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Objective: To examine associations between three clinically significant sleep disorders (chronic insomnia, obstructive sleep apnoea, restless legs syndrome) and workplace productivity losses among young Australian adults. Design, setting: Prospective, observational study; 22‐year follow‐up of participants in the longitudinal birth cohort Raine Study (Perth, Western Australia). Participants: Currently employed 22‐year‐old Raine Study participants who underwent in‐laboratory sleep disorder screening for moderate to severe obstructive sleep apnoea (apnoea–hypopnea index of more than fifteen events/hour or obstructive sleep apnoea syndrome) and were assessed for insomnia and restless legs syndrome using validated measures. Main outcome measures: Total workplace productivity loss over twelve months, assessed with the World Health Organization Health and Work Performance Questionnaire. Results: Of 1235 contactable 22‐year‐old Raine Study cohort members, 554 people (44.9%; 294 women [53%]) underwent overnight polysomnography, completed the baseline sleep questionnaire, and completed at least three quarterly workplace productivity assessments. One or more clinically significant sleep disorders were identified in 120 participants (21.7%); 90 participants had insomnia (17%), thirty clinically significant obstructive sleep apnoea (5.4%), and two restless legs syndrome (0.4%). Seventeen people (14% of those with sleep disorders) had previously been diagnosed with a sleep disturbance by a health professional, including fourteen with insomnia. Median total workplace productivity loss was greater for participants with sleep disorders (164 hours/year; interquartile range [IQR], 0–411 hours/year) than for those without sleep disorders (30 hours/year; IQR, 0–202 hours/year); total workplace productivity loss was 40% greater for participants with sleep disorders (adjusted incidence rate ratio, 1.40; bias‐corrected and accelerated 95% confidence interval, 1.10–1.76). The estimated population total productivity loss (weighted for disorder prevalence) was 28 644 hours per 1000 young workers per year, primarily attributable to insomnia (28 730 hours/1000 workers/year). Conclusion: Insomnia is a risk factor for workplace productivity loss in young workers. Tailored interventions are needed to identify and manage sleep disorders, particularly as most of the sleep disorders detected in the Raine Study had not previously been diagnosed. [ABSTRACT FROM AUTHOR]

Antipodes Island is part of New Zealand’s World Heritage subantarctic region and hosts special biodiversity values and significant species endemism. Invasive house mice were the only introduced mammal and detrimentally impacted invertebrate and native bird communities. Eradication of mice from Antipodes Island was undertaken in 2016 and confirmed in 2018. We present the monitoring used to confirm eradication of mice and the ecological outcomes measured over the 6 years since the eradication. Result monitoring for confirmation applied a simple regime to search for mice following a delay of two mouse breeding seasons since baiting was completed. Outcome monitoring targeted endemic land bird taxa for possible changes due to operational impacts and ecological recovery following eradication of mice. The operation had no longterm negative impacts and endemic land bird taxa have recovered quickly from variable levels of non-target mortality. Estimates of abundance of Antipodes Island snipe, Antipodes Island pipit and Reischek’s parakeet showed strong long-term positive response to mouse eradication. [ABSTRACT FROM AUTHOR]

IntroductionDebriefings give healthcare workers voice through the opportunity to discuss unanticipated or difficult events and recommend changes. The typical goal of routine debriefings has been to improve clinical outcomes by learning through discussion and reflection of events and then transferring that learning into clinical practice. However, little research has investigated the effects of debriefings on the emotional experiences and well-being of healthcare workers. There is some evidence that debriefings are a multi-faceted and cost-effective intervention for minimising negative health outcomes, but their use is inconsistent and they are infrequently adopted with the specific intention of giving healthcare workers a voice. The purpose of this systematic scoping review is therefore to assess the scope of existing evidence on debriefing practices for the well-being and emotional outcomes of healthcare workers.MethodsFollowing screening, 184 papers were synthesised through keyword mapping and exploratory trend identification.ResultsThe body of evidence reviewed were clustered geographically, but diverse on many other criteria of interest including the types of evidence produced, debriefing models and practices, and outcomes captured.DiscussionThe current review provides a clear map of our existing understanding and highlights the need for more systematic, collaborative and rigorous bodies of evidence to determine the potential of debriefing to support the emotional outcomes of those working within healthcare.Systematic Review Registrationhttps://osf.io/za6rj.

