RNA therapy refers to the treatment or prevention of diseases using RNA-based molecules. The recent advent of a series of effective messenger RNA-based vaccines in response to the COVID-19 pandemic has reignited research interest in RNA therapy. Based on the accumulated results of long-term research in the field of RNA therapy spanning several decades, therapeutic agents for various diseases are being rapidly developed. These therapeutics tend to target diseases that cannot be treated with other conventional drug groups, and several clinical studies are underway for a variety of RNA-based therapeutics against various incurable diseases. This review describes the history of several important discoveries in RNA biology and their impact on key developments in RNA therapy as well as the advantages of RNA therapy. In addition, it describes the action mechanisms and examples of drugs approved for RNA therapy. Finally, this review discusses methods for RNA drug delivery to target organs and cells. Given that RNA therapy is expected to advance and play an integral role in the development of novel therapeutic agents for human diseases in the future, this review is designed to offer an updated reference point for researchers in this field. RNA-based therapies should improve the lives of many people affected by difficult-to-treat diseases, provided novel drug delivery methods are fully explored and optimized. Young-Kook Kim at Chonnam National University Medical School in Hwasun, Korea, reviewed the current status of RNA therapies, particularly given recent successes in developing messenger RNA vaccines for COVID-19. RNA therapeutics work by manipulating the expression and activity of specific target molecules, providing the means to treat diseases that do not respond to conventional drug types. Such therapies can be tweaked to cover a wide range of different forms of RNA and protein, and could open the way to personalized medicines and treatments for rare diseases. However, RNA-based drugs are larger molecules than other therapeutics, making targeted delivery within the body more difficult. Kim suggests that ensuring effective RNA drug delivery should be a paramount focus for future research. RNA therapy is a term used to describe the use of RNA-based molecules to modulate biological pathways to cure a specific condition. In general, the RNA sequence is the key to manipulating the expression or activity of its target molecules. Once the nucleic acid chemistry and the delivery method are established, the production of RNA-based drugs for a new target can be achieved in a relatively short period using these pre-established methodologies. It is this strength that has made messenger RNA (mRNA) therapy, a subcategory of RNA therapy, the most important and efficient means for developing novel vaccines used to combat coronavirus disease 2019 (COVID-19). The unprecedented efficacy of these vaccines has reignited interest in RNA-based therapy, but these successes were possible only due to the preceding 30 or more years of research and development in this field.1. However, this review will include the details of the molecular mechanisms of approved RNA-based drugs or those drugs in the last stage of clinical trials. This paper will also discuss RNA-based vaccines, which were not covered in the previous paper. Finally, the delivery methods for RNA-based drugs, one of the most actively researched questions in RNA therapy, will be presented.Here, I aim to introduce the key discoveries underlying the development of current RNA-based therapeutic technologies. I then discuss the key strengths of these RNA-based therapies compared to other drug compounds, including small molecules and antibodies, and describe the different technologies included in the term RNA therapies. This will include a description of antisense single-stranded RNA, double-stranded small interfering RNAs, aptamers, and mRNAs, their mechanism of action, and a discussion of the representative drugs in each category. This categorization is based on my previous paper discussing a similar topic2 and was later confirmed by the discovery of mRNA, which highlighted the importance of these molecules as key messengers in the translation of genetic information4.Numerous discoveries have established RNA therapy as an indispensable technology for treating human disease Fig. . RNA was5. This discovery formed the basis for the subsequent discovery of microRNAs (miRNAs) in 1993 and of RNA interference in 1998, in which the production of RNA duplexes is a key step in RNA silencing8.However, there was a second equally important, but less commonly discussed, discovery critical to the field of RNA, which was the discovery that two RNAs can base-pair with each other. Although this information is now taken