While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We ...present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.
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•CRISPRi and CRISPRa provide complementary information for mapping complex pathways•CRISPRi/a expression series (up to ∼1,000-fold) reveal how gene dose controls function•CRISPRi provides strong (typically 90%–99%) knockdown with minimal off-target effects•Genome-scale screens elucidate pathways controlling cholera/diphtheria toxicity
Genome-scale-specific targeting of transcriptional repressors (CRISPRi) and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9 have been applied to growth and toxin-resistance screens, establishing CRISPRi and CRISPRa as powerful tools that provide rich and complementary information.
The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 ...protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.
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•CRISPRi enables robust gene repression and activation in human cells•CRISPRi knockdown is specific with minimal off-target effects in human cells•CRISPRi can effectively repress endogenous genes in human and yeast•dCas9 enables modular and programmable RNA-guided genome regulation in eukaryotes
Catalytically inactive CRISPR can be targeted to specific loci in human and yeast cells to specifically repress and activate transcription. The study demonstrates the potential for adapting CRISPRi for multiple modes of transcriptional control, chromatin modification, and regulatory element mapping in a broad range of eukaryotes.
Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle ...to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.
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•CRISPR scaffold RNAs (scRNAs) encode both target locus and regulatory function•scRNAs function efficiently in mammalian and yeast cells•scRNAs enable transcription programs with simultaneous activation and repression•Combinatorial control of multiple genes enables flexible pathway manipulation
Modular CRISPR RNA scaffolds engineered to encode both guides to a target locus and recruitment of transcriptional regulators allow simultaneous gene activation and repression of multiple different genes in eukaryotic cells, greatly expanding the synthetic biology toolkit.
Acute mechanical circulatory support (MCS) devices are widely used in cardiogenic shock (CS) despite a lack of high-quality clinical evidence to guide their use. Multiple devices exist across a ...spectrum from modest to complete support, and each is associated with unique risks. In this review, we summarize existing data on complications associated with the three most widely used acute MCS platforms: the intra-aortic balloon pump (IABP), Impella systems, and veno-arterial extracorporeal membrane oxygenation (VA-ECMO). We review evidence from available randomized trials and highlight challenges comparing complication rates from case series and comparative observational studies where a lack of granular data precludes appropriate matching of patients by CS severity. We further offer a series of best practices to help shock practitioners minimize the risk of MCS-associated complications and ensure the best possible outcomes for patients.
BACKGROUNDRisk-stratifying patients with cardiogenic shock (CS) is a major unmet need. The recently proposed Society for Cardiovascular Angiography and Interventions (SCAI) staging system for CS ...severity lacks uniform criteria defining each stage. OBJECTIVESThe purpose of this study was to test parameters that define SCAI stages and explore their utility as predictors of in-hospital mortality in CS. METHODSThe CS Working Group registry includes patients from 17 hospitals enrolled between 2016 and 2021 and was used to define clinical profiles for CS. We selected parameters of hypotension and hypoperfusion and treatment intensity, confirmed their association with mortality, then defined formal criteria for each stage and tested the association between both baseline and maximum Stage and mortality. RESULTSOf 3,455 patients, CS was caused by heart failure (52%) or myocardial infarction (32%). Mortality was 35% for the total cohort and higher among patients with myocardial infarction, out-of-hospital cardiac arrest, and treatment with increasing numbers of drugs and devices. Systolic blood pressure, lactate level, alanine transaminase level, and systemic pH were significantly associated with mortality and used to define each stage. Using these criteria, baseline and maximum stages were significantly associated with mortality (n = 1,890). Lower baseline stage was associated with a higher incidence of stage escalation and a shorter duration of time to reach maximum stage. CONCLUSIONSWe report a novel approach to define SCAI stages and identify a significant association between baseline and maximum stage and mortality. This approach may improve clinical application of the staging system and provides new insight into the trajectory of hospitalized CS patients. (Cardiogenic Shock Working Group Registry CSWG; NCT04682483).
Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created ...knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.
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•CRISPRi knockdown of essential genes enables discovery of direct antibiotic targets•An essential gene network reveals functional connections between core processes•A cell morphology screen identifies essential genes intimately tied to cell shape•A majority of essential genes show morphological defects as a terminal phenotype
A systematic analysis of all essential genes in Bacillus subtilis using a CRISPR-based knockdown approach established the essential gene network, identified modes of action for antibiotics, and discerned fundamental underpinnings of growth and morphological characteristics of cells.
Background:
Right ventricular failure (RVF) is associated with increased mortality among patients receiving left ventricular mechanical circulatory support (LV-MCS) for cardiogenic shock and requires ...prompt recognition and management. Increased central venous pressure (CVP) is an indicator of potential RVF.
