An Integrated Genome-wide CRISPRa Approach to Functionalize lncRNAs in Drug Resistance.

Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful against COVID-19 (refs. 1,2). However, because SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here we examined this possibility by in vitro passaging of SARS-CoV-2 in nirmatrelvir using two independent approaches, including one on a large scale. Indeed, highly resistant viruses emerged from both and their sequences showed a multitude of 3CL protease mutations. In the experiment peformed with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Nevertheless, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones showed that these mutations mediated only low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (around 100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next-generation protease inhibitors.

Molecular mechanisms of SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir is a specific antiviral drug that targets the main protease (Mpro) of SARS-CoV-2 and has been approved to treat COVID-191,2. As an RNA virus characterized by high mutation rates, whether SARS-CoV-2 will develop resistance to nirmatrelvir is a question of concern. Our previous studies have shown that several mutational pathways confer resistance to nirmatrelvir, but some result in a loss of viral replicative fitness, which is then compensated for by additional alterations3. The molecular mechanisms for this observed resistance are unknown. Here we combined biochemical and structural methods to demonstrate that alterations at the substrate-binding pocket of Mpro can allow SARS-CoV-2 to develop resistance to nirmatrelvir in two distinct ways. Comprehensive studies of the structures of 14 Mpro mutants in complex with drugs or substrate revealed that alterations at the S1 and S4 subsites substantially decreased the level of inhibitor binding, whereas alterations at the S2 and S4' subsites unexpectedly increased protease activity. Both mechanisms contributed to nirmatrelvir resistance, with the latter compensating for the loss in enzymatic activity of the former, which in turn accounted for the restoration of viral replicative fitness, as observed previously3. Such a profile was also observed for ensitrelvir, another clinically relevant Mpro inhibitor. These results shed light on the mechanisms by which SARS-CoV-2 evolves to develop resistance to the current generation of protease inhibitors and provide the basis for the design of next-generation Mpro inhibitors.

Targeting IDH2R140Q and other neoantigens in acute myeloid leukemia.

ABSTRACT: For patients with high-risk or relapsed/refractory acute myeloid leukemia (AML), allogeneic stem cell transplantation (allo-HSCT) and the graft-versus-leukemia effect mediated by donor T cells, offer the best chance of long-term remission. However, the concurrent transfer of alloreactive T cells can lead to graft-versus-host disease that is associated with transplant-related morbidity and mortality. Furthermore, ∼60% of patients will ultimately relapse after allo-HSCT, thus, underscoring the need for novel therapeutic strategies that are safe and effective. In this study, we explored the feasibility of immunotherapeutically targeting neoantigens, which arise from recurrent nonsynonymous mutations in AML and thus represent attractive targets because they are exclusively present on the tumor. Focusing on 14 recurrent driver mutations across 8 genes found in AML, we investigated their immunogenicity in 23 individuals with diverse HLA profiles. We demonstrate the immunogenicity of AML neoantigens, with 17 of 23 (74%) reactive donors screened mounting a response. The most immunodominant neoantigens were IDH2R140Q (n = 11 of 17 responders), IDH1R132H (n = 7 of 17), and FLT3D835Y (n = 6 of 17). In-depth studies of IDH2R140Q-specific T cells revealed the presence of reactive CD4+ and CD8+ T cells capable of recognizing distinct mutant-specific epitopes restricted to different HLA alleles. These neo-T cells could selectively recognize and kill HLA-matched AML targets endogenously expressing IDH2R140Q both in vitro and in vivo. Overall, our findings support the clinical translation of neoantigen-specific T cells to treat relapsed/refractory AML.

Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks.

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.

Focused ultrasound-mediated brain genome editing.

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.

Population-specific responses to developmental temperature in the arboviral vector Aedes albopictus: Implications for climate change.