for granted, early RNA scholars did not believe that RNA could produce a double helix structure. However, in 1956, Rich and Davies published the first nucleic acid hybridization reactions, which demonstrated that RNA could form a similar configuration to DNA based on the same complementarity in its base-pairing9, with the design of an antisense oligonucleotide targeting the 35S RNA sequence of the Rous sarcoma virus (RSV) to inhibit viral replication. Approximately 20 years later, the first drug using an antisense oligonucleotide was approved by the United States Food and Drug Administration (US FDA) for the treatment of cytomegalovirus retinitis10.However, the first application of RNA base-pairing for therapeutic purposes was first described by Stephenson and Zamecnik in 197812 and is now a well-known mechanism for disease, with splicing variations being linked to various human diseases14. The restoration or modulation of these splicing defects is almost impossible using conventional small molecule-based drugs but can be accomplished using RNA-based drugs, especially antisense oligonucleotides. In fact, a study from 1993 was the first to note that alternative splicing can be modulated by the application of antisense oligos15. In this study, a set of antisense oligonucleotides targeting the splice sites and branch points of thalassemic pre-mRNA were used to correct its abnormal splicing and reduce symptoms. This paper now serves as one of the cornerstones in the development of novel therapies for several difficult-to-treat neurological diseases described below.RNA splicing, the posttranscriptional step joining exons and removing introns from initial RNA transcripts, was first described in 1977Caenorhabditis elegans embryos with a mix of sense and antisense RNAs led to the potent and specific inhibition of targeted endogenous mRNAs8. Because RNAi is simple and powerful, it was rapidly adopted by the scientific community as a whole and applied extensively in a very short period. One example of this is a paper from 2002 that described the use of RNAi to inhibit hepatitis C virus replication in mice, which resulted in the widespread evaluation of RNAi for therapeutic purposes16. This facilitated the first clinical trials based on RNAi technologies in 2010, where a siRNA targeting the M2 subunit of ribonucleotide reductase was used to treat a patient with widespread melanoma17. This trial reported the successful cleavage of the target mRNA when the siRNA was delivered using a targeted nanoparticle delivery system. Following this trial, several siRNA-based drugs were evaluated for a diverse array of diseases, with the first siRNA drug for patients with hereditary transthyretin-mediated amyloidosis being approved in 201818.In contrast to the long history of the development of antisense oligo-based drugs, it took a relatively short time to proceed from the discovery of small interfering RNAs (siRNAs) to the utilization of these molecules as drugs. RNA interference (RNAi) was first characterized in a seminal paper in 1998, which demonstrated that treating 20, in which the authors found that these viruses were not sensitive to an inhibitor of DNA-dependent RNA polymerase, actinomycin D. Currently, RdRp is used as an essential factor in the production of self-amplifying RNA vaccines21. Another key enzyme, reverse transcriptase, was also first identified in viruses and is the essential enzyme in the life cycle of most retroviruses. In practice, researchers exploit this enzyme to clone the complementary DNA template to facilitate the production of the desired mRNA. Thus, the discovery of reverse transcriptase in 1970 has been critical to the manufacture of almost all mRNA-based drugs23.Despite the critical identification of mRNA as a messenger for genetic translation in 1961, it took nearly 30 years for researchers to establish a way to exploit this molecule for therapeutic purposes. In particular, the discovery of several RNA-related enzymes a few decades ago has facilitated the broader development of our current mRNA-based therapies. In the early 1960s, RNA-dependent RNA polymerase  was first discovered in a series of studies on viruses (mengovirus and poliovirus)24 and observed persistent gene expression in these tissues despite the lack of a delivery system. This later led to the evaluation of mRNA transcripts as a vaccine, which was an approach first tested in 199325. Here, the researchers synthesized the mRNAs for the influenza nucleoprotein using in vitro transcription and encapsulated them into liposomes. These were then injected into mice and were shown to elicit the production of virus-specific cytotoxic T lymphocytes in these animals. Subsequently, Conry et al. designed the first mRNA vaccine for the treatment of cancer in 1995. They