Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea
. Target interference relies on a multi-subunit, RNA-guided complex called Cascade
, which recruits a ...trans-acting helicase-nuclease, Cas3, for target degradation
. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain
and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.
The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they ...exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2′-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.
Display omitted
•chRDNAs improve Cas9 specificity while preserving on-target editing efficiency•2′-Deoxynucleotide positioning affects guide specificity in a target-dependent manner•chRDNAs cause distorted guide-target DNA duplex geometry and R-loop destabilization•chRDNAs slow Cas9 cleavage rates and promote dissociation of off-target substrates
Cas9 off-target activity remains a concern for therapeutics. Donohoue, Pacesa, et al. demonstrate that selective 2′-deoxynucleotide modifications of the Cas9 guide reduce off-target activity in primary T cells. These CRISPR hybrid RNA-DNAs (chRDNAs) increase specificity by distorting the guide-substrate duplex, which disfavors the binding and cleavage of off-target substrates.
Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the ...development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.
We used diffusion tensor imaging to investigate fractional anisotropy (FA), a measure of fiber tract integrity, in attention-deficit hyperactivity disorder (ADHD). Using a tract-based atlasing ...approach on six-direction diffusion tensor imaging data, we examined FA within the cingulum, corpus callosum, corticospinal tract, fornix, optic radiations, superior longitudinal fasciculus, uncinate fasciculus, and the superior and inferior occipitofrontal fasciculi in an all-male sample of 17 children and adolescents with ADHD and 16 age-matched controls. ADHD patients had significantly lower FA in the corticospinal tract (P=0.02) and the superior longitudinal fasciculus (P=0.017) compared with controls. Results support that disruptions in motor and attentional networks may contribute toward ADHD pathophysiology. Future research may clarify how ADHD subtype and psychiatric comorbidities affect diffusion measures.
Multiple sclerosis (MS) is the most common causes of non-traumatic disability in young adults worldwide. MS pathophysiologies include the formation of inflammatory lesions, axonal damage and ...demyelination, and blood brain barrier (BBB) disruption. Coagulation proteins, including factor (F)XII, can serve as important mediators of the adaptive immune response during neuroinflammation. Indeed, plasma FXII levels are increased during relapse in relapsing-remitting MS patients, and previous studies showed that reducing FXII levels was protective in a murine model of MS, experimental autoimmune encephalomyelitis (EAE). Our objective was to determine if pharmacological targeting of FXI, a major substrate of activated FXII (FXIIa), improves neurological function and attenuates CNS damage in the setting of EAE. EAE was induced in male mice using murine myelin oligodendrocyte glycoprotein peptides combined with heat-inactivated
Mycobacterium tuberculosis
and pertussis toxin. Upon onset of symptoms, mice were treated every other day intravenously with anti-FXI antibody, 14E11, or saline. Disease scores were recorded daily until euthanasia for ex vivo analyses of inflammation. Compared to the vehicle control, 14E11 treatment reduced the clinical severity of EAE and total mononuclear cells, including CD11b
+
CD45
high
macrophage/microglia and CD4
+
T cell numbers in brain. Following pharmacological targeting of FXI, BBB disruption was reduced, as measured by decreased axonal damage and fibrin(ogen) accumulation in the spinal cord. These data demonstrate that pharmacological inhibition of FXI reduces disease severity, immune cell migration, axonal damage, and BBB disruption in mice with EAE. Thus, therapeutic agents targeting FXI and FXII may provide a useful approach for treating autoimmune and neurologic disorders.
Agricultural expansion is a primary driver of biodiversity decline in forested regions of the tropics. Consequently, it is important to understand the conservation value of remnant forests in ...production landscapes. In a tropical landscape dominated by oil palm (Elaeis guineensis), we characterized faunal communities across eight taxa occurring within riparian forest buffers, which are legally protected alongside rivers, and compared them to nearby recovering logged forest. Buffer width was the main predictor of species richness and abundance, with widths of 40–100 m on each side of the river supporting broadly equivalent levels of biodiversity as compared to logged forest. However, width responses varied markedly among taxa, and buffers often lacked forest‐dependent species. Much wider buffers than are currently mandated are needed to safeguard most species. The largest biodiversity gains are achieved by increasing relatively narrow buffers. To provide optimal conservation outcomes in tropical production landscapes, we encourage policy makers to prescribe width requirements for key taxa and different landscape contexts.
