A novel method for mammalian large genetic circuit assembly and delivery

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, February 2012."February 2012." Cataloged from PDF version of thesis.Includes bibliographical references (p. 65-75).Genetic manipulation of mammalian cells provides a foundation for contemporary biological research both basic and applied. Existing methods for construction and introduction of large scale exogenous genetic information into mammalian cells and for creating stable cell lines are not efficient and suffer from limitations in terms of cost, speed, flexibility, and reliability. In this thesis, a novel method is presented for the efficient construction and delivery of complex genetic circuits into mammalian cells. Multi-gene circuits are assembled with high efficiency from a validated modular library into single pieces. The assembled circuits can be used for transient expression and each individual circuit can be integrated into a cellular genome to create a stable cell line. Genetic circuits were constructed that contain several expression units, including inducible control units and fluorescent markers. These circuits were delivered into Human Embryonic Kidney 293 (HEK293) cells for both transient and stable expression cases. Results show that the introduced genetic circuits performed as designed and that stable cell lines, each with the desired phenotype could be created efficiently. Several factors affecting the assembly efficiency and the performance of resulting circuits are also discussed.by Yinqing Li.S.M

Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons

Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation of rare neurons from adult tissue is challenging and markers for each phase are limited. Here, we develop Div-Seq, which combines scalable single-nucleus RNA-Seq (sNuc-Seq) with pulse labeling of proliferating cells by 5-ethynyl-2′-deoxyuridine (EdU) to profile individual dividing cells. sNuc-Seq and Div-Seq can sensitively identify closely related hippocampal cell types and track transcriptional dynamics of newborn neurons within the adult hippocampal neurogenic niche, respectively. We also apply Div-Seq to identify and profile rare newborn neurons in the adult spinal cord, a noncanonical neurogenic region. sNuc-Seq and Div-Seq open the way for unbiased analysis of diverse complex tissues.National Institute of Mental Health (U.S.) (Grant U01MH105960)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049

Crystal Structure of Staphylococcus aureus Cas9

Summary The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets with a protospacer adjacent mot if (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.National Institute of General Medical Sciences (U.S.) (Grant T32GM007753)National Institutes of Health (U.S.) (Award 1DP1-MH100706

Formation and optogenetic control of engineered 3D skeletal muscle bioactuators

Densely arrayed skeletal myotubes are activated individually and as a group using precise optical stimulation with high spatiotemporal resolution. Skeletal muscle myoblasts are genetically encoded to express a light-activated cation channel, Channelrhodopsin-2, which allows for spatiotemporal coordination of a multitude of skeletal myotubes that contract in response to pulsed blue light. Furthermore, ensembles of mature, functional 3D muscle microtissues have been formed from the optogenetically encoded myoblasts using a high-throughput device. The device, called “skeletal muscle on a chip”, not only provides the myoblasts with controlled stress and constraints necessary for muscle alignment, fusion and maturation, but also facilitates the measurement of forces and characterization of the muscle tissue. We measured the specific static and dynamic stresses generated by the microtissues and characterized the morphology and alignment of the myotubes within the constructs. The device allows testing of the effect of a wide range of parameters (cell source, matrix composition, microtissue geometry, auxotonic load, growth factors and exercise) on the maturation, structure and function of the engineered muscle tissues in a combinatorial manner. Our studies integrate tools from optogenetics and microelectromechanical systems (MEMS) technology with skeletal muscle tissue engineering to open up opportunities to generate soft robots actuated by a multitude of spatiotemporally coordinated 3D skeletal muscle microtissues.National Science Foundation (U.S.) (Science and Technology Center—Emergent Behaviors of Integrated Cellular Systems (EBICS) grant No. CBET-0939511)National Institutes of Health (U.S.) (EB00262)National Science Foundation (U.S.) (GM74048)National Science Foundation (U.S.) (HL90747)National Institute for Biomedical Imaging and Bioengineering (U.S.) (RESBIO, Integrapted Technologies for Polymeric Biomaterial)University of Pennsylvania. Center for Engineering Cells and RegenerationSingapore-MIT Alliance for Research and Technolog

A Ti3C2Tx-Based Composite as Separator Coating for Stable Li-S Batteries

The nitrogen-doped MXene carbon nanosheet-nickel (N-M@CNi) powder was successfully prepared by a combined process of electrostatic attraction and annealing strategy, and then applied as the separator coating in lithium-sulfur batteries. The morphology and structure of the N-M@CNi were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Raman spectrum, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and nitrogen adsorption-desorption method. The strong LiPS adsorption ability and high conductivity are associated with the N-doped carbon nanosheet-Ni modified surface. The modified separator offers the cathode of Li-S cell with greater sulfur utilization, better high-rate adaptability, and more stable cycling performance compared with the pristine separator. At 0.2 C the cell with N-M@CNi separator delivers an initial capacity of 1309 mAh g-1. More importantly, the N-M@CNi separator is able to handle a cathode with 3.18 mg cm-2 sulfur loading, delivering a capacity decay rate of 0.043% with a high capacity retention of 95.8%. Therefore, this work may provide a feasible approach to separator modification materials towards improved Li-S cells with improved stability

Rapid, modular and reliable construction of complex mammalian gene circuits

We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.Synthetic Biology Engineering Research Center (SA5284-11210)United States. Defense Advanced Research Projects Agency (HR0011-12-C-0067)United States. Defense Advanced Research Projects Agency (DARPA-BAA-11-23)National Science Foundation (U.S.) (CBET-0939511)National Institutes of Health (U.S.). (5-R01-CA155320-02

BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks

Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.National Institute of General Medical Sciences (U.S.) (Grant T32GM007753)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049