The applications of CRISPR/Cas-mediated microRNA and lncRNA editing in plant biology: shaping the future of plant non-coding RNA research.

MAIN CONCLUSION: CRISPR/Cas technology has greatly facilitated plant non-coding RNA (ncRNA) biology research, establishing itself as a promising tool for ncRNA functional characterization and ncRNA-mediated plant improvement. Throughout the last decade, the promising genome editing tool clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas; CRISPR/Cas) has allowed unprecedented advances in the field of plant functional genomics and crop improvement. Even though CRISPR/Cas-mediated genome editing system has been widely used to elucidate the biological significance of a number of plant protein-coding genes, this technology has been barely applied in the functional analysis of those non-coding RNAs (ncRNAs) that modulate gene expression, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Nevertheless, compelling findings indicate that CRISPR/Cas-based ncRNA editing has remarkable potential for deciphering the biological roles of ncRNAs in plants, as well as for plant breeding. For instance, it has been demonstrated that CRISPR/Cas tool could overcome the challenges associated with other approaches employed in functional genomic studies (e.g., incomplete knockdown and off-target activity). Thus, in this review article, we discuss the current status and progress of CRISPR/Cas-mediated ncRNA editing in plant science in order to provide novel prospects for further assessment and validation of the biological activities of plant ncRNAs and to enhance the development of ncRNA-centered protocols for crop improvement.

CRISPR-based assays for rapid detection of SARS-CoV-2.

COVID-19 pandemic posed an unprecedented threat to global public health and economies. There is no effective treatment of the disease, hence, scaling up testing for rapid diagnosis of SARS-CoV-2 infected patients and quarantine them from healthy individuals is one the best strategies to curb the pandemic. Establishing globally accepted easy-to-access diagnostic tests is extremely important to understanding the epidemiology of the present pandemic. While nucleic acid based tests are considered to be more sensitive with respect to serological tests but present gold standard qRT-PCR-based assays possess limitations such as low sample throughput, requirement for sophisticated reagents and instrumentation. To overcome these shortcomings, recent efforts of incorporating LAMP-based isothermal detection, and minimizing the number of reagents required are on rise. CRISPR based novel techniques, when merge with isothermal and allied technologies, promises to provide sensitive and rapid detection of SARS-CoV-2 nucleic acids. Here, we discuss and present compilation of state-of-the-art detection techniques for COVID-19 using CRISPR technology which has tremendous potential to transform diagnostics and epidemiology.

Functional Implications and Clinical Potential of MicroRNAs in Irritable Bowel Syndrome: A Concise Review.

MicroRNAs (miRNAs) are tiny (20-24 nucleotides long), non-coding, highly conserved RNA molecules that play a crucial role within the post-transcriptional regulation of gene expression via sequence-specific mechanisms. Since the miRNA transcriptome is involved in multiple molecular processes needed for cellular homeostasis, its altered expression can trigger the development and progression of several human pathologies. In this context, over the last few years, several relevant studies have demonstrated that dysregulated miRNAs affect a wide range of molecular mechanisms associated with irritable bowel syndrome (IBS), a common gastrointestinal disorder. For instance, abnormal miRNA expression in IBS patients is related to the alteration of intestinal permeability, visceral hyperalgesia, inflammatory pathways, and pain sensitivity. Besides, specific miRNAs are differentially expressed in the different subtypes of IBS, and therefore, they might be used as biomarkers for precise diagnosis of these pathological conditions. Accordingly, miRNAs have noteworthy potential as theragnostic targets for IBS. Hence, in this current review, we present an overview of the recent discoveries regarding the clinical relevance of miRNAs in IBS, which might be useful in the future for the development of miRNA-based drugs against this disorder.

Regulatory roles of noncoding RNAs in callus induction and plant cell dedifferentiation.