The increase of environmental temperature due to current global warming is not only favouring the expansion of the distribution range of many insect species, but it is also changing their phenology. Insect phenology is tightly linked to developmental timing, which is regulated by environmental temperatures. However, the degree to which the effects of developmental temperatures extend across developmental stages and their inter-stage relationships have not been thoroughly quantified in mosquitoes. Here, we used the mosquito Aedes albopictus, which is an aggressive invasive species and an arboviral vector, to study how developmental temperature influences fitness across developmental stages, thermal traits, energy reserves, transcriptome and Wolbachia prevalence in laboratory-reared populations originally collected from either temperate or tropical regions. We show that hatchability, larval and pupal viability and developmental speed are strongly influenced by temperature, and these effects extend to wing length, body mass, longevity and content of water, protein and lipids in adults in a population-specific manner. On the contrary, neither adult thermal preference nor heat resistance significantly change with temperature. Wolbachia density was generally lower in adult mosquitoes reared at 18°C than at other tested temperatures, and transcriptome analysis showed enrichment for functions linked to stress responses (i.e. cuticle proteins and chitin, cytochrome p450 and heat shock proteins) in mosquitoes reared at both 18 and 32°C. Our data showed an overall reduced vector fitness performance when mosquitoes were reared at 32°C, and the absence of isomorphy in the relationship between developmental stages and temperature in the laboratory population deriving from larvae collected in northern Italy. Altogether, these results have important implications for reliable model projections of the invasion potentials of Ae. albopictus and its epidemiological impact.

The Effect of Ultrasound on the Extraction and Functionality of Proteins from Duckweed (Lemna minor).

The inclusion of protein in the regular human diet is important for the prevention of several chronic diseases. In the search for novel alternative protein sources, plant-based proteins are widely explored from a sustainable and ecological point of view. Duckweed (Lemna minor), also known as water lentil, is an aquatic plant with potential applications for human consumption due to its protein content and carbohydrate contents. Among all the conventional and novel protein extraction methods, the utilization of ultrasound has attracted the attention of scientists because of its effects on improving protein extraction and its functionalities. In this work, a Box-Behnken experimental design was proposed to optimize the alkaline extraction of protein from duckweed. In addition, an exploration of the effects of ultrasound on the morphological, structural, and functional properties of the extracted protein was also addressed. The optimal extraction parameters were a pH of 11.5 and an ultrasound amplitude and processing time of 60% and 20 min, respectively. These process conditions doubled the protein content extracted in comparison to the value from the initial duckweed sample. Furthermore, the application of ultrasound during the extraction of protein generated changes in the FTIR spectra, color, and structure of the duckweed protein, which resulted in improvements in its solubility, emulsifying properties, and foaming capacity.

Adaptive and optimized COVID-19 vaccination strategies across geographical regions and age groups.

We evaluate the efficiency of various heuristic strategies for allocating vaccines against COVID-19 and compare them to strategies found using optimal control theory. Our approach is based on a mathematical model which tracks the spread of disease among different age groups and across different geographical regions, and we introduce a method to combine age-specific contact data to geographical movement data. As a case study, we model the epidemic in the population of mainland Finland utilizing mobility data from a major telecom operator. Our approach allows to determine which geographical regions and age groups should be targeted first in order to minimize the number of deaths. In the scenarios that we test, we find that distributing vaccines demographically and in an age-descending order is not optimal for minimizing deaths and the burden of disease. Instead, more lives could be saved by using strategies which emphasize high-incidence regions and distribute vaccines in parallel to multiple age groups. The level of emphasis that high-incidence regions should be given depends on the overall transmission rate in the population. This observation highlights the importance of updating the vaccination strategy when the effective reproduction number changes due to the general contact patterns changing and new virus variants entering.

Measuring the Effectiveness of a Multicomponent Program to Manage Academic Stress through a Resilience to Stress Index.

