RNA-based therapeutics have shown great promise in various medical applications, including cancers, infectious diseases, and metabolic diseases. The recent success of mRNA vaccines for combating the COVID-19 pandemic has highlighted the medical value of RNA drugs. However, one of the major challenges in realizing the full potential of RNA drugs is to deliver RNA into specific organs and tissues in a targeted manner, which is crucial for achieving therapeutic efficacy, reducing side effects, and enhancing overall treatment efficacy. Numerous attempts have been made to pursue targeting, nonetheless, the lack of clear guideline and commonality elucidation has hindered the clinical translation of RNA drugs. In this review, we outline the mechanisms of action for targeted RNA delivery systems and summarize four key factors that influence the targeting delivery of RNA drugs. These factors include the category of vector materials, chemical structures of vectors, administration routes, and physicochemical properties of RNA vectors, and they all notably contribute to specific organ/tissue tropism. Furthermore, we provide an overview of the main RNA-based drugs that are currently in clinical trials, highlighting their design strategies and tissue tropism applications. This review will aid to understand the principles and mechanisms of targeted delivery systems, accelerating the development of future RNA drugs for different diseases. The world has recently witnessed the remarkable impact of messenger RNA (mRNA) vaccines in effectively combating the COVID-19 pandemic, opening a new era of RNA drugs for therapeutic applications. mRNA therapeutics have demonstrated immense potential in the fields of protein replacement therapies, vaccines, and gene editing, as it can regulate gene expression and produce specialized proteins To achieve the intended therapeutic effects, RNA drugs should be precisely delivered to specific organs, tissues, or even cells. Targeted delivery endows several acknowledged advantages. Firstly, delivering RNA cargoes to diseased cells is the prerequisite to realize treatment. Secondly, targeting enables us to address the issue of off-target effects, which often limits RNA therapy by causing toxicity and affecting drug safety as well as efficacy. Precise delivery aims to enhance the bioavailability of RNA drugs at the target site while minimizing their distribution to non-target tissues. Lastly, targeted delivery can lower the required dosage of RNA for administration, improve the biosafety of drugs, and increase the clinical tolerance Despite the enthusiastic potential of RNA-based drugs for targeted treatment of various diseases, multiple obstacles remain to be addressed to fully realize their clinical applications To address the limitations of RNA-based drugs in practical use, a variety of non-viral vectors have been employed for therapeutics delivery. Until now, to develop targeting materials and strategies remains the critical challenge for RNA drug clinical translation. Although several targeted delivery systems have arisen, the guideline behind organ/tissue tropism remains unclear Different RNA drugs demonstrate distinct characteristics and therapeutic mechanisms 7GpppN, can bind to the multi-subunit initiation factor eIF4F to promote mRNA binding with ribosomes and translation of mRNA mRNA is a single-stranded ribonucleotide transcribed from a template strand of DNA, which carries the genetic code and directs the synthesis of proteins. Natural mRNA is composed of the following parts: 5' and 3' untranslated regions (UTRs), 3' poly (A) tail, 5' cap. Among these, UTRs do not encode proteins, but can regulate locations of mRNA and translation efficiency in vitro transcribed (IVT) mRNA is delivered into the desired cells by a suitable vector, such as lipid nanoparticle (LNP), the mRNA is released from the vector. Then the mRNA directs protein synthesis with the help of ribosomes and transfer RNA (tRNA), which can be used for protein replacement to treat protein deficiency (e.g. haemophilia B) or protein malfunction (e.g. muscular dystrophy) Compared to DNA drugs, mRNA does not require entry into the nucleus and can function directly in the cytoplasm. This advantage avoids the risk of gene insertion. Additionally, mRNA has transient expression and will be degraded by enzymes in a limited time after entering the body. This property endows mRNA high safety for therapeutic use + and CD8+ T cells and B cells, killing and eliminating viruses or tumor cells et al. used LNPs to deliver mRNA encoding melanoma-related antigens, which induced a strong immune response and prolonged the survival time of mice bearing melanoma To date, one of the most significant applications of mRNA is to be used as the vaccines in the field of cancer immunotherapy and viral infections. The immune system plays a crucial role in fighting cancer and protecting the body from bacteria invasion. However, tumor-associated cells can secrete a variety of cytokines and chemokines through different mechanisms to inhibit immune system activities Gene editing is another important therapeutic field of mRNA-based drugs, leveraging the development of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)) technology siRNA, also known as short interfering RNA or silencing RNA, is a type of short double-stranded RNA with the length of 21-23 nucleotides containing 2 nucleotides overhang miRNA is a single-stranded non-coding RNA molecule that plays a crucial role in the regulation of gene expression and is involved in a series of essential cellular processes, such as cell proliferation, differentiation and apoptosis in vitro, demonstrating the antisense function of synthetic oligodeoxynucleotides for the first time In 1978, Zamecnik and Stephenson reported the use of synthetic oligodeoxynucleotides to inhibit the replication of virus in vivo. To fully realize the potential of RNA-based drugs for disease treatment, further research and strategies are in urgent demand to overcome the problems associated with targeted delivery.The abundance of RNA species and functions endows huge potential in biological and medical field. However, the efficacy of RNA-based drugs can be affected by the challenge of delivering them to specific organs and sites RNA drugs open an era for treating chronic diseases, nonetheless, their full realization is seriously hindered by lack of targeted delivery technology. Despite the fact that RNA delivery systems have been studied for decades, the precise gene regulation in specific tissues or cells remains a huge challenging. Furthermore, understanding the guideline of organ/tissue tropism of nanoparticles will further the advance of next-generation gene delivery technology, facilitating future RNA-based drug development. Herein, we summarize current progress in targeted RNA delivery, from the point of four key factors: the category of vector materials, chemical structures of vectors, administration routes, and physicochemical properties of RNA vectors Figure .et al. found that ionizable cationic lipids with unsaturated tails could improve the delivery efficiency of LNPs due to the increased lipid fusion ability in vivoLNPs are one of the widely utilized non-viral vectors for delivering RNA-based drugs. Typically, LNPs are composed of four components with distinct functions, including permanently charged cationic or ionizable cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids et al. demonstrated that LNPs encapsulating HNF4A mRNA could attenuate liver fibrosis in preclinical mouse model et al. used a degradable lipid to formulate LNPs to deliver Cas9 mRNA/sgRNA for gene editing in vivo. The results showed that the editing efficiency was highly considerable for transthyretin gene editing in the liver, reducing serum protein levels by over 97% with a single dose Despite some studies show that lipid materials may cause liver or lung injuries and the cytotoxicity of LNPs is related to the dosage of administration and the properties of lipids used, LNPs offer several advantages compared to viral vectors, including ease of production, relatively low immunogenicity, high RNA loading capacity, and flexible design options Despite the great potential in clinical translation, extrahepatic RNA delivery by LNPs remains challenging. Alternatively, polymers provide an option for delivery out of the liver. Polymers are divided into different classifications, such as cationic polymers, zwitterionic polymers, polymeric micelles, and dendrimers To further improve the functionalities of vectors, numerous hybrid copolymers, such as lipid-polymer hybrid nanoparticles (LPNs), graphene oxide (GO)-cationic PEI polymers (GO-PEI complexes), polymer-dendrimer hybrids, were developed et al. utilized chitosan-coated PLGA nanoparticles to encapsulate chemically-modified mRNA encoding human cystic fibrosis transmembrane conductance regulator (CFTR), which was efficiently delivered to lungs of CFTR deficient mice following intravenous and intratracheal administrations et al. modified cationic polymers by zwitterionic phospholipidation to selectively deliver mRNA to the spleen and lymph nodes after intravenous administration in vivo, showing great potential in immunotherapy application et al. designed charge-altering releasable transporters (CARTs) via organocatalytic ring-opening polymerization of various lipids and results demonstrated mixed-lipid CARTs could achieve effective mRNA delivery to B cells and T cells of spleen in vivo, and the delivery efficiency was higher than single-lipid CART Numerous novel polymers have been reported to deliver therapeutic cargoes to special organs, particularly the lungs. For instance, hyperbranched PBAEs were synthesized to enable mRNA delivery to the lung epithelium through inhalation, resulting in consistent and controlled protein production without causing pulmonary or systemic toxicity via ligands, intercellular adhesion molecules on membranes or encapsulated cargo inside the exosomes via B cell-derived exosomes to hepatocytes or macrophages, with higher delivery efficiency and lower cytotoxicity in contrast to regular transfection methods et al. developed a technique to track the exosomes from mesenchymal stem cells of bone marrow (MSC-exo), which could accumulate in mouse brains in different pathological models, including Alzheimer's disease, autism, stroke, and Parkinson's disease. To be more specific, the homing mechanism was driven by inflammation in pathological brains and MSC-exo could be selectively taken up by neuronal cells Exosomes are extracellular vesicles (EVs) with diameters between 30 and 100 nm, and are naturally secreted by cells et al. utilized copper sulfide, which was metabolizable in liver to improve the excretion of Au. And the results showed that the conjugation of copper sulfide and Au could accelerate the excretion of AuNPs in hepatocytes Inorganic nanoparticles (INPs) are synthesized from inorganic particles, biodegradable polycations, and typical inorganic materials such as gold, silica, metallic oxide, and others. Among INPs, gold nanoparticles (AuNPs) have been extensively studied in inorganic chemistry and nanomaterials