KEY MESSAGE: Plant regulatory noncoding RNAs (ncRNAs) have emerged as key modulators of gene expression during callus induction. Their further study may promote the design of innovative plant tissue culture protocols. The use of plants by humans has recently taken on a new and expanding insight due to the advent of genetic engineering technologies. In this context, callus cultures have shown remarkable potential for synthesizing valuable biomolecules, crop improvement, plant micropropagation, and biodiversity preservation. A crucial stage in callus production is the conversion of somatic cells into totipotent cells; compelling evidence indicates that stress factors, transcriptional regulators, and plant hormones can trigger this biological event. Besides, posttranscriptional regulators of gene expression might be essential participants in callus induction. However, research related to the analysis of noncoding RNAs (ncRNAs) that modulate callogenesis and plant cell dedifferentiation in vitro is still at an early stage. During the last decade, some relevant studies have enlightened the fact that different classes of ncRNAs, such as microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs) are implicated in plant cell dedifferentiation through regulating the expression levels of diverse gene targets. Hence, understanding the molecular relevance of these ncRNAs in the aforesaid biological processes might represent a promising source of new biotechnological approaches for callus culture and plant improvement. In this current work, we review the experimental evidence regarding the prospective roles of ncRNAs in callus induction and plant cell dedifferentiation to promote this field of study.

Closer vein spacing by ectopic expression of nucleotide-binding and leucine-rich repeat proteins in rice leaves.

KEY MESSAGE: Elevated expression of nucleotide-binding and leucine-rich repeat proteins led to closer vein spacing and higher vein density in rice leaves. To feed the growing global population and mitigate the negative effects of climate change, there is a need to improve the photosynthetic capacity and efficiency of major crops such as rice to enhance grain yield potential. Alterations in internal leaf morphology and cellular architecture are needed to underpin some of these improvements. One of the targets is to generate a "Kranz-like" anatomy in leaves that includes decreased interveinal spacing close to that in C4 plant species. As C4 photosynthesis has evolved from C3 photosynthesis independently in multiple lineages, the genes required to facilitate C4 may already be present in the rice genome. The Taiwan Rice Insertional Mutants (TRIM) population offers the advantage of gain-of-function phenotype trapping, which accelerates the identification of rice gene function. In the present study, we screened the TRIM population to determine the extent to which genetic plasticity can alter vein density (VD) in rice. Close vein spacing mutant 1 (CVS1), identified from a VD screening of approximately 17,000 TRIM lines, conferred heritable high leaf VD. Increased vein number in CVS1 was confirmed to be associated with activated expression of two nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. Overexpression of the two NB-LRR genes individually in rice recapitulates the high VD phenotype, due mainly to reduced interveinal mesophyll cell (M cell) number, length, bulliform cell size and thus interveinal distance. Our studies demonstrate that the trait of high VD in rice can be achieved by elevated expression of NB-LRR proteins limited to no yield penalty.

Phytochemicals mediated modulation of microRNAs and long non-coding RNAs in cancer prevention and therapy.

MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are two main categories of noncoding RNAs (ncRNAs) that can influence essential biological functions in various ways, as well as their expression and function are tightly regulated in physiological homeostasis. Additionally, the dysregulation of these ncRNAs seems to be crucial to the pathogenesis of human diseases. The latest findings indicate that ncRNAs execute vital roles in cancer initiation and progression, and the cancer phenotype can be reversed by modulating their expression. Available scientific discoveries suggest that phytochemicals such as polyphenols, alkaloids, terpenoids, and organosulfur compounds can significantly modulate multiple cancer-associated miRNAs and lncRNAs, thereby inhibiting cancer initiation and development. However, despite promising outcomes of experimental research, only a few clinical trials are currently being conducted to evaluate the therapeutic effectiveness of these compounds. Nevertheless, understanding phytochemical-mediated ncRNA regulation in cancer and the underlying molecular mechanisms on tumor pathophysiology can aid in the development of novel therapeutic strategies to combat this deadly disease.

The Emerging Role of MicroRNAs in Bone Diseases and Their Therapeutic Potential.

MicroRNAs (miRNAs) are a class of small (20-24 nucleotides), highly conserved, non-coding RNA molecules whose main function is the post-transcriptional regulation of gene expression through sequence-specific manners, such as mRNA degradation or translational repression. Since these key regulatory molecules are implicated in several biological processes, their altered expression affects the preservation of cellular homeostasis and leads to the development of a wide range of pathologies. Over the last few years, relevant investigations have elucidated that miRNAs participate in different stages of bone growth and development. Moreover, the abnormal expression of these RNA molecules in bone cells and tissues has been significantly associated with the progression of numerous bone diseases, including osteoporosis, osteosarcoma, osteonecrosis and bone metastasis, among others. In fact, miRNAs regulate multiple pathological mechanisms, including altering either osteogenic or osteoblast differentiation, metastasis, osteosarcoma cell proliferation, and bone loss. Therefore, in this present review, aiming to impulse the research arena of the biological implications of miRNA transcriptome in bone diseases and to explore their potentiality as a theragnostic target, we summarize the recent findings associated with the clinical significance of miRNAs in these ailments.

