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Recent Advances in Understanding the Molecular Mechanisms of Multidrug Resistance and Novel Approaches of CRISPR/Cas9-Based Genome-Editing to Combat This Health Emergency.
The rapid spread of multidrug resistance (MDR), due to abusive use of antibiotics has led to global health emergency, causing substantial morbidity and mortality. Bacteria attain MDR by different means such as antibiotic modification/degradation, target protection/modification/bypass, and enhanced efflux mechanisms. The classical approaches of counteracting MDR bacteria are expensive and time-consuming, thus, it is highly significant to understand the molecular mechanisms of this resistance to curb the problem from core level. The revolutionary approach of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated sequence 9 (CRISPR/Cas9), considered as a next-generation genome-editing tool presents an innovative opportunity to precisely target and edit bacterial genome to alter their MDR strategy. Different bacteria possessing antibiotic resistance genes such as mecA, ermB, ramR, tetA, mqrB and blaKPC that have been targeted by CRISPR/Cas9 to re-sensitize these pathogens against antibiotics, such as methicillin, erythromycin, tigecycline, colistin and carbapenem, respectively. The CRISPR/Cas9 from S. pyogenes is the most widely studied genome-editing tool, consisting of a Cas9 DNA endonuclease associated with tracrRNA and crRNA, which can be systematically coupled as sgRNA. The targeting strategies of CRISPR/Cas9 to bacterial cells is mediated through phage, plasmids, vesicles and nanoparticles. However, the targeting approaches of this genome-editing tool to specific bacteria is a challenging task and still remains at a very preliminary stage due to numerous obstacles awaiting to be solved. This review elaborates some recent updates about the molecular mechanisms of antibiotic resistance and the innovative role of CRISPR/Cas9 system in modulating these resistance mechanisms. Furthermore, the delivery approaches of this genome-editing system in bacterial cells are discussed. In addition, some challenges and future prospects are also described.
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Is CRISPR/Cas9-based multi-trait enhancement of wheat forthcoming?
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies have been implemented in recent years in the genome editing of eukaryotes, including plants. The original system of knocking out a single gene by causing a double-strand break (DSB), followed by non-homologous end joining (NHEJ) or Homology-directed repair (HDR) has undergone many adaptations. These adaptations include employing CRISPR/Cas9 to upregulate gene expression or to cause specific small changes to the DNA sequence of the gene-of-interest. In plants, multiplexing, i.e., inducing multiple changes by CRISPR/Cas9, is extremely relevant due to the redundancy of many plant genes, and the time- and labor-consuming generation of stable transgenic plant lines via crossing. Here we discuss relevant examples of various traits, such as yield, biofortification, gluten content, abiotic stress tolerance, and biotic stress resistance, which have been successfully manipulated using CRISPR/Cas9 in plants. While existing studies have primarily focused on proving the impact of CRISPR/Cas9 on a single trait, there is a growing interest among researchers in creating a multi-stress tolerant wheat cultivar 'super wheat', to commercially and sustainably enhance wheat yields under climate change. Due to the complexity of the technical difficulties in generating multi-target CRISPR/Cas9 lines and of the interactions between stress responses, we propose enhancing already commercial local landraces with higher yield traits along with stress tolerances specific to the respective localities, instead of generating a general 'super wheat'. We hope this will serve as the sustainable solution to commercially enhancing crop yields under both stable and challenging environmental conditions.
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Coronary artery disease and cancer: a significant resemblance.
Cancer and coronary artery disease (CAD) are two of the most common causes of death, and they frequently coexist, especially as the world's population ages. CAD can develop prior to or following cancer diagnosis, as well as a side effect of cancer treatment. CAD develops as complex interactions of lifestyle and hereditary variables, just like the development of the most complex and non-communicable diseases. Cancer is caused by both external/acquired factors (tobacco, food, physical activity, alcohol consumption, epigenetic alterations) and internal/inherited factors (genetic mutations, hormones, and immunological diseases). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) system has recently emerged as a strong tool for gene therapy for both cancer as well as CAD treatment due to its great accuracy and efficiency. A deeper understanding of the complex link between CAD and cancer should lead to better prevention, faster detection, and safer treatment strategies.
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Transcriptional regulation by promoters with enhancer function.
UNLABELLED: Promoters with enhancer activity have been described recently. In this point of view, we will discuss current findings highlighting the commonality of this type of regulatory elements, their genetic and epigenetic characteristics, their potential biological roles in the regulation of gene expression and the underlining molecular mechanisms. ABBREVIATIONS: TSS: transcription start site; IFN: interferon; STARR-seq: Self-Transcribing Active Regulatory Region sequencing; MPRA: Massively Parallel Reporter Assay; ChIP: chromatin immunoprecipitation; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; lncRNA: long non-coding RNA.
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Application of CRISPR/Cas9 gene editing system in microalgal metabolic engineering and synthetic strategies of functional food ingredients.