in vivo, for instance, most LNPs tend to deliver mRNA into the liver. Nonetheless, minimal alteration of chemical structures of vectors in the same class might also achieve the goal of targeting, and the research on exploring targeted material structures is in full swing. Beyond Dlin-MC3-DMA, ALC-0315, and SM-102 approved by FDA for clinical use in LNP formulations, numerous advancements have been made in the development of various lipids, lipidoids, and polymers for delivering RNA to targeted organs. Here, we outline the relationship between the chemical structures of these materials and their specific targeting capabilities for different organs  study. As a result, cKK-E12 LNPs delivered siRNA to liver and silenced Phosphatase and tensin homolog (Pten) in hepatocytes with high selectivity  mice  represent one of the predominant LNP delivery systems for RNA-based drugs. Dong and colleagues designed a series of LLNs with TT3 as the core structure as depicted in Figure r Figure B. Subseqe Figure C. Furthee Figure D 116. Inet al. used linear diacrylate as the backbone and monomer E63 with two secondary amines as the end-cap to synthesize PBAE polymers, achieving preferential expression of mRNA in liver In addition to LNPs mentioned above, several other vectors with different chemical structures can also realize liver-targeting. Rui et al. discovered that the presence of the fifth component, called 'Selective Organ Targeting (SORT)' molecule, could alter the organ-targeting effect of LNPs in vivo. The organ selectivity depended on the type and amount of SORT lipid added , which achieved mRNA expression in hepatocytes, pulmonary endothelial cells, and antigen presenting cells of the spleen in vivo, showing superior delivery efficiency compared to cKK-E12 LNPs  molecules, which targeted spleen for mRNA delivery and translation, demonstrating application potential in immunotherapy and vaccines 10, Dong and colleagues optimized the position of functional groups to obtain the TNT-b10. Further optimizing the formulation of TNT-b10 LLNs resulted in a high signal of mRNA expression in the spleen, which was 10 times higher than that in the liver with intravenous injection et al. combined helper-PBAEs and DOTAP to develop five-element nanoparticles (FNPs) for lung-targeted delivery of mRNA with high stability after lyophilization et al. combined the CD4 antibody with LNPs, achieving much higher accumulation of radiolabeled mRNA in the spleen compared to non-modified LNPs after systemic administration. Further expression of a reporter gene showed CD4-targeted LNPs could specifically deliver Cre mRNA to the spleen and lymph nodes The spleen, which is rich in lymphocytes and macrophages, is an important immune organ. A library of ionizable amino-polyesters were synthesized s Figure B 125. Afs Figure C. Furthes Figure D-E 126. et al.  could co-deliver Cas9 mRNA and sgRNAs with a high efficacy in vitro and in vivo. Specifically, ZA3-Ep10 LNPs enabled gene editing in the liver, kidney, and lungs in vivo in a short time CRISPR/Cas9 gene editing technology has shown revolutionary impact on the gene therapy field. However, delivering the editing tool safely, effectively, and accurately to the target sites remains a significant challenge et al. reported that PBAE DD90-C12-122 could deliver mRNA to the lungs after intravenous administration, and PEGylation increased the delivery efficacy. Further optimizations, such as varying the carbon chain lengths of alkylamine and the molar ratios of diacrylate and amines, would lead to polymeric nanoparticles with greater delivery potential targeting different subtypes of lung. Analysis of the lung cell types after administrating the optimized formulation of polymers showed the transfected cells were mainly among pulmonary endothelial cells and few immune cells et al. synthesized a library of PBAEs and identified two polymers, D90-C12-103 and DD90-C12-103, which used diacrylate-amine as the backbone, could deliver pDNA and mRNA to the lung of mice post systemic administration. Results showed that the fluorescence peak of mRNA was much higher than that of pDNA in vivo after delivery and area analysis of radiance flux and luminescent images at different time points demonstrated that DD90-C12-103 vector was superior to D90-C12-103 for mRNA delivery in vitro and in vivo. A top polymeric vector, PE4K-A17-0.33C12, formulated with 5% F127 and FLuc mRNA was verified to show high luminescence activity in the lung after intravenous administration Researchers have also investigated the relationship between other vector structures and their targeting abilities, beyond LNPs. Kaczmarek To date, the liver, lung, and spleen are commonly targeted organs in current research, however, targeting other organs such as the eyes, skin, heart, and brain holds great potential but remains more challenging in clinical applications.et al. synthesized a series of LNPs with bisphosphonate (BP)-lipid, which showed higher affinity for bone-related fragments in vitro and higher bone-targeting in vivo compared to LNPs without BP-lipid Despite the translation potential, most LNPs tend to accumulate in the liver, and overcoming the natural liver targeting characteristics of LNPs has become a key challenge in nano-vector research. Recently, Sahay group developed LNPs decorated with an oligomer peptide to deliver mRNA to neural retina in rodents and non-human primates after intravitreal administration. This breakthrough demonstrates the potential of LNP-mRNA in the treatment of inherited retinal diseases and represents significant progress in LNP penetrating biological barriers to achieve extrahepatic targeting in vitro. Among them, P1E28 containing alkyl alcohol side chain and piperazine rings with two tertiary amines showed the highest transfected efficiency. In vivo results showed that P1E28 polymeric nanoparticles delivered Cre mRNA to skin dermal fibroblasts with a higher delivery efficiency than endothelial cells, keratinocytes and macrophages. Further mechanism analysis indicated the ability of P1E28 to target skin fibroblasts might be mediated by CD26 and FAP overexpressed in fibroblasts The skin is the largest organ in the human body and skin aging is related closely to the fibroblast in dermis. Recently, Francisco and Ferreira designed hundreds of polymers and selected six polymers with similar chemical structures composed of diacrylate, special amine, and bisacrylamide, which could transfect mouse fibroblasts with high efficiency via intravenous injection. By lipidating the neurotransmitter, the formulated lipid nanoparticles could pass through the BBB by mediating receptors and enter the central nervous system (CNS) to eventually release encapsulated small molecule drugs, macromolecules and gene editing proteins in neuronal cells. It has been demonstrated that the addition of NT1-lipidoids to facilitate vectors penetrating the BBB is suitable for various types of LNPs et al. modified liposomes with cell-penetrating peptides and transferrin ligands, achieving the targeting of brain capillary endothelial cells expressing TfR in vivovia inhibiting the expression of tau protein in a traumatic brain injury mouse model Brain represents another hard-to-target organ, and the presence of BBB limits the therapeutic effect of brain diseases  et al. developed a modular targeting platform called anchored secondary scFv enabling targeting (ASSET), which was a membrane-anchored lipoprotein integrated with LNP and could interact with Fc constant domain of the antibody. The results showed that siRNA-loaded LNPs formed by adding ASSET and RIg could be taken up by targeted cells in vitro and produce desired gene knockdown in vivo. Further research found that altering RIg could achieve targeting of various leukocyte subsets specifically, demonstrating the potential applications of this platform in different disease models et al. utilized the ASSET strategy to connect anti-Ly6c+ mAbs to LNPs, achieving targeted delivery of therapeutic mRNA to Ly6c+ inflammatory leukocytes of mice with inflammatory bowel disease. These results highlight the potential of ASSET in the field of cancers, inflammatory diseases, and rare genetic disorders Cancer is one of the leading causes of death in humans. Targeting drugs provide a precise way to target tumor cells without affecting normal cells, reducing side effects and improving overall survival rates compared to traditional chemotherapeutic drugs Cancer immunotherapy is a highly effective approach to fight against cancer, including using cancer vaccines. However, a major obstacle lies in how to target dendritic cells (DCs) to activate the immune response. In a recent study, RNA-lipoplexes (RNA-LPX) without ligand decoration were designed to target DCs for the delivery of cancer antigens and activation of immunoreaction post intravenous administration. Through adjusting the charge ratio of formulation, the positively charged RNA-LPX primarily targeted the lungs and the fluorescence expression shifted from lung to spleen with a reduction of the cationic lipid content. Notably, in a phase I clinical trial (NCT02410733), three melanoma patients were treated with RNA-LPX encoding the tumor antigens, and the produced IFNα and T-cell responses demonstrated the broad applicability of the RNA-LPX considering that antigens of any tumor cells could be encoded by RNA via intravenous (i.v.) injection or locally administrated via subcutaneous (s.c.) injection, intramuscular (i.m.) injection, intradermal (i.d.) injections, and so on Apart from vector materials, the routes and sites of administration significantly influence the therapeutic outcomes and biodistribution of RNA drugs via the intravenous injection are prone to concentrate selectively in the liver because of its abundance of ApoE, exerting an essential influence on LNP-mediated RNA drugs et al. reported that intraperitoneal administration of LNPs enabled a shift in specific mRNA expression from the liver to the pancreas Intravenous injection is commonly used for LNP-mediated delivery of mRNA or siRNA drugs. This route of administration allows the drugs to reach the target site through circulation. Notably, most LNPs administrated et al. converted LNPs from liver-targeting to lung-targeting et al. developed phosphatidylserine LNPs that could mediate effective mRNA expression in both lymph nodes and spleen after intravenous injection et al. reported a SORT strategy, which achieved the redirection of LNPs to extrahepatic organs by supplementing SORT molecules. Specifically, LNPs were shown to target the spleen by adding anionic lipids and modulating their formulations et al. showed that zwitterionic phospholipidated polymers efficiently delivered mRNA to the spleen, and these polymers with specific amphiphilic and zwitterionic structures exhibited lymph node transfection capability Numerous efforts have been made to systemically deliver RNA to the lung for therapeutic use in pulmonary diseases. For instance, by rationally designing the structure of cationic lipids, Qiu via intravenous administration, inhalation or intratracheal injection via a microneedle-based system and indicated that