Rice with reduced stomatal density conserves water and has improved drought tolerance under future climate conditions.

Much of humanity relies on rice (Oryza sativa) as a food source, but cultivation is water intensive and the crop is vulnerable to drought and high temperatures. Under climate change, periods of reduced water availability and high temperature are expected to become more frequent, leading to detrimental effects on rice yields. We engineered the high-yielding rice cultivar 'IR64' to produce fewer stomata by manipulating the level of a developmental signal. We overexpressed the rice epidermal patterning factor OsEPF1, creating plants with substantially reduced stomatal density and correspondingly low stomatal conductance. Low stomatal density rice lines were more able to conserve water, using c. 60% of the normal amount between weeks 4 and 5 post germination. When grown at elevated atmospheric CO2 , rice plants with low stomatal density were able to maintain their stomatal conductance and survive drought and high temperature (40°C) for longer than control plants. Low stomatal density rice gave equivalent or even improved yields, despite a reduced rate of photosynthesis in some conditions. Rice plants with fewer stomata are drought tolerant and more conservative in their water use, and they should perform better in the future when climate change is expected to threaten food security.

CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice.

KEY MESSAGE: CRISPR-Cas9/Cpf1 system with its unique gene targeting efficiency, could be an important tool for functional study of early developmental genes through the generation of successful knockout plants. The introduction and utilization of systems biology approaches have identified several genes that are involved in early development of a plant and with such knowledge a robust tool is required for the functional validation of putative candidate genes thus obtained. The development of the CRISPR-Cas9/Cpf1 genome editing system has provided a convenient tool for creating loss of function mutants for genes of interest. The present study utilized CRISPR/Cas9 and CRISPR-Cpf1 technology to knock out an early developmental gene EPFL9 (Epidermal Patterning Factor like-9, a positive regulator of stomatal development in Arabidopsis) orthologue in rice. Germ-line mutants that were generated showed edits that were carried forward into the T2 generation when Cas9-free homozygous mutants were obtained. The homozygous mutant plants showed more than an eightfold reduction in stomatal density on the abaxial leaf surface of the edited rice plants. Potential off-target analysis showed no significant off-target effects. This study also utilized the CRISPR-LbCpf1 (Lachnospiracae bacterium Cpf1) to target the same OsEPFL9 gene to test the activity of this class-2 CRISPR system in rice and found that Cpf1 is also capable of genome editing and edits get transmitted through generations with similar phenotypic changes seen with CRISPR-Cas9. This study demonstrates the application of CRISPR-Cas9/Cpf1 to precisely target genomic locations and develop transgene-free homozygous heritable gene edits and confirms that the loss of function analysis of the candidate genes emerging from different systems biology based approaches, could be performed, and therefore, this system adds value in the validation of gene function studies.

CRISPR: From Prokaryotic Immune Systems to Plant Genome Editing Tools.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a prokaryotic adaptive immune system that has the ability to identify specific locations on the bacteriophage (phage) genome to create breaks in it, and internalize the phage genome fragments in its own genome as CRISPR arrays for memory-dependent resistance. Although CRISPR has been used in the dairy industry for a long time, it recently gained importance in the field of genome editing because of its ability to precisely target locations in a genome. This system has further been modified to locate and target any region of a genome of choice due to modifications in the components of the system. By changing the nucleotide sequence of the 20-nucleotide target sequence in the guide RNA, targeting any location is possible. It has found an application in the modification of plant genomes with its ability to generate mutations and insertions, thus helping to create new varieties of plants. With the ability to introduce specific sequences into the plant genome after cleavage by the CRISPR system and subsequent DNA repair through homology-directed repair (HDR), CRISPR ensures that genome editing can be successfully applied in plants, thus generating stronger and more improved traits. Also, the use of the CRISPR editing system can generate plants that are transgene-free and have mutations that are stably inherited, thus helping to circumvent current GMO regulations.

Erectile dysfunction in anterior urethral strictures after urethroplasty with reference to vascular parameters.

