The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

The new frontier of genome engineering with CRISPR-Cas9

Technologies that enable targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we describe a programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. The fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers. Gene activation by the targeted acetyltransferase was highly specific across the genome. In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with an individual guide RNA. We also show that the core p300 domain can be fused to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a robust tool for manipulating gene regulation.

/pdf/epigenome-editing-by-a-crispr-cas9-based-acetyltransferase-nz3mx0pe78.pdf

Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers

https://escholarship.org/content/qt8j20f5k0/qt8j20f5k0.pdf?t=nuq33k

A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging

The solid-state lighting is revolutionizing the indoor illumination. Current incandescent and fluorescent lamps are being replaced by the LEDs at a rapid pace. Apart from extremely high energy efficiency, the LEDs have other advantages such as longer lifespan, lower heat generation, and improved color rendering without using harmful chemicals. One additional benefit of LEDs is that they are capable of switching to different light intensity at a very fast rate. This functionality has given rise to a novel communication technology (known as visible light communication—VLC) where LED luminaires can be used for high speed data transfer. This survey provides a technology overview and review of existing literature of visible light communication and sensing. This paper provides a detailed survey of 1) visible light communication system and characteristics of its various components such as transmitter and receiver; 2) physical layer properties of visible light communication channel, modulation methods, and MIMO techniques; 3) medium access techniques; 4) system design and programmable platforms; and 5) visible light sensing and application such as indoor localization, gesture recognition, screen-camera communication, and vehicular networking. We also outline important challenges that need to be addressed in order to design high-speed mobile networks using visible light communication.

Visible Light Communication, Networking, and Sensing: A Survey, Potential and Challenges

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.

/pdf/boosting-crispr-cas9-multiplex-editing-capability-with-the-1e8udwm5vf.pdf

Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

https://www.cell.com/molecular-cell/pdf/S1097-2765(14)00355-4.pdf

Multiplexed and Programmable Regulation of Gene Networks with an Integrated RNA and CRISPR/Cas Toolkit in Human Cells

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.

/pdf/interference-alignment-and-cancellation-2sr97z2dqg.pdf

Interference alignment and cancellation

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.

/pdf/tunable-and-multifunctional-eukaryotic-transcription-factors-q775mdvyfv.pdf

Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas

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.

https://www.pnas.org/content/pnas/113/9/2544.full.pdf

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

Given the abundance of cameras and LCDs in today's 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°.

/pdf/pixnet-interference-free-wireless-links-using-lcd-camera-lx8iu3qsby.pdf

Samuel D. Perli

Papers

Multiplexed and Programmable Regulation of Gene Networks with an Integrated RNA and CRISPR/Cas Toolkit in Human Cells

Interference alignment and cancellation

Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

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