Samuel David Perli

Interference alignment and cancellation

The throughput of existing MIMO LANs is limited by the number of antennas on the AP. This paper shows how to overcome this limit. It presents interference alignment and cancellation (IAC), a new approach for decoding concurrent sender-receiver pairs in MIMO networks. IAC synthesizes two signal processing techniques, interference alignment and interference cancellation, showing that the combination applies to scenarios where neither interference alignment nor cancellation applies alone. We show analytically that IAC almost doubles the throughput of MIMO LANs. We also implement IAC in GNU-Radio, and experimentally demonstrate that for 2x2 MIMO LANs, IAC increases the average throughput by 1.5x on the downlink and 2x on the uplink.

Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells

RNA-based regulation and CRISPR/Cas transcription factors (CRISPR-TFs) have the potential to be integrated for the tunable modulation of gene networks. A major limitation of this methodology is that guide RNAs (gRNAs) for CRISPR-TFs can only be expressed from RNA polymerase III promoters in human cells, limiting their use for conditional gene regulation. We present new strategies that enable expression of functional gRNAs from RNA polymerase II promoters and multiplexed production of proteins and gRNAs from a single transcript in human cells. We use multiple RNA regulatory strategies, including RNA-triple-helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based RNA processing. Using these tools, we efficiently modulate endogenous promoters and implement tunable synthetic circuits, including multistage cascades and RNA-dependent networks that can be rewired with Csy4 to achieve complex behaviors. This toolkit can be used for programming scalable gene circuits and perturbing endogenous networks for biology, therapeutic, and synthetic biology applications.

Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas.

Transcriptional regulation is central to the complex behavior of natural biological systems and synthetic gene circuits. Platforms for the scalable, tunable, and simple modulation of transcription would enable new abilities to study natural systems and implement artificial capabilities in living cells. Previous approaches to synthetic transcriptional regulation have relied on engineering DNA-binding proteins, which necessitate multistep processes for construction and optimization of function. Here, we show that the CRISPR/Cas system of Streptococcus pyogenes can be programmed to direct both activation and repression to natural and artificial eukaryotic promoters through the simple engineering of guide RNAs with base-pairing complementarity to target DNA sites. We demonstrate that the activity of CRISPR-based transcription factors (crisprTFs) can be tuned by directing multiple crisprTFs to different positions in natural promoters and by arraying multiple crisprTF-binding sites in the context of synthetic promoters in yeast and human cells. Furthermore, externally controllable regulatory modules can be engineered by layering gRNAs with small molecule-responsive proteins. Additionally, single nucleotide substitutions within promoters are sufficient to render them orthogonal with respect to the same gRNA-guided crisprTF. We envision that CRISPR-based eukaryotic gene regulation will enable the facile construction of scalable synthetic gene circuits and open up new approaches for mapping natural gene networks and their effects on complex cellular phenotypes.

PixNet: interference-free wireless links using LCD-camera pairs

Given the abundance of cameras and LCDs in todays environment, there exists an untapped opportunity for using these devices for communication. Specifically, cameras can tune to nearby LCDs and use them for network access. The key feature of these LCD-camera links is that they are highly directional and hence enable a form of interference-free wireless communication. This makes them an attractive technology for dense, high contention scenarios. The main challenge however, to enable such LCD-camera links is to maximize coverage, that is to deliver multiple Mb/s over multi-meter distances, independent of the view angle. To do so, these links need to address unique types of channel distortions, such as perspective distortion and blur. This paper explores this novel communication medium and presents PixNet, a system for transmitting information over LCD-camera links. PixNet generalizes the popular OFDM transmission algorithms to address the unique characteristics of the LCD-camera link which include perspective distortion, blur, and sensitivity to ambient light. We have built a prototype of PixNet using off-the-shelf LCDs and cameras. An extensive evaluation shows that a single PixNet link delivers data rates of up to 12 Mb/s at a distance of 10 meters, and works with view angles as wide as 120 degree°.