BACKGROUND: Relation of erectile dysfunction (ED) with urethroplasty has long been a subject of debate. Very few studies on subcontinent population are available in this regard and still rarer are studies assessing vascular parameters of ED following urethroplasty. The objective of the study was to assess the incidence and prevalence of ED in patients of urethral stricture disease, and to find out effect of urethroplasty on ED after six months of operation including vasculogenic aetiology after operation. METHODS: From January 2014 to December 2015, 35 subjects underwent urethroplasty. They were assessed pre- and postoperatively by International Index of Erectile Function (IIEF-5) and Pharmacological Colour Doppler Ultrasonography (PCDU) for a period of 6 months. RESULTS: Preoperative prevalence of ED assessed by IIEF was found to be 82.8%. Postoperative incidence of ED was 28.5% and new onset ED is 50%. There was no significant change in IIEF values and values of peak systolic velocity and resistive index of cavernosal artery over time. CONCLUSION: There is significant prevalence of ED with urethral stricture. Despite significant postoperative incidence of ED after urethroplasty, the surgical procedure per se does not result in ED.

Guide RNA library-based CRISPR screens in plants: opportunities and challenges.

Next-generation sequencing technologies have revolutionized our ability to read sequence information at the genome and transcriptome levels in a high-throughput manner. However, genetic screening at a large or genomic scale remains challenging in plants. Recently, the RNA-guided CRISPR-Cas nucleases have been optimized for high-throughput functional genomic screens combined with guide RNA (gRNA) libraries in plants. This approach has shown great promise in facilitating genetic screening, directed evolution, and quantitative trait engineering. However, this technology is still in its infancy. In this short review, we describe the recent progress in gRNA library-based CRISPR screens in plants. We provide a critical assessment of the current approaches and emerging delivery methods for CRISPR screens. We also highlight the challenges and present future perspectives on CRISPR screens in plants.

Genome Editing in Chlamydomonas reinhardtii Using Cas9-gRNA Ribonucleoprotein Complex: A Step-by-Step Guide.

Genome editing technologies have provided opportunities to manipulate literally any genomic location, opening new avenues for reverse genetics-based improvements. Among them, CRISPR/Cas9 is the most versatile tool for genome editing applications in prokaryotes and eukaryotes. Here, we provide a guide to successfully carry out high-efficiency genome editing in Chlamydomonas reinhardtii using preassembled CRISPR/Cas9-gRNA ribonucleoprotein (RNP) complexes.

CRISPR-based bioengineering in microalgae for production of industrially important biomolecules.

Microalgae, as photosynthetic organisms, have the potential to produce biomolecules for use in food, feed, cosmetics, nutraceuticals, fuel, and other applications. Faster growth rates and higher protein and lipid content make microalgae a popular chassis for many industrial applications. However, challenges such as low productivity and high production costs have limited their commercialization. To overcome these challenges, bioengineering approaches such as genetic engineering, metabolic engineering, and synthetic biology have been employed to improve the productivity and quality of microalgae-based products. Genetic engineering employing genome editing tools like CRISPR/Cas allows precise and targeted genetic modifications. CRISPR/Cas systems are presently used to modify the genetic makeup of microalgae for enhanced production of specific biomolecules. However, these tools are yet to be explored explicitly in microalgae owing to some limitations. Despite the progress made in CRISPR-based bioengineering approaches, there is still a need for further research to optimize the production of microalgae-based products. This includes improving the efficiency of genome editing tools, understanding the regulatory mechanisms of microalgal metabolism, and optimizing growth conditions and cultivation strategies. Additionally, addressing the ethical, social, and environmental concerns associated with genetic modification of microalgae is crucial for the responsible development and commercialization of microalgae-based products. This review summarizes the advancements of CRISPR-based bioengineering for production of industrially important biomolecules and provides key considerations to use CRISPR/Cas systems in microalgae. The review will help researchers to understand the progress and to initiate genome editing experiments in microalgae.

Current Advances of Plant-Based Vaccines for Neurodegenerative Diseases.

Neurodegenerative diseases (NDDs) are characterized by the progressive degeneration and/or loss of neurons belonging to the central nervous system, and represent one of the major global health issues. Therefore, a number of immunotherapeutic approaches targeting the non-functional or toxic proteins that induce neurodegeneration in NDDs have been designed in the last decades. In this context, due to unprecedented advances in genetic engineering techniques and molecular farming technology, pioneering plant-based immunogenic antigen expression systems have been developed aiming to offer reliable alternatives to deal with important NDDs, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Diverse reports have evidenced that plant-made vaccines trigger significant immune responses in model animals, supported by the production of antibodies against the aberrant proteins expressed in the aforementioned NDDs. Moreover, these immunogenic tools have various advantages that make them a viable alternative for preventing and treating NDDs, such as high scalability, no risk of contamination with human pathogens, cold chain free production, and lower production costs. Hence, this article presents an overview of the current progress on plant-manufactured vaccines for NDDs and discusses its future prospects.