Microalgae are natural and sustainable biological resources rich in high-value nutrients such as lipids, proteins, and functional pigments, which show great potential in the fields of functional foods, dietary supplements, and natural colorants. However, the yields of target components in natural microalgae are often insufficient to meet commercialization demands. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system, a revolutionary technology, provides a precise and effective means for targeted improvement of microalgae to enhance their nutritional value and yields. This review first outlines the basic principles of the CRISPR/Cas9 system, including its core components and gene editing mechanism. It then summarizes the application of this technology in microalgae, focusing on successful cases of modifying metabolic pathways to enrich specific nutrients, such as increasing the unsaturated fatty acid content of lipids, increasing the proportion of edible proteins, and enriching natural pigments with antioxidant properties. In addition, this review discusses the main challenges faced when applying this technology to microalgae, including delivery difficulties due to strong cell walls, low efficiency of genetic transformation, and the risk of off-target effects. Finally, the paper describes cutting-edge strategies to address these challenges, such as the development of high-fidelity Cas9 enzymes and the optimization of a single-guide RNA (sgRNA) design. Continued advances in these technologies are propelling microalgae into efficient and sustainable "cell factories", providing the food industry with more natural, healthy, and high-value functional ingredients.
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InVivo Cancer-Based Functional Genomics.
Pinpointing the underlying mechanisms that drive tumorigenesis in human patients is a prerequisite for identifying suitable therapeutic targets for precision medicine. In contrast to cell culture systems, mouse models are highly favored for evaluating tumor progression and therapeutic response in a more realistic in vivo context. The past decade has witnessed a dramatic increase in the number of functional genomic studies using diverse mouse models, including in vivo clustered regularly interspaced short palindromic repeats (CRISPR) and RNA interference (RNAi) screens, and these have provided a wealth of knowledge addressing multiple essential questions in translational cancer research. We compare the multiple mouse systems and genomic tools that are commonly used for in vivo screens to illustrate their strengths and limitations. Crucial components of screen design and data analysis are also discussed.
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Theoretical guarantees for phylogeny inference from single-cell lineage tracing.
Lineage-tracing technologies based on Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR-Cas9) genome editing have emerged as a powerful tool for investigating development in single-cell contexts, but exact reconstruction of the underlying clonal relationships in experiment is complicated by features of the data. These complications are functions of the experimental parameters in these systems, such as the Cas9 cutting rate, the diversity of indel outcomes, and the rate of missing data. In this paper, we develop two theoretically grounded algorithms for the reconstruction of the underlying single-cell phylogenetic tree as well as asymptotic bounds for the number of recording sites necessary for exact recapitulation of the ground truth phylogeny at high probability. In doing so, we explore the relationship between the problem difficulty and the experimental parameters, with implications for experimental design. Lastly, we provide simulations showing the empirical performance of these algorithms and showing that the trends in the asymptotic bounds hold empirically. Overall, this work provides a theoretical analysis of phylogenetic reconstruction in single-cell CRISPR-Cas9 lineage-tracing technologies.
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CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f.sp. niveum.
CRISPR/Cas9-mediated editing of Clpsk1 enhanced watermelon resistance to Fusarium oxysporum. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has proven to be an effective genome-editing tool for crop improvement. Previous studies described that Phytosulfokine (PSK) signalling attenuates plant immune response. In this work, we employed the CRISPR/Cas9 system to knockout Clpsk1 gene, encoding the PSK precursor, to confer enhanced watermelon resistance to Fusarium oxysporum f.sp. niveum (FON). Interactions between PSK and FON were analysed and it was found that transcript of Clpsk1 was significantly induced upon FON infection. Meanwhile, application of exogenous PSK increased the pathogen growth. Then, one sgRNA, which targeted the first exon of Clpsk1, was selected for construction of pRGEB32-CAS9-gRNA-Clpsk1 expression cassette. The construct was then transformed to watermelon through Agrobacterium tumefaciens-mediated transformation method. Six mutant plants were obtained and three types of mutations at the expected position were identified based on Sanger sequencing. Resistance evaluation indicated that Clpsk1 loss-of-function rendered watermelon seedlings more resistant to infection by FON. These results indicate that CRISPR/Cas9-mediated gene modification is an effective approach for watermelon improvement.
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CRISPR/Cas9 Guided Mutagenesis of Grain Size 3 Confers Increased Rice (Oryza sativa L.) Grain Length by Regulating Cysteine Proteinase Inhibitor and Ubiquitin-Related Proteins.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice.
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Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses.
CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes-derived Cas9 system (SpCas9) targeting Pten, a gene involved in NASH and a negative regulator of the PI3K-AKT pathway, in mouse liver. We found that the Ad vector mediated efficient Pten gene editing even in the presence of typical Ad vector-associated immunotoxicity in the liver. Four months after vector infusion, mice receiving the Pten gene-editing Ad vector showed massive hepatomegaly and features of NASH, consistent with the phenotypes following Cre-loxP-induced Pten deficiency in mouse liver. We also detected induction of humoral immunity against SpCas9 and the potential presence of an SpCas9-specific cellular immune response. Our findings provide a strategy to model human liver diseases in mice and highlight the importance considering Cas9-specific immune responses in future translational studies involving in vivo delivery of CRISPR/Cas9.
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