this technique could be used as a collagen replacement therapy for the treatment of skin diseases and aging via intramuscular injection, enabling the drugs to be concentrated in the draining lymph nodes and trigger a strong immune response from T cells As systemic delivery targets specific organs with intricate difficulties, local administration can be a preferable alternative for certain clinical applications. RNA-based drugs administered by topical delivery are expected to show a therapeutic effect at the specific site. In the case of treating pulmonary diseases, RNA drugs can be administrated to the lung After numerous studies presenting the challenges faced by vectors in delivering RNA, it is evident that the barriers preventing the selective accumulation of drugs may be closely associated with the physicochemical properties of designed vectors via intravenous administration, owing to the adsorption of ApoE by binding to the LDLR that are over-expressed on the hepatocytes Ka values can influence their organ targeting. More specifically, the preferred pKa range for liver, spleen, and lung targeting is 6.2~6.5, 2~6, and 7~9, respectively The formation of protein coronas is believed to be closely associated with the biodistribution of nanoparticles in the body. Upon intravenous injection, the nanoparticles come into contact with the serum, leading proteins to adsorb on their surface and form a distinctive protein corona layer in vivo. For instance, previous studies have revealed that nanoparticles with a diameter of approximately 5 nm are rapidly cleared by the renal filtration process 2), making it an appealing target for RNA therapeutics upon systemic drug delivery. To maximize the drugs' efficacy in the lungs, particles with diameters of less than 3 μm are generally preferred, as they optimize deposition in the alveoli The particle size and the dispersion of delivery vectors play a crucial role in constructing target delivery systems that meet different clinical needs. In order to overcome multiple intracellular and extracellular delivery barriers, attention should be paid to how the vector size affects the bio-interface interactions. Vectors of different sizes present different organ or tissue tropism via the interaction with negatively charged cell membranes. However, high-density surface positive charges can be correlated with corresponding toxicity, thus limiting their application to some extent. In accordance with the desired therapeutic effect, researchers have developed amphiphilic polymers or ionizable lipid nanoparticles that exhibit outstanding endosomal escape efficiency than conventional permanent positive vectors. These new materials are generally neutral under the physiological condition so as to mitigate the toxicity et al. adjusted the lipid charges in LNPs, mediating controlled pKa and specific organ tropism of nano-systems Ka may likewise exhibit a propensity to accumulate in different organ tissues, which is one of the pivotal factors in the design of vectors. Therefore, the rational design of non-viral vectors with desired charges can lead to satisfactory therapeutic effects for RNA-based drugs.Surface and vector charges of nanoparticles are significant physicochemical properties that can be engineered to enhance the site-specific accumulation of RNA vectors and affect their delivery profile Other characteristics, such as particle shape and biodegradability, should also be taken into consideration when designing and evaluating the delivery performance of vectors. Studies have demonstrated that the shape of the nano-vectors plays a crucial role in its delivery journey. For instance, disc-shaped nanoparticles interact more effectively with the vessel wall and exhibit higher specificity for endothelial cells compared to spherical ones Over 20 years have passed since the first RNA-based drug was approved by FDA, and now several RNA drugs have been approved for therapeutic applications in different diseases, including TTR-mediated amyloidosis, duchenne muscular dystrophy, hypercholesterolemia, etc. Additionally, hundreds of candidate drugs are currently under clinical studies. Here, we present a summary RNA-based drugs that have been applied in clinics Table , includiKLKB1 gene, could block production of kallikrein and further reduce bradykinin to treat hereditary angioedema. In clinical I/II trial (NCT05120830), the NTLA-2002 encapsulating Cas9 mRNA and KLKB1-specific sgRNA delivered by LNP resulted durable reductions of kallikrein protein in plasma with well tolerability.mRNA therapy has gained immense popularity since the outbreak of COVID-19. Although no mRNA drug for cancer treatment has been approved by the FDA, there are several mRNA-based therapies for cancer in various stages of clinical trials, which can be divided into two major categories roughly based on the encoded antigens All the mRNA drugs mentioned above utilize LNPs as the delivery vectors, highlighting their significance in the field of RNA-based drugs. However, one exception is AZD-8601 mRNA, which has entered clinical trial (NCT03370887) for heart failure. AZD-8601 mRNA is formulated in citrate buffered saline with biocompatibility and is delivered by epicardial injections to express vascular endothelial growth factor A protein directly. Developed by AstraZeneca and Moderna, this drug increased left ventricular ejection fraction and enhanced heart function compared to placebo group via lactate dehydrogenase (LDH) and excretion of oxalate in urine, which can cause severe kidney injury or kidney failure N-acetylgalactosamine  can achieve liver-targeting and the mechanism is that the GalNAc can combine with asialoglycoprotein receptors (ASGPRs), which are overexpressed by hepatocytes specifically via inhibiting activation of complement component 5. Alnylam declared that in phase II clinical trials (NCT03841448), Cemdisiran reduced the ratio of urinary protein and creatinine (UPCR) by an average of 37% at 24 hour without adverse effects after subcutaneous administration compared to placebo group Most of these drugs approved by FDA target the liver, particularly siRNA drugs. In addition to the five siRNA drugs receiving market authorization, there are also some siRNA drugs in clinical trials and most of them are designed to treat liver-related diseases, such as Nedosiran. Primary hyperoxaluria (PH) is a metabolic disease caused by abnormal liver metabolism, resulting in excessive oxalate production from glyoxylate et al. have demonstrated the powerful effect of CDR132L in large pig animals and the results showed that this drug significantly improved the systolic and diastolic function of the heart and reversed cardiac remodeling after repeated monthly administration via aerosol administration Similar to siRNA, ASO drugs on the market are primarily used to treat patients with rare diseases, but they have a wider range of target organs, including liver, muscle, eyes, and so on. The diseases they address include nervous system diseases, muscle diseases, cardiovascular diseases, and metabolic diseases. In addition to rare diseases, an increasing number of ASO drugs are being developed for other common diseases and are currently in middle- or late-stage clinical trials It is noteworthy that over half of the clinic trials on RNA-based drugs are focused on mRNA, highlighting the huge potential of mRNA therapeutics in RNA-based therapy at present. However, the majority of active clinic trials involving mRNA are derived from COVID-19 vaccines and for the indication of infections. If we exclude these vaccine-related trials, most remaining trials are still in the early-stage Since the discovery of the therapeutic potential of RNA drugs half a century ago, numerous delivery platforms have emerged to enable the safe and efficient delivery of RNA. Through the diligent efforts of researchers in library screening and rational design of delivery systems, several RNA-based drugs have received marketing approval or are currently undergoing clinical trials. As the focus has gradually shifted towards better adapting to clinical needs, efforts are being made to target specific sites for the treatment and reduce the side effects of RNA therapy. With a deeper understanding of the targeting mechanism, researchers are striving to achieve targeted therapies for RNA drugs by using different categories of vectors for delivery, tailoring vector chemical structures, adapting various administration routes, and altering physicochemical properties of vectors. These major advances in delivery systems provide great potential for RNA therapy.Although the first siRNA drug utilizes the LNP vector, the following four siRNA approved drugs instead employ the GalNAc technology, which has become the primary strategy for siRNA-targeted delivery to hepatocytes. While in the field of mRNA-based therapy, advanced LNPs have been developed in two widely inoculated vaccines for COVID-19, known as BNT162b2 and mRNA-1273. This breakthrough has facilitated the exploitation of mRNA-target delivery systems, which have enabled selectivity for organs or tissues by modifying the lipid chemical structures and their formulations. Despite the progress, much work remains to be undertaken to achieve better clinical translation. 1) A deeper understanding of the targeting mechanism is required to develop universal guidelines for achieving targeting to each organ or even different cell subtypes, thus facilitating the design of next-generation delivery systems. 2) Additional investigations on the physicochemical properties, morphology, stability, and other characteristics of the vectors are required to facilitate further targeting and druggability. One can design vectors with specific sizes or charges to achieve targeting delivery of RNA according to the characteristics of different organs and tissues. 3) More attention can be paid to the inherent targeting ability of some natural vectors and figuring out the mechanism of their native tropism, which can guide the future specific targeting. Identifying the exact mechanism of targeting delivery will fundamentally help to design the appropriate vectors to achieve precise delivery. 4) Biocompatibility of vector materials should be considered to reduce toxicity and avoid undesired immunogenicity. 5) Inspired by protein corona-guided and GalNAc-conjugated targeting delivery system, more efforts should be made to screen specific receptors possessed by cell subsets for cellular level targeting. 6) Stepwise targeted drug delivery strategy from organ, tissue, cell to organelle may be utilized to overcome obstacles in delivery process and improve delivery precision.Overall, the targeted RNA-based therapies have already demonstrated tremendous therapeutic potential in numerous clinical applications. As an integral part of RNA therapeutics, the development of a more precise delivery system is of undeniable significance. The prospects are promising that targeted RNA drugs have the potential of utility in diverse disease fields. Although some challenges remain for targeted RNA delivery, the enormous and continuous advances in RNA therapy will undoubtedly improve our health and lives.