MicroRNAs and long non-coding RNAs in pancreatic cancer: From epigenetics to potential clinical applications.

MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are two relevant classes of non-coding RNAs (ncRNAs) that play a pivotal role in a number of molecular processes through different epigenetic regulatory mechanisms of gene expression. As a matter of fact, the altered expression of these types of RNAs leads to the development and progression of a varied range of multifactorial human diseases. Several recent reports elucidated that miRNA and lncRNAs have been implicated in pancreatic cancer (PC). For instance, dysregulation of such ncRNAs has been found to be associated with chemoresistance, apoptosis, autophagy, cell differentiation, tumor suppression, tumor growth, cancer cell proliferation, migration, and invasion in PC. Moreover, several aberrantly expressed miRNAs and lncRNAs have the potential to be used as biomarkers for accurate PC diagnosis. Additionally, miRNAs and lncRNAs are considered as promising clinical targets for PC. Therefore, in this review, we discuss recent experimental evidence regarding the clinical implications of miRNAs and lncRNAs in the pathophysiology of PC, their future potential, as well as the challenges that have arisen in this field of study in order to drive forward the design of ncRNA-based diagnostics and therapeutics for PC.

Insight into the genome data of commercially important giant kelp Macrocystis pyrifera.

Kelps or brown algae are a wide group of marine macroalgae that play an important role in aquatic ecosystems and generally have high commercial value. To facilitate brown algal studies, we report the complete genome sequence of the largest kelp Macrocystis pyrifera. The whole genome is ∼428 Mb in size, comprises 44,307 scaffolds with an average GC content of 47%, and is predicted to contain a total of 24,778 genes. 18S sequence-based phylogenetic analysis revealed that littoral brown seaweed Scytosiphon lomentaria is the closest species of M. pyrifera. Numerous genes identified in this dataset are involved in genetic information processing, signaling, and cellular processes, carbohydrate metabolism, and terpenoids biosynthesis.

A Brief Review on the Regulatory Roles of MicroRNAs in Cystic Diseases and Their Use as Potential Biomarkers.

miRNAs are small endogenous conserved non-coding RNA molecules that regulate post-transcriptional gene expression through mRNA degradation or translational inhibition, modulating nearly 60% of human genes. Cystic diseases are characterized by the presence of abnormal fluid-filled sacs in the body, and though most cysts are benign, they can grow inside tumors and turn malignant. Recent evidence has revealed that the aberrant expression of a number of miRNAs present in extracellular fluids, including plasma or serum, urine, saliva, follicular fluid, and semen, contribute to different cystic pathologies. This review aims to describe the role of different miRNAs in three worldwide relevant cystic diseases: polycystic ovarian syndrome (PCOS), polycystic kidney disease (PKD), and pancreatic cyst tumors (PCTs), as well as their potential use as novel biomarkers.

The role of microRNAs in solving COVID-19 puzzle from infection to therapeutics: A mini-review.

Nowadays, one of the major global health concerns is coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Even though numerous treatments and vaccines to combat this virus are currently under development, the detailed molecular mechanisms underlying the pathogenesis of this disease are yet to be elucidated to design future therapeutic tools against SARS-CoV-2 variants. MicroRNAs (miRNAs) are small (20-24 nucleotides), non-coding RNA molecules that regulate post-transcriptional gene expression. Recently, it has been demonstrated that both host and viral-encoded miRNAs are crucial for the successful infection of SARS-CoV-2. For instance, dysregulation of miRNAs that modulate multiple genes expressed in COVID-19 patients with comorbidities (e.g., type 2 diabetes, lung adenocarcinoma, and cerebrovascular disorders) could affect the severity of the disease. Thus, altered expression levels of circulating miRNAs might be helpful to diagnose this illness and forecast whether a COVID-19 patient could develop a severe state of the disease. Besides, researchers have found a number of miRNAs could inhibit the expression of proteins, such as ACE2, TMPRSS2, spike, and Nsp12, involved in the life cycle of SARS-CoV-2. Accordingly, miRNAs represent potential biomarkers and therapeutic targets for this devastating viral disease. Therefore, in this current review, we present the recent discoveries regarding the clinical relevance and biological roles of miRNAs in COVID-19.