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

Significance The systematic discovery of new gene and drug combinations that modulate complex biological phenotypes and human diseases requires scalable and multiplexed screening technologies. We leverage the programmability of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of the combinatorial genetics en masse (CombiGEM) technology to rapidly assemble barcoded combinatorial genetic perturbation libraries that can be tracked with high-throughput sequencing. CombiGEM-CRISPR enables simple, massively parallel screening of barcoded combinatorial gene perturbations in human cells, and the translation of these hits into effective drug combinations. This approach is broadly applicable for performing pooled combinatorial genetic perturbations to map out how the orchestrated action of genes controls complex phenotypes and to translate these findings into novel drug combinations. The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas–based knockouts and RNA-interference–based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.

Continuous genetic recording with self-targeting CRISPR-Cas in human cells

INTRODUCTION Technologies that enable the longitudinal tracking and recording of molecular events into genomic DNA would be useful for the detailed monitoring of cellular state in artificial and native contexts. Although previous systems have been used to memorize digital information such as the presence or absence of biological signals, tools for recording analog information such as the duration or magnitude of biological activity in human cells are needed. Here, we present Mammalian Synthetic Cellular Recorders Integrating Biological Events (mSCRIBE), a memory system for storing analog biological information in the form of accumulating DNA mutations in human cells. mSCRIBE leverages self-targeting guide RNAs (stgRNAs) that are engineered to direct Streptococcus pyogenes Cas9 cleavage against DNA loci that encode the stgRNAs, thus accumulating mutations at stgRNA loci as a record of stgRNA or Cas9 expression. RATIONALE The RNA-guided DNA endonuclease Cas9 introduces a double-stranded break in target DNA containing a 5′-NGG-3′ protospacer-adjacent motif (PAM) and homology to the specificity-determining sequence (SDS) of a small guide RNA (sgRNA). Once a double-strand break is introduced, the targeted DNA can be repaired via error-prone DNA repair mechanisms in human cells. We hypothesized that if a PAM sequence were introduced in the DNA locus encoding the sgRNA, the transcribed sgRNA would direct Cas9 to cleave its own encoding DNA, thus acting as a stgRNA. After error-prone repair, the mutagenized stgRNA locus should continue to be transcribed and enact additional rounds of continuous, self-targeted mutagenesis. Thus, the stgRNA locus should acquire mutations corresponding to the level of activity of the Cas9-stgRNA complex. We hypothesized that by linking the expression of stgRNA or Cas9 to biological events of interest, one could then record the duration and/or intensity of such events in the form of accumulated mutations at the stgRNA locus. The recorded information could be read by sequencing the stgRNA locus or by other related strategies. RESULTS We first built a stgRNA by engineering a sgRNA-encoding DNA locus to contain a 5′-NGG-3′ PAM immediately downstream of the SDS-encoding region. We then validated that the stgRNA could undergo multiple rounds of self-targeted mutagenesis by building a mutation-based toggling reporter system in which the progressive accumulation of mutations at the stgRNA locus is reported by individual cells toggling between green and red fluorescent protein expression. Next, we analyzed the sequence-evolution properties of stgRNAs in order to devise a sequence-based recording metric that conveys information on the duration and/or magnitude of stgRNA activity. We showed that computationally designed stgRNAs that contain longer SDSs of length 30, 40, and 70 nucleotides are able to accumulate mutations over longer durations of time. We demonstrated the analog nature of mSCRIBE by building a tumor necrosis factor–α (TNFα)–inducible Cas9 expression system and observing graded increases in the recording metric as a function of increasing TNFα concentration and/or duration of exposure in vitro. By designing doxycycline and isopropyl-β-dthiogalactoside-inducible stgRNA expression systems, we also showed inducible, multiplexed recording at two independent DNA loci. Last, we confirmed that human cells containing TNFα-responsive mSCRIBE units can record lipopolysaccharide (LPS)–induced acute inflammation events over time in mice. CONCLUSION We demonstrate that sgRNAs can be engineered to function as stgRNAs. By linking stgRNA or Cas9 expression to specific biological events of interest—such as the presence of small molecules, exposure to TNFα, or LPS-induced inflammation—we validated mSCRIBE as an analog memory device that records information about the duration and/or magnitude of biological events. Moreover, we demonstrated that multiple biological events can be simultaneously monitored by using independent stgRNA loci. We envision that this platform for genomically encoded memory in human cells should be broadly useful for studying biological systems and longitudinal and dynamic events in vitro and in situ, such as signaling pathways, gene regulatory networks, and tissue heterogeneity involved in development, healthy cell function, and disease pathogenesis. Continuously evolving stgRNAs. The Cas9-stgRNA complex cleaves the DNA locus from which the stgRNA is transcribed, leading to error-prone DNA repair. Multiple rounds of transcription and DNA cleavage can occur, resulting in progressive mutagenesis of the DNA encoding the stgRNA. The accumulation of mutations in the stgRNA locus provides a molecular record of cellular events that regulate stgRNA or Cas9 expression. The ability to record molecular events in vivo would enable monitoring of signaling dynamics within cellular niches and critical factors that orchestrate cellular behavior. We present a self-contained analog memory device for longitudinal recording of molecular stimuli into DNA mutations in human cells. This device consists of a self-targeting guide RNA (stgRNA) that repeatedly directs Streptococcus pyogenes Cas9 nuclease activity toward the DNA that encodes the stgRNA, enabling localized, continuous DNA mutagenesis as a function of stgRNA expression. We demonstrate programmable and multiplexed memory storage in human cells triggered by exogenous inducers or inflammation, both in vitro and in vivo. This tool, Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE), provides a distinct strategy for investigating cell biology in vivo and enables continuous evolution of targeted DNA sequences.