On 8 May 2018 at 23:15 h physical contact was observed between an adult carnivorous land snail (Powelliphanta hochstetteri obscura) and an adult Maud Island frog (Leiopelma pakeka) on Te Pākeka/Maud Island, Marlborough Sounds, New Zealand. The land snail (40-45 mm diameter) was on its back with its foot under the frog (38.5 mm snout-vent length), raising the possibility that this was a predation attempt by the snail. Carnivorous endemic land snails are only known to consume a range of invertebrate species, especially earthworms. Our observation suggests that these snails might also opportunistically attempt to consume small ground vertebrates, although definitive evidence is required. [ABSTRACT FROM AUTHOR]

Plasma cells (PCs) in bone marrow (BM) play an important role in both protective and pathogenic humoral immune responses, e.g. in various malignant and non-malignant diseases such as multiple myeloma, primary and secondary immunodeficiencies and autoimmune diseases. Dedicated microenvironmental niches in the BM provide PCs with biomechanical and soluble factors that support their long-term survival. There is a high need for appropriate and robust model systems to better understand PCs biology, to develop new therapeutic strategies for PCs-related diseases and perform targeted preclinical studies with high predictive value. Most preclinical data have been derived from in vivo studies in mice, as in vitro studies of human PCs are limited due to restricted survival and functionality in conventional 2D cultures that do not reflect the unique niche architecture of the BM. We have developed a microphysiological, dynamic 3D BM culture system (BM-MPS) based on human primary tissue (femoral biopsies), mechanically supported by a hydrogel scaffold casing. While a bioinert agarose casing did not support PCs survival, a photo-crosslinked collagen-hyaluronic acid (Col-HA) hydrogel preserved the native BM niche architecture and allowed PCs survival in vitro for up to 2 weeks. Further, the Col-HA hydrogel was permissive to lymphocyte migration into the microphysiological system´s circulation. Long-term PCs survival was related to the stable presence in the culture of soluble factors, as APRIL, BAFF, and IL-6. Increasing immunoglobulins concentrations in the medium confirm their functionality over culture time. To the best of our knowledge, this study is the first report of successful long-term maintenance of primary-derived non-malignant PCs in vitro . Our innovative model system is suitable for in-depth in vitro studies of human PCs regulation and exploration of targeted therapeutic approaches such as CAR-T cell therapy or biologics., (Creative Commons Attribution license.)

Abstract: Chimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in nonphysiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR expression and redirection of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T cells exhibited comparable leukemia control to TCRα chain constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Agroforestry is a management strategy for mitigating the negative impacts of climate and adapting to sustainable farming systems. The successful implementation of agroforestry strategies requires that climate risks are appropriately assessed. The spatial scale, a critical determinant influencing climate impact assessments and, subsequently, agroforestry strategies, has been an overlooked dimension in the literature. In this study, climate risk impacts on robusta coffee production were investigated at different spatial scales in coffee-based agroforestry systems across India. Data from 314 coffee farms distributed across the districts of Chikmagalur and Coorg (Karnataka state) and Wayanad (Kerala state) were collected during the 2015/2016 to 2017/2018 coffee seasons and were used to quantify the key climate drivers of coffee yield. Projected climate data for two scenarios of change in global climate corresponding to (1) current baseline conditions (1985-2015) and (2) global mean temperatures 2 °C above preindustrial levels were then used to assess impacts on robusta coffee yield. Results indicated that at the district scale rainfall variability predominantly constrained coffee productivity, while at a broader regional scale, maximum temperature was the most important factor. Under a 2 °C global warming scenario relative to the baseline (1985-2015) climatic conditions, the changes in coffee yield exhibited spatial-scale dependent disparities. Whilst modest increases in yield (up to 5%) were projected from district-scale models, at the regional scale, reductions in coffee yield by 10-20% on average were found. These divergent impacts of climate risks underscore the imperative for coffee-based agroforestry systems to develop strategies that operate effectively at various scales to ensure better resilience to the changing climate., (© 2024. The Author(s).)