Cellular plasticity defines the capacity of cells to adopt distinct identities during development, tissue homeostasis and regeneration. Dynamic fluctuations between different states, within or across lineages, are regulated by changes in chromatin accessibility and in gene expression. When deregulated, cellular plasticity can contribute to cancer initiation and progression. Cancer cells are remarkably plastic which contributes to phenotypic and functional heterogeneity within tumours as well as resistance to targeted therapies. It is for these reasons that the scientific community has become increasingly interested in understanding the molecular mechanisms governing cancer cell plasticity. The purpose of this mini-review is to discuss different examples of cellular plasticity associated with metaplasia and epithelial-mesenchymal transition with a focus on therapy resistance. In 1957, the biologist Conrad Waddington described embryonic development as a series of distinct paths that stem cells follow before reaching an irreversible terminal differentiation stage . This prSince then, a growing number of studies conducted on different organs have demonstrated the ability of committed cells to be reprogrammed in response to various stimuli. Currently the term “plasticity” refers to the cell’s ability to adopt different identities through multiple mechanisms, such as dedifferentiation or transdifferentiation, particularly when tissue homeostasis is perturbed. Interestingly, cancer cells can alter their identity by adopting alternative developmental lineages. Lineage plasticity is a mechanism commonly employed by tumour cells to evade chemotherapy treatment, leading to intra-tumour heterogeneity, therapy resistance and metastasis. The development of single cell sequencing technologies has profoundly contributed to gathering new insight into tumour cell plasticity. This has led to the identification of previously uncharacterized cell states and allowed us to trace transitions between different states. As a result, we have gained a better understanding of the mechanisms underlying tumour heterogeneity and resistance to therapy offering us new therapeutic prospects.Here, we will discuss concisely recent literature on metaplasia and EMT, two biological processes that regulate the emergence of distinct cell identities in response to specific stimuli, with a final emphasis on lineage plasticity and therapy resistance.Metaplasia is an adaptive process characterized by the replacement of a differentiated cell type with another cell type not normally observed in the tissue where it occurs. It is mainly characterized by changes in cell morphology and expression of lineage-specific markers, including transcription factors defining tissue-specific identity. Metaplasia affects organs constantly exposed to external damaging agents or suffering from chronic inflammation, such as the lungs, intestinal tract, and pancreas. It is generally considered a protective process to limit tissue injury. However, metaplasia is also considered a precursor to dysplasia, and can be associated with an increased risk of malignancy. Here, we will discuss the most common forms of inflammatory-driven metaplasia which includes the specialized intestinal metaplasia (SIM) reported in Barrett’s esophagus (BE) and the acinar-ductal metaplasia (ADM) observed in the pancreas.in vivo model able to recapitulate many aspects of human disease has left the question about its origin and the association to EAC unanswered for several years. In 2012, M. +ve progenitor cells in the gastric cardia as the origin of BE. In 2017, M. +ve, KRT5+ve, KRT7+ve basal progenitor cells, localized in the squamous/columnar junction of the upper gastrointestinal tract, can differentiate into intestinal cells, including goblet cells, recapitulating SIM. Few years later, BE is a metaplastic condition in which the normal stratified squamous epithelium, covering the distal esophagus, is gradually replaced with a columnar intestinal-like epithelium, including goblet cells, in response to chronic exposure to acid and bile in patients with gastroesophageal reflux disease (GERD). It has been suggested that the goblet cells’ cytoprotective role makes the epithelium more resistant to GERD-induced damage. Nevertheless, BE can progress into low-grade and high-grade dysplasia and culminate in the development of esophageal adenocarcinoma (EAC) . BE pathCdx2 from early endoderm affects the expression of additional TFs that define the intestinal identity, such as CDX1 and HNF1α, and leads to squamous differentiation through the activation of the esophageal differentiation program  such as Twist1, Zeb1, Snail1, and Slug  Figure . DespiteVarious degrees of dynamic hybrid states have been reported among cancer cells, indicating their ability to shift between multiple states. Compelling work by I. Pastushenko and co-workers, performed on a mouse model of skin squamous cell carcinomas undergoing spontaneous EMT and on metaplastic-like mammary tumours, led to the identification of different tumour subpopulations with a spectrum of intermediate EMT states which contribute to tumour heterogeneity. Interestingly, the different intermediate subpopulations show varying degrees of invasive and metastatic capacity, with hybrid epithelial-mesenchymal cells showing an increased capacity for entering the circulation and forming lung metastases . A numbein vivo, where cells gradually lose epithelial markers and gradually acquire EMT markers with late hybrid EMT transcriptional signatures associated with higher risk of death in patients with PDAC and lung cancer (Intriguing discussion has arisen regarding the existence of a phenotypic continuum rather than the presence of stable discrete EMT states. MAGIC, a computational method that recovers missing gene expression in individual cells, has significantly improved scRNA seq quality data and, when applied to an EMT model, has revealed a transcriptional continuum gradient with the majority of cells residing in intermediate states . This alg cancer . The grag cancer .Finally, it is worth mentioning that alternative EMT programmes can be adopted during tumour progression. Using a lineage-labelled murine model of ductal pancreatic cancer, in vitro and in vivo tumour models. For instance, EMT has been associated with resistance to gemcitabine treatment in models of PDACs (During cancer progression, cancer cells undergo extensive genetic and epigenetic reprogramming which reveals their latent ability to change lineages. The expansion of emerging subclones with a new identity is highly context specific, can contribute to intra-tumour cell heterogeneity and adversely affect treatment response as new cell types have different sensitivity to the original drug target, which may even be no longer present. Thus, targeting communal regulatory mechanisms of the epithelial-mesenchymal plasticity could prove to be a more efficient therapeutic strategy  Figure . Severalof PDACs . Accordiof PDACs . Furtherof PDACs . Debaugnof PDACs . More reof PDACs .The process of therapy resistance is further complicated because chemotherapy treatment itself can trigger lineage plasticity and promotes drug resistance. As an example, EGFR mutant lung adenocarcinomas that are repeatedly treated with tyrosine kinase inhibitors develop resistance within months of treatment. In a subgroup of patients with resistance to treatment, evidence of small-cell lung cancer transition has been reported, indicating that treatment can trigger the transformation of the tumour into a distinct histological subtype . TransdiThe studies discussed in this mini-review highlight the cells’ remarkable capacity to adapt and change their fate and identity in response to stimuli and cancer. Perturbations of adult tissue integrity result in a prompt adaptive response characterized by extensive cellular reprogramming. This is mainly regulated by a complex network of TFs that define cell identity. In this context, cell plasticity may serve as a repair mechanism, which, at least initially, may be beneficial since reprogrammed cells can adopt new functions to limit damage. However, it is now widely accepted that even this form of “advantageous” cell plasticity can predispose to cancer. Therefore, defining the molecular mechanisms that govern this functional switch is imperative to stop tumour progression. Considering this, several questions remain open including: how do specific insults, like, for example, inflammation, regulate the expression of TFs that determine cell identity? What are the long-term consequences of early changes in chromatin accessibility for tumour initiation and progression?In regard to EMT, as discussed in this mini-review, the transition from an epithelial to a mesenchymal state can manifest through two distinct models: a continuum spectrum of transitional states or multiple discrete states. Despite this apparent discrepancy, perhaps caused by different technical approaches, what overall emerges from these studies is the extraordinary capacity of EMT cells to dynamically shift between multiple different states. However, in response to specific cell intrinsic and extrinsic factors, cells within the EMT continuum can stall and reside in intermediate and more stable states. This considerably contributes to tumour heterogeneity and therapy resistance. Open questions that arise from these observations are: which model plays a more critical role in tumour development and metastasis formation? Can we target a stable discrete state or a range of multiple states? Can we shift EMT cells toward a less aggressive or more targetable state?In addition to EMT, distinct forms of cell plasticity have been reported in cancer posing new challenges in investigating cell heterogeneity and therapeutic approaches. However, given the rapid progress with single-cell sequencing technologies and methods of analysis the function of novel cell plasticity regulators will be described soon potentially unveiling new therapeutic avenues.


