Negative DNA supercoiling induces genome-wide Cas9 off-target activity.

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.

CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy.

The discovery of CRISPR/Cas9 as an easily programmable endonuclease heralds a new era of genetic manipulation. With this comes the prospect of novel gene therapy approaches, and the potential to cure previously untreatable genetic diseases. However, reports of spurious off-target editing by CRISPR/Cas9 pose a significant hurdle to realizing this potential. A deeper understanding of the factors that affect Cas9 specificity is vital for development of safe and efficient therapeutics. Here, we describe methods for the use of optical tweezers combined with confocal fluorescence microscopy and microfluidics for the analysis of on- and off-target activity of Cas9 activity.

Gastral Drumming in Vespula germanica (Hymenoptera: Vespidae): Vibrational Communication at Night Suggests Additional Roles in Colony Organization.

Gastral drumming (GD) is a type of vibrational communication that has been reported in several species of yellowjackets and hornets. Despite early claims that it acts as a hunger signal, a more recent study found evidence that it acts as a nest-based food-recruitment signal, the first reported for eusocial wasps. Early studies also claimed, without supporting data, that it is produced most often in the early morning hours when the sun rises. Here, I recorded drumming continuously in colonies of Vespula germanica (Fabricius) to assess whether production was highest in the morning. Although I found no evidence in support of greater early morning production, I found, surprisingly, that it is produced at night, a time when foraging does not occur. When these results are combined with the results from previous studies on this species and similar findings in honey bees, they suggest that GD may be a modulatory signal, which acts by increasing general activity levels and by increasing the rate that individuals come into contact with social cues.

Universal toxin-based selection for precise genome engineering in human cells.

Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.

CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs.

Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.

Systematic Evaluation of CRISPRa and CRISPRi Modalities Enables Development of a Multiplexed, Orthogonal Gene Activation and Repression System.

The ability to manipulate the expression of mammalian genes using synthetic transcription factors is highly desirable in both fields of basic research and industry for diverse applications, including stem cell reprogramming and differentiation, tissue engineering, and drug discovery. Orthogonal CRISPR systems can be used for simultaneous transcriptional upregulation of a subset of target genes while downregulating another subset, thus gaining control of gene regulatory networks, signaling pathways, and cellular processes whose activity depends on the expression of multiple genes. We have used a rapid and efficient modular cloning system to build and test in parallel diverse CRISPRa and CRISPRi systems and develop an efficient orthogonal gene regulation system for multiplexed and simultaneous up- and downregulation of endogenous target genes.

DNA stretching induces Cas9 off-target activity.

CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.

BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B.

BACKGROUND: Base Editing is a precise genome editing method that uses a deaminase-Cas9 fusion protein to mutate cytidine to thymidine in target DNA in situ without the generation of a double-strand break. However, the efficient enrichment of genetically modified cells using this technique is limited by the ability to detect such events. RESULTS: We have developed a Base Editing FLuorescent Activity REporter (BE-FLARE), which allows for the enrichment of cells that have undergone editing of target loci based on a fluorescence shift from BFP to GFP. We used BE-FLARE to evaluate the editing efficiency of APOBEC3A and APOBEC3B family members as alternatives deaminase domains to the rat APOBEC1 domain used in base editor 3 (BE3). We identified human APOBEC3A and APOBEC3B as highly efficient cytidine deaminases for base editing applications with unique properties. CONCLUSIONS: Using BE-FLARE to report on the efficiency and precision of editing events, we outline workflows for the accelerated generation of genetically engineered cell models and the discovery of alternative base editors.

Decoding non-random mutational signatures at Cas9 targeted sites.

The mutation patterns at Cas9 targeted sites contain unique information regarding the nuclease activity and repair mechanisms in mammalian cells. However, analytical framework for extracting such information are lacking. Here, we present a novel computational platform called Rational InDel Meta-Analysis (RIMA) that enables an in-depth comprehensive analysis of Cas9-induced genetic alterations, especially InDels mutations. RIMA can be used to quantitate the contribution of classical microhomology-mediated end joining (c-MMEJ) pathway in the formation of mutations at Cas9 target sites. We used RIMA to compare mutational signatures at 15 independent Cas9 target sites in human A549 wildtype and A549-POLQ knockout cells to elucidate the role of DNA polymerase θ in c-MMEJ. Moreover, the single nucleotide insertions at the Cas9 target sites represent duplications of preceding nucleotides, suggesting that the flexibility of the Cas9 nuclease domains results in both blunt- and staggered-end cuts. Thymine at the fourth nucleotide before protospacer adjacent motif (PAM) results in a two-fold higher occurrence of single nucleotide InDels compared to guanine at the same position. This study provides a novel approach for the characterization of the Cas9 nucleases with improved accuracy in predicting genome editing outcomes and a potential strategy for homology-independent targeted genomic integration.