reported that their vector, which expressed human carcinoembryonic antigen (CEA), could safely be injected into CEA-expressing tumor cells in a mouse model26. However, it took a long time for these animal experiments to yield sufficient data to allow this technology to move to human clinical trials. In 2008, Weide and colleagues reported the first set of results for their clinical trial using mRNA vaccines in patients with metastatic melanoma27. These studies used protamine-protected mRNAs coding for tumor-associated antigens, and the results showed that the delivery of these vectors induced an increase in vaccine-directed T cells28. The first clinical trial of an mRNA vaccine against infectious disease was conducted in 2013 (NCT02241135) and was designed to evaluate the efficacy of a novel rabies vaccine designed to deliver an mRNA encoding its glycoprotein. These evaluations revealed that treatment with this vaccine elicited functional antibodies targeting these viral antigens, demonstrating a clear proof of principle for this technology29. Finally, the first mRNA-based vaccine against an infectious disease, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was approved by the majority of the world in 2020. This brief description demonstrates the rich history of RNA therapeutics and shows that recent achievements in mRNA vaccines were made possible by the results of many studies completed over more than a four-decade span.Attempts to produce specific proteins via the introduction of exogenous mRNA began in the 1990s. In 1990, Wolff and colleagues injected the mRNAs for several reporter genes directly into the skeletal muscle of miceRNA-based drugs are known to exhibit a wide variety of advantageous traits, making them ideal candidates for the development of various novel therapeutic strategies. Here, I will discuss some of the major advantages of these technologies over more conventional therapeutic compounds.30, and because recent studies have confirmed the essential roles of these noncoding RNAs in the pathogenesis of various human diseases33, the importance of RNA drugs that can target these molecules will only increase.One of the greatest advantages of RNA-based drugs is their ability to target almost any genetic component within the cell, many of which may be considered undruggable using other technologies, including both small molecules and antibodies Fig. . In the 35. Since many proteins have similar structures, it is not easy to directly target a single specific target protein. In addition, membrane-integrated proteins are even harder to target using small molecules or antibodies due to their limited interactions with the cytoplasm. However, because RNA-based drugs can block the biogenesis of these proteins, they may be better suited to inhibiting their production, improving therapeutic efficacy.Moreover, previous studies have suggested that less than one-third of all human proteins can be effectively targeted by small molecules36.In general, the development process to produce novel small molecule or antibody-based drugs takes years. However, once the chemical structure of the RNA and the means of delivery into the body are established, RNA-based drugs can be designed and synthesized rapidly for clinical tests. For example, if a siRNA-based drug is developed to treat a disease caused by the overexpression of a gene in a specific organ, simply changing the sequence of the siRNA has the potential to treat other diseases within the same organ. This is why siRNA drugs using liver cell-specific conjugates are under widespread development as therapeutic agents for various liver metabolism-related diseases38. These companies are able to quickly adapt this technology, as their production chain does not vary, making these items ideal for pandemic preparedness.One of the reasons that we were able to curb the COVID-19 pandemic and significantly reduce the severity of the disease in infected people was that vaccines against COVID-19 were developed so quickly. Both BioNTech/Pfizer and Moderna were able to design mRNA sequences for their vaccine candidates, apply them in animal experiments, and conduct clinical trials within a short period of time after the sequence of the novel coronavirus was released from China39. In addition, when these RNAs are encapsulated within carriers such as liposomes, the RNA is efficiently protected from attack by nucleases following systemic administration40, improving its longevity. The long-acting effect of RNA-based drugs is exemplified by the recently developed drug inclisiran. This drug is a synthetic siRNA targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA. Notably, the silencing effect of inclisiran lasts more than six months after only a single injection41, allowing significant