Despite the improvements in endovascular techniques during the last decades, there is still an increase in the prevalence of peripheral artery disease (PAD) with limited practical treatment, which timeline impact of any intervention for critical limb ischemia (CLI) is poor. Most common treatments are not suitable for many patients due to their underlying diseases, including aging and diabetes. On the one hand, there are limitations for current therapies due to the contraindications of some individuals, and on the other hand, there are many side effects caused by common medications, for instance, anticoagulants. Therefore, novel treatment strategies like regenerative medicine, cell-based therapies, Nano-therapy, gene therapy, and targeted therapy, besides other traditional drugs combination therapy for PAD, are newly considered promising therapy. Genetic material encoding for specific proteins concludes with a potential future for developed treatments. Novel approaches for therapeutic angiogenesis directly used the angiogenetic factors originating from key biomolecules such as genes, proteins, or cell-based therapy to induce blood vessel formation in adult tissues to initiate the recovery process in the ischemic limb. As PAD is associated with high mortality and morbidity of patients causing disability, considering the limited treatment choices for these patients, developing new treatment strategies to prevent PAD progression and extending life expectancy, and preventing threatening complications is urgently needed. This review aims to introduce the current and the novel strategies for PAD treatment that lead to new challenges for relief the patient’s suffered from the disorder. According to recent reports, 237 million of 25 years old and older people were suffered from PAD in the last decade. Chronic limb-threatening ischemia, defined as the most violent form of PAD, originated from narrowing or occlusion of arteries, and there is a significant risk of limb amputation, mortality. It estimated costs to the healthcare system to range from $84 billion to $380 billion annually.9 Current common therapies for PAD patients consist of treatment strategies, including lifestyle modifications, exercise, smoking cessation, and pharmacologic treatments like antiplatelet therapy, anticoagulation, management of blood pressure, cholesterol reduction, and peripheral vasodilators, endovascular repair, and surgery.14 The common side effects have reported after administrating medications for PAD are from severe hemorrhages in some cases to gastrointestinal dysfunction, major bleeding events, osteoporosis, thrombocytopenia, hypersensitivity reaction and skin necrosis16 Difficulty in controlling the lifestyle in different classes of society on the one hand and the detrimental side effects of common medications, such as bleeding, on the other hand, emphasize the need for the emergence of new, advanced, and accurate therapies. Moreover, due to the high rate of mortality and morbidity that lead to the disability of individuals and considering the limited treatment choices for patients suffering from severe PAD, developing new treatment strategies to prevent the disease progression and control the life-threatening complications is urgently needed. Nowadays, the promising role of regenerative medicine, cell therapy, targeted therapy, gene therapy, Nano-therapy, and combination drug therapies in PAD management is highlighted  discusses the effect of clopidogrel on the background of low-dose aspirin therapy for a clinically demonstrated duration compared to 12 months in patients receiving endovasculartherapy.24 The LONGDAPTPAD  compares 3 months DAPT compared with 12 months.25 In addition to chemical drugs, supportive therapies to relieve pain are common in patients. The mechanism of the action of traditional medicines on pain has been previously investigated.27 Although the role of DAPT in acute MI was well established, the impact of DAPT in PAD is unclear. Researchers conducted an observational cohort study of 629 patients with claudication or critical limb ischemia (CLI) treated either with DAPT or aspirin monotherapy and followed them for 3 years. There was a significant reduction in major adverse cardiovascular events (MACE) in patients receiving DAPT vs. aspirin alone.28 This combination therapy signifies an important breakthrough in the treatment of patients with PAD.29 In a double-blind trial, patients with PAD undergoing revascularization were randomized to take Rivaroxaban plus aspirin or placebo plus aspirin. Compared to aspirin monotherapy, this combination therapy significantly reduced the composite outcome of acute limb ischemia (ALI), major amputation for vascular causes, MI, ischemic stroke, or cardiovascular causes-related death.30 Rivaroxaban is a direct OAC that inhibits Factor Xa. Although it is a relatively new medication, it was widely used to treat venous thromboembolism and stroke prevention in non-valvar atrial fibrillation. In a double-blind trial of 27 395 participants with stable atherosclerotic vascular disease, those given Rivaroxaban plus aspirin had better cardiovascular outcomes and the most severe bleeding events than those given aspirin alone. Not only Rivaroxaban monotherapy does not lead to better cardiovascular outcomes than aspirin monotherapy but also it resulted in the most severe bleeding events.31 Administration of ACEI/ARBs is associated with reducing MACE as well as death rates in patients with CLI. There was no correlation between ACEI or ARB administration and limb-related outcomes. Paying more attention to secondary prevention measures in CLI patients can significantly improve long-term outcomes.33 Among the most suitable effects of statins in PAD are decreased platelet activation, endothelial dysfunction, and inflammatory responses to the atherothrombotic progression. Statins cause beneficial changes in atherosclerotic plaque composition, reducing their necrotic core and overall atheroma volume. Initiation of statin therapy in patients with the peripheral arterial occlusive disease after index revascularization is effective and safe with an effect size comparable to previous studies.34 Evolocumab reduces the low-density lipoprotein (LDL)-Cholesterol plasma levels by inhibiting the function of proprotein convertase subtilisin-Kexin type 9 (PCSK9). A combination of evolocumab and statins was compared with placebo plus statins in 27 564 patients with cardiovascular disease in a double-blind, randomized trial. This study noted a 15% reduction in composite cardiovascular events at 2.2 years for evolocumab-treated patients, with a low frequency of side effects for both groups.35 Patients with PAD are at high possibility of cardiovascular events, and inhibition of PCSK9 with evolocumab significantly reduces this risk. Besides, lowering LDL cholesterol by evolocumab reduces the risk of major adverse limb events.7 This is called therapeutic angiogenesis, which means directly using angiogenesis factors such as genes, proteins, or cell-based therapy to induce blood vessel formation in adult tissues and initiate the recovery process in the ischemic limb , fibroblast growth factor, and hepatocyte growth factor (HGF), for instance, would cause hemangioma formation.38 Therefore, the long-expression of proangiogenic factors should be avoided when finding effective treatment options.39 While several studies used naked plasmid DNA expressing HGH and VEGF, researchers decided to use internal ribosome plasmid vectors in patients with PAD to examine its safety and efficacy.40 Their data confirm that IM administration of internal ribosome carried VEGF165/HGF is safe, feasible, and effective for patients who suffer from ischemia in the limb body. Using a lentivirus-mediated vector, others investigate the expression of endothelial Per-Arnt-Sim domain protein 1 (EPAS1) on PAD in a rat model. The hypoxia-inducible factors-2α or originally named EPAS1 associated with pathologic vascular wall remodeling, and identifying the HIF pathway is very important in the line of introducing novel therapeutic strategies.41 Recent studies revealed that mRNA expressions of EPAS1, HGF, basic fibroblast growth factor (bFGF), and VEGF were up-regulated in the lenti-EPAS1-treated sites resulted in recovered limb function and circulation after 7 days.42 In addition, during tissue ischemia in PDA, lipid oxidation is associated with inflammation, leading to the protein adducts inducing angiogenesis. Recent reports indicated that CEP (x-[2-carboxyethyl] pyrrole) as one of these proteins has a profound positive effect on angiogenesis.43 CEP (x-[2-carboxyethyl] pyrrole) protein, a product of lipid oxidation, induces angiogenesis in PAD.38 Furthermore, some cells can take up spontaneously naked plasmid DNA.29 To identify novel therapeutic targets for gene therapy and any other specific new techniques, in silico analysis was conducted by researchers to identify the molecular basis of PAD. They found a significant enhancement in the inflammatory response, immune response, chemokine-mediated signaling pathway, and JAK-STAT signaling pathway. Interleukin 6 (IL-6), C-X-C Motif chemokine ligand 12 (CXCL12), interleukin 1 beta (IL1B) are some of the chemokines that participate in the progression of the disease. IL6, CXCL12, IL1B are some of the chemokines that participate in the progression of the disease, and signal transducer and activator of transcription 3 were differentially expressed and regulated 27 potential target miRNAs in PAD. Although this data needs to be further verified in experimental studies, this makes a new vision regarding new targets for gene therapy. This indicates that gene therapy should be considered an extra tool for PAD treatment, while there is room for another technical improvement. On the other side, one problem with gene therapy is the fact that it is strongly linked to dose-dependent microvascular permeability. Around half of the patients faced moderate or severe edema during clinical trials, indicating that long-term and slow-release are more effective. This is the main reason that some experts suggest cell therapy instead of gene therapy, while its effectiveness is still the place of debate. During PAD and the subsequence, CLI as its end-stage redox biology and oxidative stress play an important role in this setting. Hence, some of the studies pay attention to strategies that inhibit ischemia through pharmacological intervention, such as statins, angiotensin-converting enzyme inhibitors, and phosphodiesterase inhibition. Numerous studies provided us encouraging data about the potential role of several agents, but in targeted therapy, underlying conditions that may contribute to an improvement of PAD or failure in treatment must be considered. Although the use of gene therapy to treat apple disease is an attractive and promising method, it is also associated with the general limitations of gene therapy methods. In gene therapy methods, the use of silencers is one of the most common methods of gene therapy. Proper design, fabrication, transmission, stability, and targeting of silencers are some of the major challenges facing gene therapy methods. For some of these issues, solutions have been proposed however are not currently definitively accepted. For example, the development in altered bases that are created to increase the stability of silencers such as siRNA within the body system. Other limitations of gene therapy are the development of immunological responses and innate immune system stimulation that can lead to unwanted and severe symptoms. Another challenge in the treatment of various diseases with gene therapy is Accelerated blood clearance, which leads to the rapid elimination of gene therapy cases from the bloodstream.44 Cell therapy is known to be curative for disease conditions like diabetes mellitus, vascular complications, ischemic stroke, and trauma which offered novel treatment options for regenerative medicine, including the engineering and regenerating de novo generated cells considered the emerging field of medicine and helped substitute current treatment with newly developed treatment strategies. The beneficial focus on cell therapy for PAD is not limited to the injection of cells into the vascular network to effectuate a medical impact. Cell therapy could activate unknown cytoprotective/ regenerative pathways to recover limb activity via its independent effect on the neo-vessel formation.45 Alternatively, most clinical studies reported that the cell transplantation effectiveness in patients suffers from CLI.47 Experimental studies have been used the hindlimb ischemia models to investigate the effectiveness of cell therapy in angiogenesis promoting and reducing skeletal muscle damages. Identifying ideal endothelial progenitor cells’ source for therapeutic application in PAD needs further investigations. Cell therapy projects using various cell types are investigated in different phases of preclinical/clinical trials for PAD treatment. In these studies, each cell type indicated a promising approach for PAD treatment in preclinical studies. Several randomized, controlled trials were carried out in this field from 2010, which are discussed below .54 Intramuscular (IM) injection has been the most common method used in preclinical and clinical studies. MSCs have also been injected intravenously in both preclinical and patient models. In addition, several clinical trials performed on bone marrow-derived mononuclear cells have successfully used intra-arterial injections. Some investigations have shown that MSCs are therapeutically effective when injected immediately after induction of hind limb ischemia. In a similar study, a 24-hour delay after ischemia induction has also been shown to be more effective than immediate