The evidence regarding the influence of sedentary behavior (SB) on brain health – namely cognitive performance, dementia risk, and brain-related biological markers – is mixed, and most studies focus ...on middle-aged and older adults.Type of SB (e.g., cognitively active SB vs cognitively passive SB) appears to be differentially linked to brain health, but further studies considering dose-response relationships and the context are needed to verify such a relationship.Evidence is needed on how interrupting prolonged sitting with different types of physically active breaks may mitigate potential adverse impacts of SB on specific aspects of brain health.Future studies should utilize innovative approaches to capture SB within the 24-h activity cycle in ecologically valid free-living settings, assess the characteristics and context of SB, and consider different levels of analysis, to reach robust conclusions on relationships between SB and brain health.
Higher levels of physical activity are known to benefit aspects of brain health across the lifespan. However, the role of sedentary behavior (SB) is less well understood. In this review we summarize and discuss evidence on the role of SB on brain health (including cognitive performance, structural or functional brain measures, and dementia risk) for different age groups, critically compare assessment approaches to capture SB, and offer insights into emerging opportunities to assess SB via digital technologies. Across the lifespan, specific characteristics of SB (particularly whether they are cognitively active or cognitively passive) potentially act as moderators influencing the associations between SB and specific brain health outcomes. We outline challenges and opportunities for future research aiming to provide more robust empirical evidence on these observations.
Higher levels of physical activity are known to benefit aspects of brain health across the lifespan. However, the role of sedentary behavior (SB) is less well understood. In this review we summarize and discuss evidence on the role of SB on brain health (including cognitive performance, structural or functional brain measures, and dementia risk) for different age groups, critically compare assessment approaches to capture SB, and offer insights into emerging opportunities to assess SB via digital technologies. Across the lifespan, specific characteristics of SB (particularly whether they are cognitively active or cognitively passive) potentially act as moderators influencing the associations between SB and specific brain health outcomes. We outline challenges and opportunities for future research aiming to provide more robust empirical evidence on these observations.
Objective: This cross-sectional study sought to confirm the presence and regional profile of previously reported changes in laminar cortical thickness in children and adolescents with ...attention-deficit/hyperactivity disorder (ADHD) compared with typically developing control subjects. Method: High-resolution magnetic resonance images were obtained from 22 (19 male and 3 female subjects; mean age 11.7 years) children and adolescents with ADHD and 22 age- and sex-matched control subjects (mean age 11.7 years). Brain tissue volumes were estimated for each subject. Cortical pattern matching methods were used to sample measures of laminar thickness at high spatial frequency across homologous regions of the cortex. Volume and thickness measures were compared across diagnostic groups with and without controlling for general intelligence. False discovery rate correction confirmed regional results. Results: The subjects with ADHD exhibited significant reductions in overall brain volume, gray matter volume, and mean cortical thickness compared with the controls, whereas white matter volumes were significantly increased in ADHD. Highly significant cortical thinning (false discovery rate-corrected p less than 0.0006) was observed over large areas of the frontal, temporal, parietal, and occipital association cortices and aspects of motor cortex but not within the primary sensory regions. Conclusions: Cortical thickness reductions present a robust neuroanatomical marker for child and adolescent ADHD. Observations of widespread cortical thinning expand on earlier cross-sectional findings and provide further evidence to support that the neurobiological underpinnings of ADHD extend beyond prefrontal and subcortical circuits. (Contains 1 table and 2 figures.)
The acquisition of thermally tolerant algal symbionts by corals has been proposed as a natural or assisted mechanism of increasing coral reef resilience to anthropogenic climate change, but the ...cell-level processes determining the performance of new symbiotic associations are poorly understood. We used liquid chromatography-mass spectrometry to investigate the effects of an experimentally induced symbiosis on the host proteome of the model sea anemone Exaiptasia pallida. Aposymbiotic specimens were colonised by either the homologous dinoflagellate symbiont (Breviolum minutum) or a thermally tolerant, ecologically invasive heterologous symbiont (Durusdinium trenchii). Anemones containing D. trenchii exhibited minimal expression of Niemann-Pick C2 proteins, which have predicted biochemical roles in sterol transport and cell recognition, and glutamine synthetases, which are thought to be involved in nitrogen assimilation and recycling between partners. D. trenchii-colonised anemones had higher expression of methionine-synthesising betaine-homocysteine S-methyltransferases and proteins with predicted oxidative stress response functions. Multiple lysosome-associated proteins were less abundant in both symbiotic treatments compared with the aposymbiotic treatment. The differentially abundant proteins are predicted to represent pathways that may be involved in nutrient transport or resource allocation between partners. These results provide targets for specific experiments to elucidate the mechanisms underpinning compensatory physiology in the coral-dinoflagellate symbiosis.
PDF
