Development of a genetic sensor that eliminates p53 deficient cells.

The TP53 gene fulfills a central role in protecting cells from genetic insult. Given this crucial role it might be surprising that p53 itself is not essential for cell survival. Indeed, TP53 is the single most mutated gene across different cancer types. Thus, both a theoretical and a question of significant practical applicability arise: can cells be programmed to make TP53 an essential gene? Here we present a genetic p53 sensor, in which the loss of p53 is coupled to the rise of HSV-TK expression. We show that the sensor can distinguish both p53 knockout and cells expressing a common TP53 cancer mutation from otherwise isogenic TP53 wild-type cells. Importantly, the system is sensitive enough to specifically target TP53 loss-of-function cells with the HSV-TK pro-drug Ganciclovir both in vitro and in vivo. Our work opens new ways to programming cell intrinsic transformation protection systems that rely on endogenous components

Purified Cas9 Fusion Proteins for Advanced Genome Manipulation.

The CRISPR/Cas9 system (CRISPR = clustered regularly interspaced short palindromic repeats) has rapidly become one of the most versatile genome manipulation technologies, and different methods to introduce the Cas9 nuclease activity into cells have been developed. The direct delivery of purified Cas9 protein complexed with a guide RNA as a ribonucleoprotein (RNP) has emerged as an advantageous approach, as it provides instant, but limited activity of the enzyme, thereby reducing off-target cleavage. The usefulness of the CRISPR/Cas9 system has recently been extended by the generation of Cas9 or dead (d) Cas9 fusion genes. However, these systems have so far been mainly explored when delivered by expression plasmids. Here, a variety of purified Cas9 fusion proteins are generated, and their utility is tested in a number of assays. This work illustrates that Cas9 fused to green-or redfluorescent proteins can be usefully employed to increase the frequency of targeted cells when transfected as RNPs. Furthermore, it is demonstrated that purified dCas9 fused to a dual transactivation domain can potently activate gene expression when transfected as an RNP into embryonic stem cells. The results show that purified Cas9 fusion proteins are versatile and efficient reagents that facilitate advanced genome manipulation

Dependence of saturation effects on electron confinement and injector doping in GaAs/Al0.45Ga0.55As quantum-cascade lasers

We report on a detailed experimental and theoretical analysis of the role of injector doping density on both the threshold current density (J(th)) and the saturation current density (J(sat)), determining the dynamic range of the quantum cascade lasers. The experimental results were obtained from two growth series of lambda approximate to 9 mu m GaAs/Al0.45Ga0.55As quantum-cascade lasers based on single and double phonon resonance depopulation mechanisms. We derive a quasilinear dependence of J(th) on the injector doping density of both designs for doping range as wide as (4-10)x10(11) cm(-2). Despite threshold current increase the faster rise of saturation current with doping results in an enhanced dynamic range for injector doping up to similar to 8x10(11) cm(-2). For higher doping levels, J(sat) itself saturates. Furthermore, our investigations yield that single phonon resonance devices exhibit clear current saturation simultaneously with a decrease of the optical power, whereas two phonon resonance devices show only power saturation, which we attribute to increased leakage currents. These deteriorate the laser performance of the latter design at higher operational temperatures. (c) 2006 American Institute of Physics.</p

Inactivation of Cancer Mutations Utilizing CRISPR/Cas9.

Although whole-genome sequencing has uncovered a large number of mutations that drive tumorigenesis, functional ratification for most mutations remains sparse. Here, we present an approach to test functional relevance of tumor mutations employing CRISPR/Cas9. Combining comprehensive sgRNA design and an efficient reporter assay to nominate efficient and selective sgRNAs, we establish a pipeline to dissect roles of cancer mutations with potential applicability to personalized medicine and future therapeutic use

lncRNA Panct1 Maintains Mouse Embryonic Stem Cell Identity by Regulating TOBF1 Recruitment to Oct-Sox Sequences in Early G1.