Gastral drumming: a nest-based food-recruitment signal in a social wasp.

Many social insect species produce signals that either recruit foragers to a specific food source or simply activate more nestmates to become foragers. Both are means of enhancing resource exploitation by increasing the number of individuals devoted to gathering profitable resources. Gastral drumming (GD) has been documented in several species of yellowjackets and hornets (Vespidae: Vespinae). It has been hypothesized that it is a hunger signal, but there is little empirical evidence to support this claim. An alternative hypothesis is that GD recruits workers to forage for food. Here, we report the results of a test between the hunger-signal and food-recruitment hypotheses in the German yellowjacket wasp, Vespula germanica. We show that the rate of performance of GD decreased when colonies were deprived of food and increased when supplemental food was provided. Playback of GD caused increased rates of (1) movement in the nest, (2) trophallaxis, and (3) worker departures from the nest. Together, these results support the conclusion that GD is not a hunger signal as previously asserted but instead is a nest-based food-recruitment signal, the first to be reported for a social wasp.

Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease.

The CRISPR-Cas9 RNA-guided endonuclease system allows precise and efficient modification of complex genomes and is continuously developed to enhance specificity, alter targeting and add new functional moieties. However, one area yet to be explored is the base chemistry of the associated RNA molecules. Here we show the design and optimisation of hybrid DNA-RNA CRISPR and tracr molecules based on structure-guided approaches. Through careful mapping of the ribose requirements of Cas9, we develop hybrid versions possessing minimal RNA residues, which are sufficient to direct specific nuclease activity in vitro and in vivo with reduced off-target activity. We identify critical regions within these molecules that require ribose nucleotides and show a direct correlation between binding affinity/stability and cellular activity. This is the first demonstration of a non-RNA-guided Cas9 endonuclease and first step towards eliminating the ribose dependency of Cas9 to develop a XNA-programmable endonuclease.

Tunable single-mode coupled-cavity laser in a standard InP photonics platform.

We present a wavelength tunable, coupled-cavity laser in a standard indium phosphide multiproject wafer shuttle which did not support distributed feedback gratings. The single-mode operation was enabled by reflections from slots in the laser cavity. The wavelength of the laser emission was tunable over 20 nm near a wavelength of 1560 nm via the currents applied to each section of the laser. A maximum side-mode suppression ratio of 46 dB was observed. The delayed self-heterodyne spectrum of the laser showed a Voigt line shape, corresponding to optical linewidths of 3.7 MHz for the Lorentzian and 88 MHz for the Gaussian contributions.

Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes.

Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors.

Wide bandwidth and high coupling efficiency Si3N4-on-SOI dual-level grating coupler.

We propose and experimentally demonstrate fiber-to-chip grating couplers with aligned silicon nitride (Si(3)N(4)) and silicon (Si) grating teeth for wide bandwidths and high coupling efficiencies without the use of bottom reflectors. The measured 1-dB bandwidth is a record 80 nm, and the measured peak coupling efficiency is -1.3 dB, which is competitive with the best Si-only grating couplers. The grating couplers are integrated in a Si(3)N(4) on silicon-on-insulator (SOI) integrated optics platform with aligned waveguides in both the Si(3)N(4) and Si, and we demonstrate a 1 × 4 tunable multiplexer/demultiplexer using the Si(3)N(4)-on-SOI dual-level grating couplers and thermally-tuned Si microring resonators.

Polarization rotator-splitters and controllers in a Si3N4-on-SOI integrated photonics platform.

We demonstrate novel polarization management devices in a custom-designed silicon nitride (Si(3)N(4)) on silicon-on-insulator (SOI) integrated photonics platform. In the platform, Si(3)N(4) waveguides are defined atop silicon waveguides. A broadband polarization rotator-splitter using a TM0-TE1 mode converter in a composite Si(3)N(4)-silicon waveguide is demonstrated. The polarization crosstalk, insertion loss, and polarization dependent loss are less than -19 dB, 1.5 dB, and 1.0 dB, respectively, over a bandwidth of 80 nm. A polarization controller composed of polarization rotator-splitters, multimode interference couplers, and thin film heaters is also demonstrated.