Quantum algorithms provide an exponential speedup for solving certain classes of linear systems, including those that model geologic fracture flow. However, this revolutionary gain in efficiency does not come without difficulty. Quantum algorithms require that problems satisfy not only algorithm-specific constraints, but also application-specific ones. Otherwise, the quantum advantage carefully attained through algorithmic ingenuity can be entirely negated. Previous work addressing quantum algorithms for geologic fracture flow has illustrated core algorithmic approaches while incrementally removing assumptions. This work addresses two further requirements for solving geologic fracture flow systems with quantum algorithms: efficient system state preparation and efficient information extraction. Our approach to addressing each is consistent with an overall exponential speed-up., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Environmental pathogen reservoirs exist for many globally important diseases and can fuel epidemics, influence pathogen evolution, and increase the threat of host extinction. Species composition can be an important factor that shapes reservoir dynamics and ultimately determines the outcome of a disease outbreak. However, disease-induced mortality can change species communities, indicating that species responsible for environmental reservoir maintenance may change over time. Here we examine the reservoir dynamics of Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome in bats. We quantified changes in pathogen shedding, infection prevalence and intensity, host abundance, and the subsequent propagule pressure imposed by each species over time. We find that highly shedding species are important during pathogen invasion, but contribute less over time to environmental contamination as they also suffer the greatest declines. Less infected species remain more abundant, resulting in equivalent or higher propagule pressure. More broadly, we demonstrate that high infection intensity and subsequent mortality during disease progression can reduce the contributions of high-shedding species to long-term pathogen maintenance., (© 2023 The Authors. Ecology published by Wiley Periodicals LLC on behalf of The Ecological Society of America.)

Graft-versus-host disease (GVHD) is a major risk of the administration of allogeneic chimeric antigen receptor (CAR)-redirected T cells to patients who are HLA unmatched. Gene editing can be used to disrupt potentially alloreactive T-cell receptors (TCRs) in CAR T cells and reduce the risk of GVHD. Despite the high knockout rates achieved with the optimized methods, a subsequent purification step is necessary to obtain a safe allogeneic product. To date, magnetic cell separation (MACS) has been the gold standard for purifying TCRα/β- CAR T cells, but product purity can still be insufficient to prevent GVHD. We developed a novel and highly efficient approach to eliminate residual TCR/CD3+ T cells after TCRα constant (TRAC) gene editing by adding a genetically modified CD3-specific CAR NK-92 cell line during ex vivo expansion. Two consecutive cocultures with irradiated, short-lived, CAR NK-92 cells allowed for the production of TCR- CAR T cells with <0.01% TCR+ T cells, marking a 45-fold reduction of TCR+ cells compared with MACS purification. Through an NK-92 cell-mediated feeder effect and circumventing MACS-associated cell loss, our approach increased the total TCR- CAR T-cell yield approximately threefold while retaining cytotoxic activity and a favorable T-cell phenotype. Scaling in a semiclosed G-Rex bioreactor device provides a proof-of-principle for large-batch manufacturing, allowing for an improved cost-per-dose ratio. Overall, this cell-mediated purification method has the potential to advance the production process of safe off-the-shelf CAR T cells for clinical applications., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out or targeted transgene knock-in. However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells., Results: Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted knock-in and Cas9-derived base editing technology for DSB free knock-outs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two knock-outs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduces translocations to 1.4% of edited cells. Small insertions and deletions at the base editing target sites indicate guide RNA exchange between the editors. This is overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR knock-in and a Cas9-derived base editor enables the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting TCR- and MHC-negative CAR T cells resist allogeneic T cell targeting in vitro., Conclusions: We outline a solution for non-viral CAR gene transfer and efficient gene silencing using different CRISPR enzymes for knock-in and base editing to prevent translocations. This single-step procedure may enable safer multiplex-edited cell products and demonstrates a path towards off-the-shelf CAR therapeutics., (© 2023. The Author(s).)

