Profiling and Improving the Specificity of Site-Specific Nucleases

Programmable site-specific endonucleases are useful tools for genome editing and may lead to novel therapeutics to treat genetic diseases. TALENs can be designed to cleave chosen DNA sequences. To better understand TALEN specificity and engineer TALENs with improved specificity, we profiled 30 unique TALENs with varying target sites, array length, and domain sequences for their ability to cleave any of 1012 potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results collectively suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches, yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. We engineered a TALEN variant, Q3, that exhibits equal on-target cleavage activity but 10-fold lower average off-target activity in human cells. Our results demonstrate that identifying and mutating residues that contribute to non-specific DNA-binding can yield genome engineering agents with improved DNA specificities

Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity

Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have been shown to have activity against related off-target sequences. To better understand TALEN specificity and engineer TALENs with improved specificity, we profiled 30 unique TALENs with varying target sites, array length, and domain sequences for their ability to cleave any of 1012 potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results collectively suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches, yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant, Q3, that exhibits equal on-target cleavage activity but 10-fold lower average off-target activity in human cells. Our results demonstrate that identifying and mutating residues that contribute to non-specific DNA-binding can yield genome editing reagents with improved DNA specificities

Predicting Postfire Sediment Yields of Small Steep Catchments Using Airborne Lidar Differencing

Predicting sediment yield from recently burned areas remains a challenge but is important for hazard and resource management as wildfire impacts increase. Here we use lidar-based monitoring of two fires in southern California, USA to study the movement of sediment during pre-rainfall periods and postfire periods of flooding and debris flows over multiple storm events. Using a data-driven approach, we examine the relative importance of terrain, vegetation, burn severity, and rainfall amounts through time on sediment yield. We show that incipient fire-activated dry sediment loading and pre-fire colluvium were rapidly flushed out by debris flows and floods but continued erosion occurred later in the season from soil erosion and, in ∼9% of catchments, from shallow landslides. Based on these observations, we develop random forest regression models to predict dry ravel and incipient runoff-driven sediment yield applicable to small steep headwater catchments in southern California

Evaluation of the porous silicon capacitor as a moisture sensor for vacuum applications

A growing demand exists for inexpensive and reliable sensors for moisture detection in reduced pressure processing environments. Sandia`s Porous Silicon Capacitor (PSC) sensor appears to be an ideal candidate for this application. This sensor is a solid state device that detects moisture through changes in dielectric constant with water adsorption. Standard microelectronic fabrication techniques are used in its production affording low cost production and ready integration into complex sensor and electronic arrays. This sensor has previously been investigated for moisture detection in fluid streams, however, little effort has been placed on its behavior in a vacuum environment. Sandia`s Sensors in Vacuum (SIV) test facility has been employed to evaluate the performance characteristics of this sensor in vacuum. In addition, a vacuum-based study allows for a more controlled environment in which the intrinsic lower limit for moisture detection and response times to moisture changes can be easily determined quantitatively. This report describes the performance characteristics of a series of sensors from a single production lot. Calibration of these sensors to moisture levels from part per billion to part per hundred concentrations has been performed. The concentration-dependent sensitivity of these sensors is documented. The response time and drift characteristics of these sensors are also discussed. The investigation of a preliminary method for increasing the recovery time of the sensor after moisture exposure is presented. The role of hydrocarbon contamination, a potential problem in some vacuum schemes, is also evaluated. Specific recommendations are made on how to implement this sensor for vacuum applications

High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity

The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight Cas9:guide RNA complexes to cleave each of 10^12 potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two Cas9:guide RNA complexes. In contrast to previous models, our results show that Cas9:guide RNA specificity extends past a 7- to 12-base pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific then a longer, more-active guide RNA. High concentrations of Cas9:guide RNA complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting

Continuous directed evolution of DNA-binding proteins to improve TALEN specificity

Nucleases containing programmable DNA-binding domains can alter the genomes of model organisms and have the potential to become human therapeutics. Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity. We used this system to generate TALE nucleases with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents

Hypogene Calcitization: Evaporite Diagenesis in the Western Delaware Basin

Evaporite calcitization within the Castile Formation of the Delaware Basin is more widespread and diverse than originally recognized. Coupled field and GIS studies have identified more than 1000 individual occurrences of calcitization within the Castile Formation outcrop area, which includes both calcitized masses (limestone buttes) and laterally extensive calcitized horizons (limestone sheets). Both limestone buttes and sheets commonly contain a central brecciated zone that we attribute to hypogene dissolution. Lithologic fabric of calcitized zones ranges from little alteration of original varved laminae to fabrics showing extensive laminae distortion as well as extensive vuggy and open cavernous porosity. Calcitization is most abundant in the western portion of the Castile outcrop region where surface denudation has been greatest. Calcitization often forms linear trends, indicating fluid migration along fractures, but also occurs as dense clusters indicating focused, ascending, hydrocarbon-rich fluids. Native sulfur, secondary tabular gypsum (i.e. selenite) and hypogene caves are commonly associated with clusters of calcitization. This assemblage suggests that calcium sulfate diagenesis within the Castile Formation is dominated by hypogene speleogemesis

Use of designer nucleases for targeted gene and genome editing in plants

The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single-celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/ RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/ RNA oligonucleotides) to create a double-stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technology’s longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production