1775

    View details for Web of Science ID 000287023000022

    View details for PubMedID 21248753

    View details for PubMedCentralID PMC3084533