injection. The delay between the onset of disease and therapeutically effective administration of MSCs seems to be wide. For example, bone marrow-derived MSCs transplanted one hour, one week, or two weeks after MI were all effective, although injection approximately one week after infarction showed the highest recovery of myocardial function as the acute inflammatory reaction is almost complete by this time, the success in delayed injections may be a consequence of enhancement in the survival of transplanted cells. More concern is about clarifying the optimal dose for cell therapy because in general, only a small fraction of MSCs survives and can exert a therapeutic effect through paracrine mechanisms or final determination. The tendency to use large numbers of MSCs for transplantation has to be weighed against reports of complications from such efforts. However, further research is needed to optimize the concentration, timing and delivery of MSCs that are critical for effective neovascularization in PAD55 as described in  Mesenchymal stem cells (MSCs) are promising as a treatment for peripheral arterial diseases. MSCs can be derived from a variety of potential sources and administered by several techniques.57 There are many challenges to using human embryonic stem cells, as obtaining these cells requires the destruction of ballast cells, which is not accepted by some ethical and religious schools. To solve this problem, the use of other types of stem cells such as adult stem cells, amniotic stem cells, and induced pluripotent stem cells was suggested. It can be said that the most important limitations related to cell therapy are technical limitations about obtaining, differentiating, and transferring these cells to the intended destination.58 However, due to these molecules’ short distribution half-life, applying these naked molecules has not resulted in an appropriate clinical effect. Different forms of NPs were produced to address this problem and are being used to transport nucleic acids and proteins.61 Nono-technology and nanoparticles (NPs) are important strategies that can help clinicians deliver the drug agents to the specific site, finding the new route of administration and constant treatment of diseases. Over the previous century, nucleic acids and protein distribution have been widely used in scientific and clinical trials to increase the effectiveness of PAD therapy.64 In addition, these nanostructures are commonly known to be effective in transduction due to electrostatic interactions between the primary amine terminal groups with incorporated DNA or siRNA.67 Liposomes are often used as vascular transmission vectors because of their biological compatibility and wide range of vascular uses.69 Proteins are encapsulated within NPs to shield them from enzyme degradation and ensure regulated release into cells used for greater therapeutic efficacy. These carriers can help improve drug reaction by targeting and adhering to the affected areas, thus synergizing the drug’s effectiveness.70 By increasing permeability and retention, nanoliposomes can be useful in treating myocardial ischemia by improving angiogenesis and treating ischemic myocardium as a carrier of therapeutic agents.71 Researchers improved liposomal blood supply and promote drug transfer by altering the surface of liposomes with polyethylene glycol (PEG)  and complementary DNA (cDNA) plasmid distribution due to their exceptional characteristics, including strongly branched polymers with three major regions  where alteration can happen. This structure was studied in the treatment of coronary artery disease and gene transfer to dysfunctional smooth muscle cells (SMCs) in rabbit vessels. It has strong effectiveness and therapeutic potential for PAD.ol (PEG) .7274 Polymer NPs have been used to transfer genes for targeted ischemic treatment in peripheral arteries and may be used to substitute viral vectors and fix their safety issues. Loading cDNA VEGF on NP compared to naked plasmid can stimulate angiogenesis in rabbit ischemic models. NPs containing chitosan, thioglycolic acid, and encapsulated the secretoneurin peptide to improve angiogenesis in the hind leg of mice, resulting in controlled release for optimum effects and dense arteriole formation. Probucol is an anti-angiogenesis and apoptosis-inducing agent used to control hypercholesterolemia. The drug was synthesized in PLGA polymer carriers and liposomes. According to the findings, probucol in PLGA nanocarrier successfully prevented seizures through injection into rabbit arteries following angioplasty. Growth factors in combination with NPs have emerged as a promising strategy for treating patients with PAD. bFGF can be used as long-term angiogenic therapy in heparin-bound poly(L-lactide-co-glycolide) nanospheres (HCPNs). This compound was studied to treat a mouse ischemic limbs model, in which microvasculature developed in ischemic organs treated with bFGF delivery, and it was discovered that sustained release of bFGF from HCPN could enhance the angiogenic effect.75 For PAD therapy, cyclic arginylglycylasparticacid (RGD) peptide conjugated liposomes were engineered to interact with P-selectin and integrin receptors.99mTc was used, which demonstrated a reduction in drug activity. The therapeutic effects of angiogenic peptides loaded in PEGylated nano liposomes improve perfusion defects. (72 According to recent report, angiogenic peptide-loaded Nano liposomes improve vascular density. The perfusion depicts an autoradiogram of the myocardium after 30 minutes of ischemia and reperfusion. To detect the result of therapy, tetrofosmin defects. .72 The beneficial effects of PAD cell therapy are not limited to the cells in the vascular network; the paracrine effects induced by the secretion of antigenic mediators are so important. During PAD and the subsequence, CLI as its end-stage redox biology and oxidative stress play an important role in this setting. Hence, some of the studies pay attention to strategies that inhibit ischemia through pharmacological intervention, such as statins, angiotensin-converting enzyme inhibitors, and phosphodiesterase inhibition. Numerous studies provided encouraging data about the potential role of several agents, but in targeted therapy, underlying conditions that may contribute to an improvement of PAD or failure in treatment must be considered. Scientific researchers have indicated that novel treatment strategies like regenerative medicine, cryotherapy provides the growth of new healthy tissues, safe and effective autologous therapy, fast recovery of patients from injuries, and therapeutic surgeries, and that’s benefits consist for many years. Gene and targeted therapy can resolve and eliminate the compared problems related to the side effects of current drug treatments like anticoagulants, including bleeding, abdominal pain, flatulence, headache, lethargy, dizziness, and fever. Novel emerging treatment strategies for PAD may be a bright prospect to replace new and practical treatments with fewer side effects besides new, more efficient, and comfortable treatment options for patients and clinicians to advance clinical decisions. The authors thank Ardabil University of Medical Sciences, Deputy of Research and Technology, for their collaboration with this review. The authors declare no conflict of interest. Not applicable.


Recently, cognitive deficits occurring in rheumatic diseases have attracted scientific attention. Cognitive symptoms in patients with Rheumatoid Arthritis (RA) and Systemic Sclerosis (SSc) have not been thoroughly studied. This study aimed to assess cognitive function and its relationship with depressive symptoms in RA and SSc and compare it to mild neurocognitive disorder due to Alzheimer’s disease (MiND) and to individuals without cognitive impairment.Cognitive function and depressive symptoms were tapped with the Cognitive Telephone Screening Instrument plus (COGTEL+), the Serial Seven Test (SST), the Mini-Mental State Examination (MMSE) and the Geriatric Depression scale-15 (GDS), respectively. Statistical analyses included between groups-, correlation- and regression analyses. Demographic characteristics were considered in the regression models.The study included 30 individuals with RA, 24 with SSc, 26 adults without cognitive impairment and 33 individuals with MiND. Lower performance in verbal short-term memory, concentration/attention, verbal fluency and MMSE in patients with RA compared to individuals without cognitive impairment was detected. Of note, performance on verbal fluency, concentration/attention, inductive reasoning and MMSE was lower in RA compared to MiND. Individuals with SSc performed worse in verbal fluency and in MMSE in comparison to adults without cognitive deficits. Verbal fluency deficits in SSc exceeded that in MiND. Performance on MMSE, COGTEL+, prospective memory, working memory, verbal fluency and concentration/attention was related to GDS scores, which did not vary across the groups.Patients with RA and SSc encountered cognitive dysfunction, which partially pertains to depressive symptoms. Of note, the severity of cognitive dysfunction in many cases exceeded that of MiND. Rheumatoid arthritis (RA) and Systemic sclerosis (SSc) are systemic autoimmune rheumatic diseases. The former approximately affects 1% of the global population, while the latter with a global prevalence of approximately 17.6 per 100.000 is less common . RegardiCognitive deficits and depressive symptoms have been reported in individuals suffering from RA or SSc. Cognitive dysfunction in RA has a prevalence ranging from 38 to 71% and is mainly reflected in difficulties with divided/sustained attention, learning, memory, inhibition, mental flexibility, executive functions and visuo-spatial processing –6. FurthAlthough RA and SSc have been related to cognitive impairment, potential differences in their cognitive phenotypes as well as the associations of depressive symptoms with cognitive function have not been thoroughly investigated yet. The aims of the present study were (i) to study cognitive function and its relationship with depressive symptoms in patients with RA or SSc, (ii) to compare it to patients with mild neurocognitive disorder due to Alzheimer’s disease (MiND), an oligosymptomatic stage of Alzheimer’s disease  negativePatients suffering from SSc and RA, who attended follow-up appointments at the outpatient clinic of the Department of Rheumatology of the Patras University Hospital between January and September 2019, and patients with MiND and individuals without cognitive impairment who were assessed at the psychogeriatric outpatient clinic of the Department of Psychiatry of the above hospital between January and July 2021 (convenience sample) were asked to participate in the study. Inclusion criteria for the entire sample were (i) diagnosis of RA, SSc, MiND or absence of both neurocognitive- and rheumatic disorders and (ii) treatment/assessment at outpatient units of Patras University Hospital. Exclusion criteria were (i) diagnosis of major neurocognitive disorder (ii) diagnosis of MiND caused by a disease other than Alzheimer’s disease , (iii) diagnosis of a rheumatic disease other than RA or SSc, (iv) coexistence of RA or SSc with MiND, (v) mental or neurological disorder or unstable medical condition potentially affecting cognitive function , (vi) hearing or visual difficulties, being potential sources of bias in diagnostic accuracy, (vii) insufficient knowledge of the Greek language (viii) unwillingness to participate in the study. RA was diagnosed according to the updated classification criteria published in 2010 by the American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) . SSc diaThe description of clinical phenotypes and treatment of patients with RA and SSc was based on several parameters. Disease activity was assessed in patients with RA with the Disease Activity Score 28 (DAS28) . Anti-ciCognitive function was assessed with the Cognitive Telephone Screening Instrument plus (COGTEL+), the Serial Seven Test (SST) and the Mini Mental State Examination (MMSE), while depressive symptoms were tapped with the Geriatric Depression scale-15 (GDS) –30. COGTData normal distribution was tested with the Shapiro-Wilk W test. Demographic, clinical and cognitive performance differences were studied with one way analysis of variance (ANOVA) Kruskal–Wallis, Wilcoxon rank-sum (Mann-Whitney) test or chi-square test as appropriate. Post hoc comparisons were performed using Bonferroni post-hoc for the ANOVA, Dunn’s post-hoc test for the Kruskal–Wallis case and adjusted residuals and Bonferroni correction for the chi-square test. Relationships between depressive symptoms and cognitive function on the one side and DAS28- or SHAQ- score and treatment on the other were investigated with the Spearman rank-order correlation coefficient. Stepwise linear, logistic and ordered logistic regression models were employed for studying the relationship between both cognitive function and depressive symptoms, which were included in the models as dependent variables, diagnostic status, demographic - and clinical data, which were the independent variables.The study included 30 consecutive individuals with RA, 24 with SSc, 26 adults without cognitive impairment and 33 individuals with MiND. The demographic and clinical characteristics of the four groups are shown in Table Cognitive function was related to the diagnoses of RA and SSc as well as to depressive symptoms. Group comparisons unveiled significant differences in several cognitive domains between patients with RA and individuals without cognitive deficits, while only performance on verbal fluency tasks significantly differed between patients with SSc and adults without cognitive impairment Table ; Fig. 1.The present