Long noncoding RNAs (lncRNAs) have been implicated in diverse biological processes, including embryonic stem cell (ESC) maintenance. However, their functional mechanisms remain largely undefined. Here, we show that the lncRNA Panct1 regulates the transient recruitment of a putative X-chromosome-encoded protein A830080D01Rik, hereafter referred to as transient octamer binding factor 1 (TOBF1), to genomic sites resembling the canonical Oct-Sox motif. TOBF1 physically interacts with Panct1 and exhibits a cell-cycle-specific punctate localization in ESCs. At the chromatin level, this correlates with its recruitment to promoters of pluripotency genes. Strikingly, mutating an octamer-like motif in Panct1 RNA abrogates the strength of TOBF1 localization and recruitment to its targets. Taken together, our data reveal a tightly controlled spatial and temporal pattern of lncRNA-mediated gene regulation in a cell-cycle-dependent manner and suggest that lncRNAs might function as barcodes for identifying genomic addresses for maintaining cellular states

CRISPR/Cas9 screen in gastric cancer patient-derived organoids reveals KDM1A-NDRG1 axis as a targetable vulnerability

Viability CRISPR screens have proven indispensable in parsing genome function. However, their application in new, more physiologically relevant culturing systems like patient-derived organoids (PDOs) has been much slower. To probe epigenetic contribution to gastric cancer (GC), the third leading cause of cancer-related deaths worldwide, the first negative selection CRISPR screen in GC PDOs that faithfully preserve primary tumor characteristics is performed. Extensive quality control measurements showing feasibility of CRISPR screens in primary organoid culture are provided. The screen reveals the histone lysine demethylase-1A (KDM1A) to constitute a GC vulnerability. Both genetic and pharmacological inhibition of KDM1A cause organoid growth retardation. Further, it is shown that most of KDM1A cancer-supporting functions center on repression of N-myc downstream regulates gene-1 (NDRG1). De-repression of NDRG1 by KDM1A inhibitors (KDM1Ai) causes inhibition of Wnt signaling and a strong G1 cell cycle arrest. Finally, by profiling 20 GC PDOs, it is shown that NDRG1 upregulation predicts KDM1Ai response with 100% sensitivity and 82% specificity in the tested cohort. Thus, this work pioneers the use of negative selection CRISPR screens in patient-derived organoids, identifies a marker of KDM1Ai response, and accordingly a cohort of patients who may benefit from such therapy

Differences between familial and sporadic dilated cardiomyopathy: ESC EORP Cardiomyopathy & Myocarditis registry

Aims: Dilated cardiomyopathy (DCM) is a complex disease where genetics interplay with extrinsic factors. This study aims to compare the phenotype, management, and outcome of familial DCM (FDCM) and non-familial (sporadic) DCM (SDCM) across Europe. Methods and results: Patients with DCM that were enrolled in the prospective ESC EORP Cardiomyopathy & Myocarditis Registry were included. Baseline characteristics, genetic testing, genetic yield, and outcome were analysed comparing FDCM and SDCM; 1260 adult patients were studied (238 FDCM, 707 SDCM, and 315 not disclosed). Patients with FDCM were younger (P\ua0<\ua00.01), had less severe disease phenotype at presentation (P\ua0<\ua00.02), more favourable baseline cardiovascular risk profiles (P\ua0 64\ua00.007), and less medication use (P\ua0 64\ua00.042). Outcome at 1\ua0year was similar and predicted by NYHA class (HR 0.45; 95% CI [0.25\u20130.81]) and LVEF per % decrease (HR 1.05; 95% CI [1.02\u20131.08]. Throughout Europe, patients with FDCM received more genetic testing (47% vs. 8%, P\ua0<\ua00.01) and had higher genetic yield (55% vs. 22%, P\ua0<\ua00.01). Conclusions: We observed that FDCM and SDCM have significant differences at baseline but similar short-term prognosis. Whether modification of associated cardiovascular risk factors provide opportunities for treatment remains to be investigated. Our results also show a prevalent role of genetics in FDCM and a non-marginal yield in SDCM although genetic testing is largely neglected in SDCM. Limited genetic testing and heterogeneity in panels provides a scaffold for improvement of guideline adherence