increases in the administration intervals and potentially reducing toxicity compared to small molecule-based drugs whose effectiveness is halved within a few days.Although natural RNAs are easily degraded by native nucleases, the stability of RNA greatly increases when various modifications are applied to their synthesis42, which would have been impossible using other technologies. Because the technologies related to the development of this antisense drug were well established, it was possible to develop this unprecedented therapy due to reduced processes and development costs.Pharmaceutical companies are often reluctant to produce new drugs for very rare diseases because the investment is not warranted by the potential profit. However, in the case of RNA-based drugs, once the chemistry of the RNA and its delivery system are optimized, the cost of developing novel variants of these drugs for new diseases is greatly reduced. One important example of this is the recent development of a splicing-modulatory antisense RNA drug designed to treat a single patient with a rare genetic diseaseRNA therapy also has no significant genotoxic effects compared to DNA therapy. In DNA-based therapies, the DNA molecule is delivered to the cells using a viral vector, and there is a possibility that this vector may integrate into the genome and cause a mutation. This potential risk can be avoided when using RNA instead of DNA.RNA-based drugs can be classified into four unique categories based on their structural characteristics and mode of action Table . Here, I39.Antisense oligonucleotides modulate the expression of target RNAs via sequence-specific binding, and although the structure of these antisense oligonucleotides is determined primarily by their specific sequence, their chemistry can be modulated to produce novel effects. These modifications can endow antisense oligonucleotides with increased specificity and stability10. However, this drug was withdrawn by the FDA in 2001 because the antiretroviral therapies used in patients with HIV significantly reduced the demand for fomivirsen.Fomivirsen was the first antisense oligonucleotide drug approved by the FDA in 1998 and is used in the treatment of cytomegalovirus retinitis, especially in patients coinfected with human immunodeficiency virus (HIV). Fomivirsen binds to the cytomegalovirus (CMV) mRNA encoding the immediate-early 2 protein, which is essential for CMV replication44, providing an advantage in some situations compared to siRNA-based drugs that operate primarily in the cytoplasm45.Antisense oligonucleotides use several diverse mechanisms of action, but approved antisense oligonucleotide drugs can be divided into two broad categories based on their mechanism Fig. . The fir46 . Mipomersen, the second antisense oligonucleotide drug approved by the FDA in 2013, is used for the treatment of homozygous familial hypercholesterolemia. Mipomersen binds to the mRNA sequence of apolipoprotein B-100 (ApoB-100) and cleaves its sequence46 Fig. . Because48. This second group of antisense oligonucleotides drugs targets these sequences in pre-mRNAs, where alternative splicing may result in the inhibition of disease. The ability to modulate this alternative splicing is unique to antisense oligonucleotides, making them a potentially valuable source of therapeutics for a variety of inherited diseases. Examples of FDA-approved antisense oligonucleotide drugs designed to modulate the splicing of their target pre-mRNAs include nusinersen, eteplirsen, and golodirsen.The second group of antisense oligonucleotide drugs is primarily used to regulate the splicing of pre-mRNAs via a steric hindrance-based mechanism Fig. . DiverseSMN1 gene, which encodes the survival motor neuron (SMN) protein49 , a severe type of muscular dystrophy. In patients with DMD, the mRNA encoding the dystrophin protein usually contains a mutation that causes an alteration within the reading frameein Fig. . Eteplirnts Fig. . Golodir54, and tofersen, which targets the SOD1 protein, a frequently mutated protein in a familial form of amyotrophic lateral sclerosis55. The preliminary data for these drugs suggest that antisense oligonucleotide-based therapeutics may provide a rich source of novel therapies for previously difficult-to-treat diseases.In addition to these approved drugs, various clinical trials using antisense oligonucleotides are currently underway for several neurological diseases that have proven to be difficult to treat using more conventional therapies. These include IONIS-HTTRx, which targets the mutant form of the huntingtin protein in Huntington’s disease56. siRNA-based drugs use this same machinery to exert a similar effect. siRNA duplexes join the native RNAi pathway following their incorporation into RISC, where only the