study sheds light on cognitive performance and its relationship with depressive symptoms in individuals suffering from RA or SSc compared to individuals without cognitive deficits and people with MiND. The novelty of the study comprises (i) the evaluation of cognitive domains with a COGTEL + capturing interindividual differences in cognition across the full range of adult cognitive functioning; (ii) the consideration of the impact of depressive symptoms in the analyses, since depression can manifest with cognitive deficits or accentuate cognitive impairment ; (iii) tIn patients with RA, performance on verbal short-term memory, verbal fluency, concentration/attention and MMSE was significantly lower compared to that of individuals without cognitive impairment, while differences in inductive reasoning and COGTEL + tended to reach statistical significance according to the final selected regression models, which included age, education and sex and independent variables. Despite the previously prevailing attitude to the absence of cognitive deficits in RA , mountinAccording to the results of the final selected regression models, SSc pertains to worse performance on verbal fluency and MMSE compared to cognitively healthy individuals. It is noteworthy that verbal fluency performance in SSc was lower compared to patients with MiND and better compared to RA. The past few reports focused on SSc cognitive function point to lower performance on memory, verbal fluency, impaired attention, working memory, visual-spatial abilities, executive functions , albeit inconsistently –11. HereDepressive symptoms were found to associate with performance on several cognitive domains. Depressive symptoms were inversely related to performance on tasks assessing prospective memory, working memory, verbal fluency, attention/concentration, as well as with MMSE- and COGTEL + total scores. GDS scores tended to be inversely associated with inductive reasoning performance. Interestingly, in RA the positive association between GDS- and DAS28 reached statistical significance. Depressive phenotypes are closely linked to cognitive impairment . Common The discrepancy between the lack of differences in depressive symptoms between adults without cognitive deficits and patients with either RA or SSc in our study and past reports , 39 may Patients with MiND were found to tend to encounter more depressive symptoms compared to individuals without cognitive impairment and patients with either RA or SSc. Depressive symptoms have recently attracted attention as parts of the neuropsychiatric symptoms that shape the clinical phenotype of MiND . DespiteThe present study has several limitations. First, the size of each diagnostic group was relatively small. Nonetheless, differences in performance on several cognitive domains between the groups attained statistical significance. Second, several factors which had been shown to pertain to the presence of cognitive deficits and/or depressive symptoms in rheumatic diseases   were notCompared to cognitively healthy individuals, the clinical phenotype of RA is related to worse functioning in verbal short-term memory, verbal fluency and concentration/attention, while that of SSc is linked to pooper performance on verbal fluency tasks. Of note, in both RA and SSc verbal fluency performance was lower than in MiND. Thus, the clinical significance of low cognitive function in RA and SSc becomes evident and warrants further investigation in larger samples, so that light is shed not only on the cognitive domains that are affected in RA and SSc and should possibly be regularly screened when these diseases are diagnosed, but also on the pathogenesis of these deficits, in order to develop adequate therapeutic strategies.


This paper provides a concise review of learning-based motion artifacts (MA) processing methods in functional near-infrared spectroscopy (fNIRS), highlighting the challenges of maintaining optimal contact during subject movement, which can lead to MA and compromise data integrity. Traditional strategies often result in reduced reliability of the hemodynamic response and statistical power. Recognizing the limited number of studies focusing on learning-based MA removal, we examine 315 studies, identifying seven pertinent to our focus area. We discuss the current landscape of learning-based MA correction methods and highlight research gaps. Noting the absence of standard evaluation metrics for quality assessment of MA correction, we suggest a novel framework, integrating signal and model quality considerations and employing metrics like ΔSignal-to-Noise Ratio (ΔSNR), confusion matrix, and Mean Squared Error. This work aims to facilitate the application of learning-based methodologies to fNIRS and improve the accuracy and reliability of neurovascular studies. Functional near-infrared spectroscopy (fNIRS), a non-invasive neuroimaging technique, leverages near-infrared light measurements scattered through cortical tissues to estimate changes in oxy-hemoglobin (HbO) and deoxy-hemoglobin (HbR) concentrations, yielding insights into neurovascular coupling and cortical activities , such as high-frequency spikes  have recently become prominent tools in biomedical engineering due to their resilience, real-time capabilities, and ability to handle large amounts of data. This section summarizes current research on learning-based MA removal methods, particularly for fNIRS datasets. Our review methodically examined literature based on criteria in Lee et al.  introducSiddiquee et al.  investigKim et al.  employedGao et al.  proposedHuang et al.  put forwA diverse range of evaluation metrics has been established to assess the performance of artifacts removal solutions, providing insights into noise suppression and signal distortion.The ΔSignal-to-Noise Ratio (ΔSNR) in MA removal involves analyzing the SNRs at both the input and estimation stages, and utilizing their difference as a performance indicator  refers to the reduction or suppression of unwanted noise or artifacts present in a signal, typically achieved through filtering techniques, with the aim of enhancing the quality of the underlying information  evaluation metric determines the percentage difference between the square roots of two signals, serving as a measure of dissimilarity or deviation between them  for Learning-based methods is crucial to systematically gauge the efficacy of learning-based solutions tailored for MA removal. Such a metric would be independent of the particular dataset employed, thereby allowing for an efficient and holistic comparison among various methodologies. The GEM should not merely evaluate the suppression of artifacts within the signal but should also incorporate an assessment of the adapted learning model in terms of robustness, adaptability and accuracy, especially given the rising significance of such models in this domain.When scrutinizing learning-based models tailored for fNIRS MA rectification, it is indispensable to amalgamate modeling-specific metrics with conventional methodologies. Two paramount tools in this arena are the Confusion Matrix and the MSE. The Confusion Matrix offers a nuanced performance evaluation for MA detection tasks by bifurcating instances into “clean” and “contaminated by MA” based on the model's prognostications. This metric doesn't merely clarify the model's performance but also reveals pertinent insights that could be pivotal for model refinement, including potential enhancements in model architecture or judicious alterations to the dataset to ensure a balanced representation.In parallel, other metrics, endowed with their distinct attributes and utilities, are instrumental for a thorough evaluation of fNIRS signals. The MSE stands out as a pivotal metric for MA rectification endeavors. It gains prominence when the rectified fNIRS signal is juxtaposed with the reference signal. Particularly, MSE is the preferred evaluation tool for contexts transcending mere categorical classifications, such as when the output is continuous or regression-oriented. By computing the mean squared deviation between the predicted and the reference values, the MSE offers an acute insight into the model's precision and accuracy. In tandem, the Confusion Matrix and the MSE proffer a comprehensive comprehension of the efficiency of learning-based fNIRS MA rectification models, elucidating potential pathways for optimization.While certain metrics shed light on the overall proficiency of correction techniques, others might encounter impediments in particular scenarios. For instance, representing the CNR's results in a 2D paradigm complicates its numerical integration with other metrics for innovative evaluations. This method is typically adept for inspecting spatial-filtering methodologies, but care must be exercised in the selection of filters. Applying non-uniform filters might inculcate biases, jeopardizing fair comparisons. The PRD demands auxiliary metrics like the Root-mean-square deviation (RMSE) to discern the congruence between signals, and CC to fathom their resemblance. Introducing these metrics augments computational intricacy, which isn't propitious in this context. The within- and between-subject standard deviation methodology, though capable of differentiating neural response variations from MA fluctuations, makes assumptions that may not always hold, rendering it less than ideal as a standalone approach.Acknowledging the complexity of the evaluation landscape, the ΔSNR metric stands out as especially pertinent for tasks focused on signal regression. In the context of the GEM framework, ΔSNR serves as an instrumental metric for quantitatively assessing the efficacy of artifact removal techniques, thereby facilitating numerically grounded comparisons among various methods. Importantly, ΔSNR is optimally suited for evaluating performance on simulated data, given that these datasets come with ground-truth information, enabling more accurate and reliable assessment. Conversely, for the evaluation of classification models, the ΔSNR metric is not applicable. Instead, the use of a confusion matrix is advocated, as it provides a comprehensive and tailored evaluation criterion explicitly designed for classification tasks. It is crucial to recognize that classification models typically require ground-truth datasets for training and validation, necessitating evaluation methodologies that are synergistic with the intrinsic properties of simulated data.In the realm of learning-based techniques for mitigating MAs, the evaluation of both signal fidelity and model efficacy stands as a pivotal pursuit. In the work delineated by Yamada et al. , paramouWhile the MSE metric is frequently utilized to assess the performance of learning-based models, it may also exhibit a correlation with the change in SNR, thereby possibly serving as an ancillary indicator of signal fidelity. The decoupling of these metrics might be considered to mitigate the risk of skewed evaluations of a learning algorithm's effectiveness, especially when both metrics carry positive weightings. For tasks not oriented toward classification, MSE may suffice as the singular evaluation criterion. Considering the scarcity of well-established guidelines regarding the proportional weighting of these evaluation aspects in the realm of classification problems, we suggest the possible introduction of a novel parameter, denoted as “α”. This parameter could be allocated the role of balancing the assessment of signal fidelity, while the remaining weightings may dictate the proportion of focus given to the model's performance evaluation. This nuanced approach might offer a way to harmonize essential facets of signal quality and model performance within the scope of MA removal techniques. Based on these considerations, we tentatively propose a novel evaluation metric, which might be termed the GEM for learning-based methods, and would be mathematically formulated as follows:α is in range where This tentative evaluation criterion outlines a possibly balanced framework for considering both signal refinement and model effectiveness. However, achieving a universal comparison across various learning-based approaches may remain an elusive goal, considering the plausible variability in weight allocations between the two evaluation dimensions. It is our hope that future research might explore standardized weighting schemes to enable more direct performance comparisons among diverse learning-based methods.This review accentuates the burgeoning interest in employing deep learning techniques for mitigating MAs in fNIRS data analyses, attributed to their adaptability and capabilities for real-time processing. These methodologies have shown potential effectiveness across a range of fNIRS datasets, spanning both empirical and simulated data. However, the task of evaluation is rendered complex by the metrics designed for traditional signal processing. This work cautiously suggests an exploratory evaluation approach that amalgamates signal and model metrics through a weighted formula. This may facilitate concurrent assessment of MA suppression and model performance. Despite these advances, the lack of a universally accepted evaluation framework underscores an important avenue for future research. Accordingly, it is imperative that future investigations undertake quantitative evaluation and rigorous statistical analysis to validate the efficacy of this newly proposed metric, thereby fostering a more robust, universally accepted framework for assessment.YZ: Writing—original draft, Writing—review & editing, Methodology, Resources. HL: Writing—original draft, Writing—review & editing. JC: Writing—review & editing. RL: Project administration, Writing—review & editing. SY: Writing—review & editing. HZ: Writing—review & editing.


