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Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays. Paralog synthetic lethals have been assessed with multiple CRISPR-based methods, but systematic comparison among these platforms is unavailable. Here, the authors systematically compare combinatorial perturbation platforms and establish the in4mer CRISPR/Cas12a multiplex knockout platform.

The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly-described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly-described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.

Genetic interactions mediate the emergence of phenotype from genotype, but initial technologies for combinatorial genetic perturbation in mammalian cells suffer from inefficiency and are challenging to scale. Recent focus on paralog synthetic lethality in cancer cells offers an opportunity to evaluate different approaches and improve on the state of the art. Here we report a meta-analysis of CRISPR genetic interactions screens, identifying a candidate set of background-independent paralog synthetic lethals, and find that the Cas12a platform provides superior sensitivity and assay replicability. We demonstrate that Cas12a can independently target up to four genes from a single guide array, and we build on this knowledge by constructing a genome-scale library that expresses arrays of four guides per clone, a platform we call 'in4mer'. Our genome-scale human library, with only 49k clones, is substantially smaller than a typical CRISPR/Cas9 monogenic library while also targeting more than four thousand paralog pairs, triples, and quads. Proof of concept screens in four cell lines demonstrate discrimination of core and context-dependent essential genes similar to that of state-of-the-art CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes, a capability not offered by any extant library. Importantly, the in4mer platform offers a fivefold reduction in the number of clones required to assay genetic interactions, dramatically improving the cost and effort required for these studies.

Base editing is a CRISPR-based technology that enables high-throughput, nucleotide-level functional interrogation of the genome, which is essential for understanding the genetic basis of human disease and informing therapeutic development. Base editing screens have emerged as a powerful experimental approach, yet significant cell-to-cell variability in editing efficiency introduces noise that may obscure meaningful results. Here, we develop a co-selection method that enriches for cells with high base editing activity, substantially increasing editing efficiency at a target locus. We evaluate this activity-based selection method against a traditional screening approach by tiling guide RNAs across TP53, demonstrating its enhanced capacity to pinpoint specific mutations and protein regions of functional importance. We anticipate that this modular selection method will enhance the resolution of base editing screens across many applications.Competing Interest StatementJGD consults for Microsoft Research, BioNTech, PhenomicAI, Servier, and Pfizer. JGD consults for and has equity in Tango Therapeutics. JGD serves as a paid scientific advisor to the Laboratory for Genomics Research, funded in part by GSK; and the Innovative Genomics Institute, funded in part by Apple Tree Partners. JGD receives funding support from the Functional Genomics Consortium: Abbvie, Bristol Myers Squibb, Janssen, and Merck. JGD interests are reviewed and managed by the Broad Institute in accordance with its conflict of interest policies. A patent application related to this work has been filed.

Carrying a payload directly on the body is an unavoidable aspect of human life. Human bipedal locomotion knows no equal: people travel on foot to virtually every corner of the globe. Despite the efficiency and convenience of wheeled apparatus, uneven terrain, enclosed environments and accessibility limits require virtually every transportation task to include a phase in which material goods must be physically carried by a person. As of today, no artificial intelligence or programmed behavior has been able to match a human's ability to balance and maneuver in unstructured real-world environments. The Berkeley Lower Extremity Exoskeleton solves the problem of supporting and carrying heavy loads on the body and allows a person to navigate unencumbered by the weight of the payload they are carrying. The Berkeley Lower Extremity Exoskeleton is an anthropomorphic and energetically autonomous robotic device comprised of two legs, a backpack, a harness system and a control computer that provides a wearable load support platform. This thesis presents a control scheme called Sensitivity Amplification Control that enables an exoskeleton to support a payload and shadow the movement of the wearer in an intuitive and unobtrusive manner. The control algorithm developed here increases the closed-loop system sensitivity to its wearer's forces and torques without any measurement from the wearer. This strategy requires an accurate dynamic model of the system but does not require direct measurements from the human. The trade-off between not having sensors to measure human action and the sacrificed robustness due to model parameter variation is described. A modification to the controller is also explored that partially circumvents this limitation.