Polarization rotator-splitters in standard active silicon photonics platforms.

We demonstrate various silicon-on-insulator polarization management structures based on a polarization rotator-splitter that uses a bi-level taper TM0-TE1 mode converter. The designs are fully compatible with standard active silicon photonics platforms with no new levels required and were implemented in the IME baseline and IME-OpSIS silicon photonics processes. We demonstrate a polarization rotator-splitter with polarization crosstalk < -13 dB over a bandwidth of 50 nm. Then, we improve the crosstalk to < -22 dB over a bandwidth of 80 nm by integrating the polarization rotator-splitter with directional coupler polarization filters. Finally, we demonstrate a polarization controller by integrating the polarization rotator-splitters with directional couplers, thermal tuners, and PIN diode phase shifters.

Gender differences in health-related quality of life among cannabis users: results from the National Epidemiologic Survey on Alcohol and Related Conditions.

BACKGROUND: Cannabis is the most widely used illicit substance worldwide. The aim of the present study was to assess self-reported Quality of Life (QoL) among cannabis users in a large representative sample. METHODS: We analyzed data from the National Epidemiological Survey of Alcohol and Related Conditions (NESARC, n=43,093). Health-related QoL was assessed using the Short-form 12-item Health Survey (SF-12). The contribution of cannabis use and cannabis use disorders (CUD) to SF-12 scores was assessed using multiple linear regressions models. RESULTS: The prevalence of cannabis use and CUD in the last 12 months was 4.1% and 1.5%, respectively. Mean SF-12 mental summary scores were significantly lower (indicating a lower QoL) among female and male cannabis users compared to non-users (by 0.6 standard deviations (SD) and 0.3 SD, respectively), and among females and males with CUD compared to those without CUD (by 0.9 SD and 0.4 SD, respectively). Controlling for sociodemographic variables and mental illness, each joint smoked daily was associated with a greater decrease in mental QoL summary scores in females (0.1 SD) compared to males (0.03 SD). CONCLUSIONS: Cannabis use and CUD were associated with lower self-reported mental QoL. Specifically, our findings showed that cannabis use and CUD have a more significant effect on self-reported mental health QoL among female users. Assessing severity of cannabis use and impact of CUD should take into account functional and emotional outcomes. This may particularly aid in detecting the impact of cannabis use and CUD on mental health-related QoL among females.

Combining best evidence: a novel method to calculate the alcohol-attributable fraction and its variance for injury mortality.

BACKGROUND: The alcohol-attributable fraction for injury mortality is defined as the proportion of fatal injury that would disappear if consumption went to zero. Estimating this fraction has previously been based on a simplistic view of drinking and associated risk. This paper develops a new way to calculate the alcohol-attributable fraction for injury based on different dimensions of drinking, mortality data, experimental data, survey research, new risk scenarios, and by incorporating different distributions of consumption within populations. For this analysis, the Canadian population in 2005 was used as the reference population. METHODS: Binge drinking and average daily consumption were modeled separately with respect to the calculation of the AAF. The acute consumption risk was calculated with a probability-based method that accounted for both the number of binge drinking occasions and the amount of alcohol consumed per occasion. The average daily consumption was computed based on the prevalence of daily drinking at various levels. These were both combined to get an overall estimate. 3 sensitivity analyses were performed using different alcohol consumption parameters to test the robustness of the model. Calculation of the variance to generate confidence limits around the point estimates was accomplished via Monte Carlo resampling methods on randomly generated AAFs that were based on the distribution and prevalence of drinking in the Canadian population. RESULTS: Overall, the AAFs decrease with age and are significantly lower for women than men across all ages. As binge drinking increases, the injury mortality AAF also increases. Motor vehicle collisions show the largest relative increases in AAF as alcohol consumption is increased, with over a 100% increase in AAF from the lowest to highest consumption category. Among non-motor vehicle collisions, the largest change in total AAF occurred both for homicide and other intentional injuries at about a 15% increase in the AAF from the lowest to the highest binge consumption scenarios. CONCLUSIONS: This method combines the best available evidence to generate new alcohol-attributable fractions for alcohol-attributable injury mortality. Future research is needed to refine the risk function for non-motor vehicle injury types and to investigate potential interactions between binge drinking and average volume of alcohol consumption.