In this work, we evaluate the effectiveness of a multicomponent program that includes psychoeducation in academic stress, mindfulness training, and biofeedback-assisted mindfulness, while enhancing the Resilience to Stress Index (RSI) of students through the control of autonomic recovery from psychological stress. Participants are university students enrolled in a program of excellence and are granted an academic scholarship. The dataset consists of an intentional sample of 38 undergraduate students with high academic performance, 71% (27) women, 29% (11) men, and 0% (0) non-binary, with an average age of 20 years. The group belongs to the "Leaders of Tomorrow" scholarship program from Tecnológico de Monterrey University, in Mexico. The program is structured in 16 individual sessions during an eight-week period, divided into three phases: pre-test evaluation, training program, and post-test evaluation. During the evaluation test, an assessment of the psychophysiological stress profile is performed while the participants undergo a stress test; it includes simultaneous recording of skin conductance, breathing rate, blood volume pulse, heart rate, and heart rate variability. Based on the pre-test and post-test psychophysiological variables, an RSI is computed under the assumption that changes in physiological signals due to stress can be compared against a calibration stage. The results show that approximately 66% of the participants improved their academic stress management after the multicomponent intervention program. A Welch's t-test showed a difference in mean RSI scores (t = -2.30, p = 0.025) between the pre-test and post-test phases. Our findings show that the multicomponent program promoted positive changes in the RSI and in the management of the psychophysiological responses to academic stress.

A continuous process in mixers-settlers for the removal/recovery of chromium in effluents from the tanning industry.

One of the main environmental issues caused by the tanning industry is given by the high concentration of chromium contained on its effluents. The removal of this pollutant has become a technological challenge. To solve this issue, this work proposes a continuous process based on mixers-settlers for the removal of the chromium present in effluents from the tanning industry. The process involves the use of liquid-liquid extraction systems. The study includes the development of isotherms for the removal and stripping, which are further represented through a mathematical model to determine the number of theoretical extraction stages and other operational variables. The results show that a better extraction is achieved in a system with two theoretical stages using Cyanex 272 as extractant, reaching more than 94% of removal of chromium with an extractant concentration of 0.32 mol/L. For stripping, sulfuric acid is used, obtaining a maximum recovery of 94%.

Accurate analysis of genuine CRISPR editing events with ampliCan.

We present ampliCan, an analysis tool for genome editing that unites highly precise quantification and visualization of genuine genome editing events. ampliCan features nuclease-optimized alignments, filtering of experimental artifacts, event-specific normalization, and off-target read detection and quantifies insertions, deletions, HDR repair, as well as targeted base editing. It is scalable to thousands of amplicon sequencing-based experiments from any genome editing experiment, including CRISPR. It enables automated integration of controls and accounts for biases at every step of the analysis. We benchmarked ampliCan on both real and simulated data sets against other leading tools, demonstrating that it outperformed all in the face of common confounding factors.

Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity.

We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identified compounds with broad 3CLpro-inhibitory activity. We also adapted the assay for use in compound screening and in doing so uncovered additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CLpro inhibitors. We observed strong concordance between data emerging from this assay and those obtained from live-virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. We identified two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested, including 3CLpro enzymes from understudied zoonotic coronaviruses. IMPORTANCE Multiple coronavirus pandemics have occurred over the last 2 decades. This has highlighted a need to be proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus pandemics. We developed and validated a simplified cell-based assay for the identification of chemical inhibitors of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter free, does not require specialized biocontainment, and is optimized for performance in high-throughput screening. By testing reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence similarity, we identified compounds with broad activity against 3CL proteases and uncovered structural insights into features that contribute to their broad activity. Furthermore, we demonstrated that this assay is suitable for identifying chemical inhibitors of proteases from families other than 3CL proteases.

Prioritizing COVID-19 Contact Tracing During a Surge Using Chatbot Technology.

When COVID-19 cases surge, identifying ways to improve the efficiency of contact tracing and prioritize vulnerable communities for isolation and quarantine support services is critical. During a fall 2020 COVID-19 resurgence in San Francisco, California, prioritization of telephone-based case investigation by zip code and using a chatbot to screen for case participants who needed isolation support reduced the number of case participants who would have been assigned for a telephone interview by 31.5% and likely contributed to 87.5% of Latinx case participants being successfully interviewed. (Am J Public Health. 2022;112(1):43-47. https://doi.org/10.2105/AJPH.2021.306563).

Daisy-chain gene drives for the alteration of local populations.