Objectives:
We studied whether elevated CVP during LV-MCS for acute myocardial infarction complicated by cardiogenic shock is associated with higher mortality.
Methods:
Between January 2014 and June 2019, we analyzed hemodynamic parameters during Impella LV-MCS from 28 centers in the United States participating in the global, prospective catheter-based ventricular assist device (cVAD) study. A total of 132 patients with a documented CVP measurement while on Impella left-sided support for cardiogenic shock were identified.
Results:
CVP was significantly higher among patients who died in the hospital (14.0 vs. 11.7 mmHg,
p
= 0.014), and a CVP >12 identified patients at significantly higher risk for in-hospital mortality (65 vs. 45%,
p
= 0.02). CVP remained significantly associated with in-hospital mortality even after adjustment in a multivariable model (adjusted OR 1.10 95% CI 1.02–1.19 per 1 mmHg increase). LV-MCS suction events were non-significantly more frequent among patients with high vs. low CVP (62.11 vs. 7.14 events,
p
= 0.067).
Conclusion:
CVP is a single, readily accessible hemodynamic parameter which predicts a higher rate of short-term mortality and may identify subclinical RVF in patients receiving LV-MCS for cardiogenic shock.
Trans-valvular micro-axial flow pumps such as Impella are increasingly utilized in patients with cardiogenic shock CS. A number of different Impella devices are now available providing a wide range ...of cardiac output. Among these, the Impella 5.0 and recently introduced Impella 5.5 pumps can provides 5.55 L/min of flow, enabling complete left ventricular support with more favorable hemodynamic effects on myocardial oxygen consumption and left ventricular unloading. These devices require placement of a surgical conduit graft for endovascular delivery, but are increasingly being used in patients with CS due to acutely decompensated heart failure ADHF, acute myocardial infarction AMI and after cardiac surgery as a bridge to transplant or durable ventricular assist device surgery or myocardial recovery.
This review focuses on the device profile and use of the Impella 5.0 and 5.5 systems in patients with CS. Specifically; we reviewed the published literature for Impella 5.0 device to summarize data regarding safety and efficacy.
The Impella 5.0 and 5.5 are trans-valvular micro-axial flow pumps for which the current data suggest excellent safety and efficacy profiles as approaches to provide circulatory support, myocardial unloading, and axillary placement enabling patient mobilization and rehabilitation.
pMCS, Percutaneous mechanical circulatory support devices; CS, Cardiogenic shock; ADHF, Acute decompensated heart failure; AMI, Acute myocardial infarction; LVAD, Left ventricular assist deviceI; ABP, Intra-aortic balloon pump; VA-ECLS, Veno-arterial extracorporeal life support.
Understanding the prognostic impact of right ventricular dysfunction (RVD) in cardiogenic shock (CS) is a key step toward rational diagnostic and treatment algorithms and improved outcomes. Using a ...large multicenter registry, we assessed (1) the association between hemodynamic markers of RVD and in-hospital mortality, (2) the predictive value of invasive hemodynamic assessment incorporating RV evaluation, and (3) the impact of RVD severity on survival in CS.
Inpatients with CS owing to acute myocardial infarction (AMI) or heart failure (HF) between 2016 and 2019 were included. RV parameters (right atrial pressure, right atrial/pulmonary capillary wedge pressure RA/PCWP, pulmonary artery pulsatility index PAPI, and right ventricular stroke work index RVSWI) were assessed between survivors and nonsurvivors, and between etiology and SCAI stage subcohorts. Multivariable logistic regression analysis determined hemodynamic predictors of in-hospital mortality; the resulting models were compared with SCAI staging alone. Nonsurvivors had a significantly higher right atrial pressure and RA/PCWP and lower PAPI and RVSWI than survivors, consistent with more severe RVD. Compared with AMI, patients with HF had a significantly lower RA/PCWP (0.58 vs 0.66, P = .001) and a higher PAPI (2.71 vs 1.78, P < .001) and RVSWI (5.70 g-m/m2 vs 4.66 g-m/m2, P < .001), reflecting relatively preserved RV function. Paradoxically, multiple RVD parameters (PAPI, RVSWI) were associated with mortality in the HF but not the AMI cohort. RVD was more severe with advanced SCAI stage, although its prognostic value was progressively diluted in stages D and E. Multivariable modelling incorporating the RA/PCWP improved the predictive value of SCAI staging (area under the curve AUC 0.78 vs 0.73, P < .001), largely driven by patients with HF (AUC 0.82 vs 0.71, P < .001).
RVD is associated with poor outcomes in CS, with key differences across etiology and shock severity. Further studies are needed to assess the usefulness of RVD assessment in guiding therapy.
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