The use of spatially referenced data in agricultural systems modelling has grown in recent decades, however, the use of spatial modelling techniques in agricultural science is limited. In this paper, we test an effective and efficient technique for spatially modelling and analysing agricultural data using Bayesian hierarchical spatial models (BHSM). These models utilise analytical approximations and numerical integration called Integrated Nested Laplace Approximations (INLA). We critically analyse and compare the performance of the INLA and INLA-SPDE (Integrated Nested Laplace Approximation with Stochastic Partial Differential Equation) approaches against the more commonly used generalised linear model (glm), by modelling binary geostatistical species presence/absence data for several agro-ecologically significant Australian grassland species. The INLA-SPDE approach showed excellent predictive performance (ROCAUC 0.9271-0.9623) for all species. Further, the glm approach not accounting for spatial autocorrelation had inconsistent parameter estimates (switching between significantly positive and negative) when the dataset was subsetted and modelled at different scales. In contrast, the INLA-SPDE approach which accounted for spatial autocorrelation had stable parameter estimates. Using approaches which explicitly account for spatial autocorrelation, such as INLA-SPDE, improves model predictive performance and may provide a significant advantage for researchers by reducing the potential for Type I or false-positive errors in inferences about the significance of predictors., (© 2023. The Author(s).)

Introduction: The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8 + T cell exhaustion and dysregulates CD4 + T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen., Methods: We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain ( TRAC ) locus., Results: Applying upscaled methods with the ExPERT ATx ® MaxCyte system, KI efficacy was ~20% of the total ~2 × 10 8 TCR-knocked-out (KO) generated cells. KO TCR KI CAR-T cells were co-cultured in vitro with the gp350 + CD19 + BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro . CD8 + KI CAR-T cells showed higher persistency than CD4 + KI CAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 10 5 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19 KI CAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4 + CD19 KI CAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (T regs ) and TIM-3 + CD4 + T cells. Administration of gp350 KI CAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8 + PD-1 + LAG-3 + gp350 KI CAR-T cells were predominant in the bone marrow., Discussion: The two types of KO TCR KI CAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation., Competing Interests: Author AR-M is employed by MaxCyte Inc., MD, USA. Author RS has filed a patent application for generation of CAR-T cells targeting lytic herpes infections and is a founding shareholder and scientific consultant of BioSyngen/Zelltechs Lpt Ltd. Author DW has filed multiple patent applications on CRISPR-Cas gene editing and adoptive T cell therapy. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Braun, Pruene, Darguzyte, vom Stein, Nguyen, Wagner, Kath, Roig-Merino, Heuser, Riehm, Schneider, Awerkiew, Talbot, Bleich, Figueiredo, Bornhäuser and Stripecke.)

Purpose: Besides their developmental and neurological phenotype, most patients with MECP2/IRAK1 duplication syndrome present with recurrent and severe infections, accompanied by strong inflammation. Respiratory infections are the most common cause of death. Standardized pneumological diagnostics, targeted anti-infectious treatment, and knowledge of the underlying pathomechanism that triggers strong inflammation are unmet clinical needs. We investigated the influence of IRAK1 overexpression on the canonical NF-κB signaling as a possible cause for excessive inflammation in these patients., Methods: NF-κB signaling was examined by measuring the production of proinflammatory cytokines and evaluating the IRAK1 phosphorylation and degradation as well as the IκBα degradation upon stimulation with IL-1β and TLR agonists in SV40-immortalized fibroblasts, PBMCs, and whole blood of 9 patients with MECP2/IRAK1 duplication syndrome, respectively., Results: Both, MECP2/IRAK1-duplicated patients and healthy controls, showed similar production of IL-6 and IL-8 upon activation with IL-1β and TLR2/6 agonists in immortalized fibroblasts. In PBMCs and whole blood, both patients and controls had a similar response of cytokine production after stimulation with IL-1β and TLR4/2/6 agonists. Patients and controls had equivalent patterns of IRAK1 phosphorylation and degradation as well as IκBα degradation upon stimulation with IL-1β., Conclusion: Patients with MECP2/IRAK1 duplication syndrome do not show increased canonical NF-κB signaling in immortalized fibroblasts, PBMCs, and whole blood. Therefore, we assume that these patients do not benefit from a therapeutic suppression of this pathway., (© 2022. The Author(s).)