If they are able to spread in wild populations, CRISPR-based gene-drive elements would provide new ways to address ecological problems by altering the traits of wild organisms, but the potential for uncontrolled spread tremendously complicates ethical development and use. Here, we detail a self-exhausting form of CRISPR-based drive system comprising genetic elements arranged in a daisy chain such that each drives the next. "Daisy-drive" systems can locally duplicate any effect achievable by using an equivalent self-propagating drive system, but their capacity to spread is limited by the successive loss of nondriving elements from one end of the chain. Releasing daisy-drive organisms constituting a small fraction of the local wild population can drive a useful genetic element nearly to local fixation for a wide range of fitness parameters without self-propagating spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionarily stable daisy drive as well as self-propagating CRISPR-based gene drive. Especially when combined with threshold dependence, daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.

Contamination assessment and potential sources of heavy metals and other elements in sediments of a basin impacted by 500 years of mining in central Mexico.

Since the middle of the 1500 s, mining has been active in central Mexico. Total estimates for low-grade piles and mine tailing materials in the Guanajuato mining district (GMD) are in the range of 150 million tons, covering an area of 15 to 20 km2. GMD is located in the Guanajuato River sub-basin (GRB), which is part of one of the largest basins in Mexico (Lerma-Santiago). Previous studies on the GRB found unusually high concentrations of heavy metals in mining tailings and sediments. Geochemical and statistical methods were used here to determine the sediment's origin, background values, degree of contamination, and toxicity through different contamination indices. This analysis shows that Cu, Co, As, Sb, and Hg are higher than they are in the upper continental crust (UCC) overbank sediments without human and mining influence, because of the ore deposits and rock weathering in GRB. Geochemistry results in stream sediments show anomalies, where Hg, Cu, Zn, As, and Pb are higher than UCC because those heavy metals and trace elements (HMT) have been influenced by human activities and mineral recovery (smelting, amalgamation, cyanidation). The distribution of high concentrations of HMTs and contamination indices occur in the main channel of the Guanajuato River and downstream of the city of Guanajuato. Statistical analyses (cluster and principal component analysis) reveal relationships between Cr, Ni, Cu, and Pb, which are primarily of natural origin, related to rocks of the upper basin. The middle and lower basins are distinctive in their associations between As, Sb, Zn, Pb, and Hg. Additionally, it is recognized that the origins of Pb, Zn, and Hg are geogenic and anthropogenic. This study demonstrates how crucial it is to understand the geochemistry of various HMT sources, with both natural and anthropogenic contributions (stream sediments and rocks), in order to calculate a more realistic background in a basin with both natural anomalies and anthropogenic contamination. The basin is a regional aquifer recharge area, so the new geochemical data are important for improving basin environmental management.

An enhanced CRISPR repressor for targeted mammalian gene regulation.

The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits.

Precise Cas9 targeting enables genomic mutation prevention.

Here, we present a generalized method of guide RNA "tuning" that enables Cas9 to discriminate between two target sites that differ by a single-nucleotide polymorphism. We employ our methodology to generate an in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.

Rap1 relocalization contributes to the chromatin-mediated gene expression profile and pace of cell senescence.

Cellular senescence is accompanied by dramatic changes in chromatin structure and gene expression. Using Saccharomyces cerevisiae mutants lacking telomerase (tlc1Δ) to model senescence, we found that with critical telomere shortening, the telomere-binding protein Rap1 (repressor activator protein 1) relocalizes to the upstream promoter regions of hundreds of new target genes. The set of new Rap1 targets at senescence (NRTS) is preferentially activated at senescence, and experimental manipulations of Rap1 levels indicate that it contributes directly to NRTS activation. A notable subset of NRTS includes the core histone-encoding genes; we found that Rap1 contributes to their repression and that histone protein levels decline at senescence. Rap1 and histones also display a target site-specific antagonism that leads to diminished nucleosome occupancy at the promoters of up-regulated NRTS. This antagonism apparently impacts the rate of senescence because underexpression of Rap1 or overexpression of the core histones delays senescence. Rap1 relocalization is not a simple consequence of lost telomere-binding sites, but rather depends on the Mec1 checkpoint kinase. Rap1 relocalization is thus a novel mechanism connecting DNA damage responses (DDRs) at telomeres to global changes in chromatin and gene expression while driving the pace of senescence.

Comparison of Cas9 activators in multiple species.

Several programmable transcription factors exist based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems, and we assess the role of cooperativity in maximizing gene expression.