Positive affect (PA) has consistently been shown to predict meaning in life (MIL). In one of the first investigations to examine multiple predictors of MIL simultaneously, we tested in three studies the hypothesis that satisfactions associated with being benevolent and fulfilling psychological needs for autonomy, competence, and relatedness are more central predictors of MIL, and could explain the correlation between PA and MIL. Study 1, a cross-sectional survey, regressed the four suggested factors and PA simultaneously on MIL, showing that all four emerged as independent predictors, whereas PA and MIL were no longer connected. Study 2 looked at recollections of meaningful situations, showing that all four satisfactions and PA emerged as independent predictors of situational meaning. Study 3 used a diary method to show that daily fluctuations in autonomy, competence, relatedness, beneficence, and PA all simultaneously and independently predicted daily sense of meaning. However, a brief longitudinal study showed that whereas combined satisfaction of autonomy, competence, relatedness, and beneficence at T1 predicted general sense of MIL at T2, PA did not. Together, these studies show that the four satisfactions consistently emerge as independent predictors of both general and short-term meaning, in some situations even accounting for the relation between PA and general MIL.

Drug-associated stimuli (cues) can usurp potent control of behavior in individuals with substance use disorders; and these effects are often attributed to altered dopamine transmission. However, there is much debate over the way in which dopamine signaling changes over the course of chronic drug use. Here, we carried out longitudinal recording and manipulation of cue-evoked dopamine release in the core of the nucleus accumbens across phases of substance use in male rats. We show that, in a subset of individuals that exhibit increased cue reactivity and escalated drug consumption, this signaling undergoes diametrically opposed changes in amplitude, determined by the context in which the cue was presented. Dopamine evoked by non-contingent cue presentation (independent of the animal’s actions) increases over drug use, producing greater cue reactivity; whereas dopamine evoked by contingent cue presentation (dependent on the animal’s actions) decreases over drug use, producing escalation of drug consumption. Therefore, despite being in opposite directions, these dopamine trajectories each promote cardinal features of substance use disorders. Here authors demonstrate how opposing trajectories of dopamine transmission underlie changes in drug seeking and taking over chronic drug use. Increased dopamine to drug cues elevates craving, whereas decreased dopamine produces escalation of drug consumption.

We report new results of Bose-Einstein condensation of polaritons in specially designed microcavities with a very high quality factor, on the order of 106 , giving polariton lifetimes of the order of 100 ps. When the polaritons are created with an incoherent pump, a dissipationless, coherent flow of the polaritons occurs over hundreds of microns, which increases as density increases. At high density, this flow is suddenly stopped, and the gas becomes trapped in a local potential minimum, with strong coherence.

Abstract Meaningfulness of work has been cited as one of many psychological reasons for and benefits of working. Although prior research has theorized that meaningfulness of work is an antecedent of retirement, little empirical research has been conducted to investigate meaningfulness of work in relation to retirement plans and behavior. Using panel data from the HRS collected between 2012–2016, the present study investigates perceptions of meaningfulness of work among N=944 individuals who completed a unidimensional 10-item meaningfulness of work module in the 2014 wave of the HRS (M=4.37, SD=.71). We will summarize descriptive statistics about meaningfulness of work, and how meaningfulness relates to retirement expectations, retirement behavior, and retirement adjustment as well as other antecedents and outcomes of the retirement process. We will discuss the findings in relation to extant retirement research and describe implications for workers, their employers, and society in general.

In career counseling, social justice is typically integrated by helping oppressed groups navigate their way around obstacles of injustice while also working to dislodge the oppressive conditions from society. The authors affirm both of these courses of action while also advocating a third strategy: inviting clients to serve as agents of change by introducing and incorporating prosocial values into career development interventions. This article makes the case that this strategy may enhance client well‐being while simultaneously promoting harmony among organizations and society. Support for this strategy comes from theory, research, and the ethical approaches for working with values in counseling.