A shift towards earlier flowering is a widely noted consequence of climate change for the world's plants. However, whether early flowering changes the way in which plants respond to climate stress, and in turn plant yield, remains largely unexplored. Using 10 years of flowering time and yield observations (Total N = 5580) from 558 robusta coffee (Coffea canephora) farms across Vietnam we used structural equation modelling (SEM) to examine the drivers of flowering day anomalies and the consequent effects of this on coffee climate stress sensitivity and management responses (i.e. irrigation and fertilization). SEM allowed us to model the cascading and interacting effects of differences in flowering time, growing season length and climate stress. Warm nights were the main driver of early flowering (i.e. flowering day anomalies <0), which in turn corresponded to longer growing seasons. Early flowering was linked to greater sensitivity of yield to temperature during flowering (i.e. early in the season). In contrast, when late flowering occurred yield was most sensitive to temperature and rainfall later in the growing season, after flowering and fruit development. The positive effects of tree age and fertilizer on yield, apparent under late flowering conditions, were absent when flowering occurred early. Late flowering models predicted yields under early flowering conditions poorly (a 50 % reduction in cross-validated R 2 of 0.54 to 0.27). Likewise, models based on early flowering were unable to predict yields well under late flowering conditions (a 75 % reduction in cross-validated R 2 , from 0.58 to 0.14). Our results show that early flowering changes the sensitivity of coffee production to climate stress and management and in turn our ability to predict yield. Our results indicate that changes in plant phenology need to be taken into account in order to more accurately assess climate risk and management impacts on plant performance and crop yield., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Our understanding of the impact of climate change on global coffee production is largely based on studies focusing on temperature and precipitation, but other climate indicators could trigger critical threshold changes in productivity. Here, using generalized additive models and threshold regression, we investigate temperature, precipitation, soil moisture and vapour pressure deficit (VPD) effects on global Arabica coffee productivity. We show that VPD during fruit development is a key indicator of global coffee productivity, with yield declining rapidly above 0.82 kPa. The risk of exceeding this threshold rises sharply for most countries we assess, if global warming exceeds 2 °C. At 2.9 °C, countries making up 90% of global supply are more likely than not to exceed the VPD threshold. The inclusion of VPD and the identification of thresholds appear critical for understanding climate change impacts on coffee and for the design of adaptation strategies., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant ( TRAC ) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome-editing method for efficient CAR insertion into the TRAC locus of primary human T cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knockin rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin rates and CAR T cell yield. Resulting TRAC -replaced CD19-CAR T cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair-modulating small molecules. With TRAC -integrated CAR + T cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T cells., Competing Interests: As part of a collaboration agreement between Charité Universitätsmedizin Berlin and Integrated DNA Technologies (IDT), IDT provided certain reagents (HDR enhancer v.2 and TRAC sgRNA used in some experiments) and performed GUIDE-seq analysis, HDR-enhancing small-molecule screen in Jurkat cells, and targeted sequencing of potential off-target sites. R.T., B.T., M.L.S., G.L.K., and A.M.J. are employees of IDT, which offers reagents for sale similar to some of the compounds described in the manuscript. Products and tools supplied by IDT are for research use only and not intended for diagnostic or therapeutic purposes. Purchaser and/or user are solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. Lonza GmbH provided 96-well 4D-Nucleofector unit and some nucleofection reagents. A.W. and L. Akyüz are part-time employees of CheckImmune GmbH. A.R. and U.E.H. filed a patent application WO 2017211900A1 “Chimeric antigen receptor and CAR T cells that bind BCMA” related to the work with the BCMA-CAR disclosed in this paper. A.R. and U.E.H. have received research funding from Fate Therapeutics for work unrelated to the data generated in the manuscript., (© 2022 The Author(s).)