Foundations and Emerging Paradigms for Computing in Living Cells

Genetic circuits, composed of complex networks of interacting molecular machines, enable living systems to sense their dynamic environments, perform computation on the inputs, and formulate appropriate outputs. By rewiring and expanding these circuits with novel parts and modules, synthetic biologists have adapted living systems into vibrant substrates for engineering. Diverse paradigms have emerged for designing, modeling, constructing, and characterizing such artificial genetic systems. In this paper, we first provide an overview of recent advances in the development of genetic parts and highlight key engineering approaches. We then review the assembly of these parts into synthetic circuits from the perspectives of digital and analog logic, systems biology, and metabolic engineering, three areas of particular theoretical and practical interest. Finally, we discuss notable challenges that the field of synthetic biology still faces in achieving reliable and predictable forward-engineering of artificial biological circuits.

Ratiometric logic in living cells via competitive binding of synthetic transcription factors

Although there have been a flurry of designs in the recent past describing implementations of digital logic in living cells, computational elements that perform analog operations such as division and subtraction are scarce. By employing the principle of competitive binding between different DNA binding proteins, we present a novel approach towards ratiometric computation in living cells. After developing a quantitative model to analyze our design, we build and experimentally characterize our system in Saccharomyces cerevisiae. Our work demonstrates the feasibility of performing analog computation in eukaryotic cells and will potentially enable the design of more sophistated gene networks.

Analog synthetic gene networks

In analog synthetic biology, the underlying mathematical principles of various biological processes is harnessed to perform complex computation. Here, we review key synthetic gene circuits that implement analog computation in living cells and propose biological constructs that can implement further novel analog computation.

An integrated RNA and CRISPR/Cas toolkit for multiplexed synthetic circuits and endogenous gene regulation in human cells

RNA-based regulation, such as RNA interference, and CRISPR/Cas transcription factors (CRISPR-TFs), can enable scalable synthetic gene circuits and the modulation of endogenous networks but have yet to be integrated together. Here, we combined multiple mammalian RNA regulatory strategies, including RNA triple helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based RNA processing in human cells. We describe three complementary strategies for expressing functional gRNAs from transcripts generated by RNA polymerase II (RNAP II) promoters while allowing the harboring gene to be translated. These architectures enable the multiplexed expression of proteins and multiple gRNAs from a single compact transcript for efficient modulation of synthetic constructs and endogenous human promoters. We used these regulatory tools to implement tunable synthetic gene circuits, including multi-stage transcriptional cascades. Finally, we show that Csy4 can rewire regulatory connections in RNA-dependent gene circuits with multiple outputs and feedback loops to achieve complex functional behaviors. This multiplexable toolkit will be valuable for the construction of scalable gene circuits and the perturbation of natural regulatory networks in human cells for basic biology, therapeutic, and synthetic-biology applications.