The constituent proteins of the condensates/aggregates are disentangled by protein disaggregation systems mainly based on Hsp70 and AAA ATPase Hsp100 chaperones prior to their handover to refolding and degradation systems. Here, we discuss the functional roles that condensate formation/aggregation and disaggregation play in protein quality control to maintain proteostasis and why it matters for understanding health and disease.While proteins populating their native conformations constitute the functional entities of cells, protein aggregates are traditionally associated with cellular dysfunction, stress and disease. During recent years, it has become clear that large aggregate-like protein condensates formed  The cellular proteome encompasses proteins that populate a plethora of conformations, from stably folded proteins to those without fixed tertiary structures, consisting completely of intrinsically-disordered regions (IDRs). For example, in human cells the majority of proteins carry both folded domains and IDRs, while 37% of the proteome is fully folded and only 5% of the proteins occupy entirely disordered conformations . The convia autophagy (Cellular protein homeostasis (proteostasis) is constantly challenged by the flow of unfolded proteins produced by protein biosynthesis as well as from protein misfolding caused by stochastic errors in gene expression and damage from proteotoxic stressors including but not limited to heat, oxidants and metabolic imbalances  Zakariy. Chaperoutophagy   and disaggregase machineries ensure that the aggregation is reversible despite the stable nature of protein aggregates [please see  for furtThe persistence of aberrantly folded and aggregated proteins is linked to cytotoxicity . The provice versa increased protein compositional heterogeneity in biomolecular condensates inhibits condensate hardening  plays a key role. HSFs induce gene regulatory programs that support the removal of misfolded and aggregated proteins, thereby counteracting proteostasis collapse . In yeasin vivo  cochaperones and Hsp110 nucleotide exchange factors (NEFs)  to thread trapped polypeptides in an ATP-dependent manner through its central pore, resolving a wide range of protein aggregates and hardened condensates  and autophagy .in vitro and in vivo. Furthermore, protease-inactivated HTRA1 converts fibrillar tau to soluble species, substantiating its disaggregation activity (Metazoan VCP/p97 (homologous to yeast and plant Cdc48) and ATP-independent HTRA1 can execute disaggregation . Human Hactivity . Interesactivity .Drosophila melanogaster and C. elegans . Prior to degradation, ubiquitin is removed by deubiquitinating enzymes (DUB)  . Ubiquites (DUB) .via LLPS and thus hindering their accumulation in the nucleus  and involves a plethora of receptors and other regulatory proteins (via cargo recognition by adaptor proteins with p62 (Cue5 in yeast) playing a key role by interacting with ubiquitin moieties on the protein aggregates, and linking them to LC3 (or Atg8 in yeast) on the autophagosome membranes. Selective degradation of aggregates via autophagy (aggrephagy) plays a critical role in limiting their accumulation in cells   . A relattophagy) . These ftophagy) . The inttophagy) .When assessing the ratio between refolding and degradation rates, PQC has a preference towards rescuing misfolded proteins with only little degradation of reporter and bulk proteins, as observed both in yeast and in mammalian cell culture during recovery from mild cellular stresses . RefoldiIncreasing age decreases the functionality of PQC, resulting, e.g., from lower expression and/or activity of proteasome subunits and ubiquitin-related enzymes, and thus leads to occurrence and accumulation of toxic protein aggregates . Mutatioin vitro under controlled and predictable conditions, a valuable tool gathering basic knowledge on these processes, nevertheless, in vivo data reflecting the dynamic nature of this process is inevitable for a complete picture. The facts that biomolecular condensates can adopt different states ranging from liquid-like to solid-like, harbor different scaffold and client proteins depending on cellular conditions and govern essential cellular processes make their full potential hard to understand. Research has just begun to explore aging as additional complexity factor, as traditionally PQC studies, especially those in model systems like yeast and bacteria, were conducted in young, immaculately working systems that instantly react to proteotoxic cues to safeguard cellular proteostasis. In light of our perpetually aging society and the fact that PQC capacity decreases over time (The strict distinction between biomolecular condensates as easily reversible and beneficial vs. aggregates as irreversibly, toxic compartment is not reflecting the fact that aging condensates can adopt solid-like, irreversible properties, while especially protein-disorder diseases show that aggregation processes producing almost inert compartments can indeed be the less toxic species compared to soluble oligomers. Thus, a more nuanced way of interpretation is required. Especially substantiated by the fact that distinction between phase-separated condensates and aggregates formed by misfolded protein species is not always the easiest task, as seen in yeast, where stress granules were originally identified as aggregated compartments. However, as discussed above, biomolecular condensates can incorporate misfolded proteins and it is hypothesized that phase separation might be a transition state preceding aggregate formation. Traditionally, LLPS has been widely studied ver time , it is vver time , it seem


The multifaceted nature of CRISPR screens has propelled advancements in the field of functional genomics. Pooled CRISPR screens involve creating programmed genetic perturbations across multiple genomic sites in a pool of host cells subjected to a challenge, empowering researchers to identify genetic causes of desirable phenotypes. These genome-wide screens have been widely used in mammalian cells to discover biological mechanisms of diseases and drive the development of targeted drugs and therapeutics. Their use in non-model organisms, especially in microbes to improve bioprocessing-relevant phenotypes, has been limited. Further compounding this issue is the lack of bioinformatic algorithms for analyzing microbial screening data with high accuracy. Here, we describe the general approach and underlying principles for conducting pooled CRISPR knockout screens in non-conventional yeasts and performing downstream analysis of the screening data, while also reviewing state-of-the-art algorithms for identification of CRISPR screening outcomes. Application of pooled CRISPR screens to non-model yeasts holds considerable potential to uncover novel metabolic engineering targets and improve industrial bioproduction.This mini-review describes experimental and computational approaches for functional genomic screening using CRISPR technologies in non-conventional microbes.  Escherichia coli and Saccharomyces cerevisiae—have identified essential genes as well as those required for conferring tolerance to biochemicals like isobutanol and furfural  and a CRISPR-associated endonuclease, typically Cas9 or Cas12a, to create mutations or alter gene expression  strain, a control (or reference) strain is also needed so that changes in guide abundances at the end of the screen can be determined. In many cases, a strain devoid of Cas endonuclease but harboring the guide RNA library is used as a control  indicate consistency between biological replicates. Upon passing this quality check, raw sgRNA read counts from control and treatment samples are provided to one or more CRISPR screen analysis methods that employ statistical approaches to identify significant genes in the screen.Most methods normalize the raw sgRNA abundances to account for varying sequencing depths across samples and ensure a fair comparison between controls and treatments. These normalized abundances are used directly or converted to log2-fold change to estimate gene essentiality scores, predominantly using Bayesian principles.The genome-wide library contains sgRNA with variable activity; failure to account for this variability could lead to inaccurate predictions of screening results. High-activity sgRNA should thus have a greater influence in determining gene essentiality compared to low-activity sgRNA. A common strategy to infer sgRNA activity involves screening across multiple conditions and applying probability-based approaches to make a prediction  is typically conducted, thus resulting in a A suitable choice for the ‘‘null’’ population depends on the host organism used to generate the screening data. Ideally, the null population is representative of the behavior of non-essential genes in the screen. For mammalian cells, the non-essential gene population overlaps well with the population of negative control sgRNA, and as a result, the negative control population serves as a suitable null model. On the other hand, non-conventional yeast datasets, in our experience, have a non-essential gene population that is relatively distant from the population of negative control sgRNA , to remove low-activity sgRNA from the analysis and correct screening outcomes based on an activity threshold, by calculating an ac-coefficient (given as the product of sgRNA activity threshold and library coverage). In the absence of experimental activity values, acCRISPR can utilize predicted activity scores for the library, if available. Gene essentiality is determined by testing against a null distribution, created using sgRNA targeting putative non-essential genes, which makes acCRISPR a suitable method for analyzing screens in non-model yeasts. This method has been shown to accurately call essential genes and genes important for environmental stress tolerance in the oleaginous yeast Y. lipolytica , promoting discovery of gene function and establishment of novel genotype–phenotype relationships. These biological findings would further improve host genetic engineering, drive enhancement of desirable phenotypes, and consequently improve the feasibility of industrial bioprocesses.