CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34+ hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

In this paper, we focus on the implementation and verification of an extended Boussinesq model for surf zone hydrodynamics in two horizontal dimensions. The time-domain numerical model is based on the fully nonlinear Boussinesq equations. As described in Part I of this two-part paper, the energy dissipation due to wave breaking is modeled by introducing an eddy viscosity term into the momentum equations, with the viscosity strongly localized on the front face of the breaking waves. Wave runup on the beach is simulated using a permeable-seabed technique. We apply the model to simulate two laboratory experiments in large wave basins. They are wave transformation and breaking over a submerged circular shoal and solitary wave runup on a conical island. Satisfactory agreement is found between the numerical results and the laboratory measurements.

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Hurricane Gustav (2008) made landfall in southern Louisiana on 1 September 2008 with its eye never closer than 75 km to New Orleans, but its waves and storm surge threatened to flood the city. Easterly tropical-storm-strength winds impacted the region east of the Mississippi River for 12–15 h, allowing for early surge to develop up to 3.5 m there and enter the river and the city’s navigation canals. During landfall, winds shifted from easterly to southerly, resulting in late surge development and propagation over more than 70 km of marshes on the river’s west bank, over more than 40 km of Caernarvon marsh on the east bank, and into Lake Pontchartrain to the north. Wind waves with estimated significant heights of 15 m developed in the deep Gulf of Mexico but were reduced in size once they reached the continental shelf. The barrier islands further dissipated the waves, and locally generated seas existed behind these effective breaking zones. The hardening and innovative deployment of gauges since Hurricane Katrina (2005) resulted in a wealth of measured data for Gustav. A total of 39 wind wave time histories, 362 water level time histories, and 82 high water marks were available to describe the event. Computational models—including a structured-mesh deepwater wave model (WAM) and a nearshore steady-state wave (STWAVE) model, as well as an unstructured-mesh “simulating waves nearshore” (SWAN) wave model and an advanced circulation (ADCIRC) model—resolve the region with unprecedented levels of detail, with an unstructured mesh spacing of 100–200 m in the wave-breaking zones and 20–50 m in the small-scale channels. Data-assimilated winds were applied using NOAA’s Hurricane Research Division Wind Analysis System (H*Wind) and Interactive Objective Kinematic Analysis (IOKA) procedures. Wave and surge computations from these models are validated comprehensively at the measurement locations ranging from the deep Gulf of Mexico and along the coast to the rivers and floodplains of southern Louisiana and are described and quantified within the context of the evolution of the storm.

Methods for rapidly assessing sequence-structure-function landscapes and developing conditional gene-regulatory devices are critical to our ability to manipulate and interface with biology. We describe a framework for engineering RNA devices from preexisting aptamers that exhibit ligand-responsive ribozyme tertiary interactions. Our methodology utilizes cell sorting, high-throughput sequencing, and statistical data analyses to enable parallel measurements of the activities of hundreds of thousands of sequences from RNA device libraries in the absence and presence of ligands. Our tertiary interaction RNA devices exhibit improved performance in terms of gene silencing, activation ratio, and ligand sensitivity as compared to optimized RNA devices that rely on secondary structure changes. We apply our method to building biosensors for diverse ligands and determine consensus sequences that enable ligand-responsive tertiary interactions. These methods advance our ability to develop broadly applicable genetic tools and to elucidate understanding of the underlying sequence-structure-function relationships that empower rational design of complex biomolecules.

[1] Hurricane Ike (2008) made landfall near Galveston, Texas, as a moderate intensity storm. Its large wind field in conjunction with the Louisiana-Texas coastline's broad shelf and large scale concave geometry generated waves and surge that impacted over 1000 km of coastline. Ike's complex and varied wave and surge response physics included: the capture of surge by the protruding Mississippi River Delta; the strong influence of wave radiation stress gradients on the Delta adjacent to the shelf break; the development of strong wind driven shore-parallel currents and the associated geostrophic setup; the forced early rise of water in coastal bays and lakes facilitating inland surge penetration; the propagation of a free wave along the southern Texas shelf; shore-normal peak wind-driven surge; and resonant and reflected long waves across a wide continental shelf. Preexisting and rapidly deployed instrumentation provided the most comprehensive hurricane response data of any previous hurricane. More than 94 wave parameter time histories, 523 water level time histories, and 206 high water marks were collected throughout the Gulf in deep water, along the nearshore, and up to 65 km inland. Ike's highly varied physics were simulated using SWAN þ ADCIRC, a tightly coupled wave and circulation model, on SL18TX33, a new unstructured mesh of the Gulf of Mexico, Caribbean Sea, and western Atlantic Ocean with high resolution of the Gulf's coastal floodplain from Alabama to the Texas-Mexico border. A comprehensive validation was made of the model's ability to capture the varied physics in the system.

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A large, unpredicted, water level increase appeared along a substantial section of the western Louisiana and northern Texas (LATEX) coasts 12–24 hrs in advance of the landfall of Hurricane Ike (2008), with water levels in some areas reaching 3 m above mean sea level. During this time the cyclonic wind field was largely shore parallel throughout the region. A similar early water level rise was reported for both the 1900 and the 1915 Galveston Hurricanes. The Ike forerunner anomaly occurred over a much larger area and prior to the primary coastal surge which was driven by onshore directed winds to the right of the storm track. We diagnose the forerunner surge as being generated by Ekman setup on the wide and shallow LATEX shelf. The longer forerunner time scale additionally served to increase water levels significantly in narrow‐entranced coastal bays. The forerunner surge generated a freely propagating continental shelf wave with greater than 1.4 m peak elevation that travelled coherently along the coast to Southern Texas, and was 300 km in advance of the storm track at the time of landfall. This was, at some locations, the largest water level increase seen throughout the storm, and appears to be the largest freely‐propagating shelf wave ever reported. Ekman setup‐driven forerunners will be most significant on wide, shallow shelves subject to large wind fields, and need to be considered for planning and forecasting in these cases.