guide strand is retained by the Ago protein while the passenger strand is discarded  protein to produce an RNA-induced silencing complex (RISC), which then suppresses the expression of their target mRNAs via sequence-specific bindingded Fig. . The gui18. This disease is caused by mutations in the transthyretin (TTR) gene, which results in the production of misfolded TTR proteins and thus in the accumulation of amyloid deposits57. Patisiran uses an LNP-based delivery system and is injected into the body through intravenous infusion. These particles are expected to associate with apolipoprotein E (ApoE) and enter hepatocytes through ApoE receptors58. Once released into the cytoplasm, patisiran is loaded into RISC and binds to the 3′ untranslated region (UTR) of both wild-type and mutant TTR mRNA, leading to the suppression of TTR protein translation and an overall reduction in amyloid deposits . Patisiran was the first FDA-approved siRNA-based drug in 2018 and was developed to treat hereditary transthyretin-mediated (hATTR) amyloidosisits Fig. .59. This rare disease is caused by an increase in the plasma levels of aminolevulinic acid (ALA) and porphobilinogen (PBG), which are both intermediates in the heme biosynthesis pathway60. Givosiran targets ALA synthase 1 (ALAS1), which is the key enzyme required for the production of ALA and PBG, thus inhibiting their expression and returning heme biosynthesis to normal levels  receptor63, inclisiran treatment directly reduces LDL cholesterol in the bloodstream , blocking its interaction with its receptor protein and thereby suppressing cell proliferation, the main downstream effect of VEGF signaling65 -mediated immune responses69, mRNAs can be difficult to deliver. These difficulties slowed their clinical application, especially in their early stages of development, but were resolved by Kariko and Weissman, who reported that this reaction could be reduced by modifying the mRNA with pseudouridine70.mRNA-based therapies exert their therapeutic effect by exploiting the fact that even exogenous mRNAs can be translated into functional proteins. These mRNAs are synthesized using in vitro transcription, and a cap analog is attached to their 5′ end during the reaction to facilitate their recognition by the translational machinery in the cell71. In the second category, the mRNA transcript is designed to act as a vaccine against infectious diseases or cancer antigens  and elasomeran , produce a modified spike protein from COVID-19, which, when administered via intramuscular injection, enters cells and facilitates spike-protein antigen presentation, allowing the maturation of a SARS-CoV-2-specific immune response in recipients.However, the utility of mRNA-based vaccines has been convincingly demonstrated in their application as vaccines against COVID-19. Because COVID-19 caused a global pandemic over a very short period, the scientific community needed to develop an effective vaccine as soon as possible. Among the various types of vaccines, mRNA vaccines have the advantage of a relatively short development cycle. In addition, because mRNA vaccines have been under development for so long, the existing knowledge base underlying their development was robust and easily adapted to this question. This meant that scientists were able to develop two highly effective mRNA vaccines against COVID-19 in a relatively short period, less than a year after the release of the genome sequence for this virus in early 202073, which suggested that these mRNAs can train the immune system to identify melanoma-specific mutations and eliminate the cells that express these mutant proteins, potentially reducing tumor size and preventing metastasis.mRNA vaccines can also be used as personalized medicines for targeting specific tumors. Here, clinicians will first identify a unique tumor mutation that is then included in an mRNA transcript. This exogenous transcript will then express this mutation, inducing a specific immune response targeting cells expressing these antigens, allowing the targeted destruction of these cells within the body. There was a recent report detailing this kind of application in the treatment of melanomaGiven the likely increase in circulation of novel infectious diseases in the future and the obvious need for rapid, cost-effective solutions to prevent their spread, there is a strong argument that mRNA vaccines will become a critical weapon in these outbreaks. In addition, mRNA-based drugs are likely to be incredibly effective in treating genetic diseases that cause some form of protein deficiency. Given this, it is likely that we will continue to see the maturation of these therapeutics in the future.The delivery of RNA-based drugs remains one of the single largest challenges for these kinds of therapies. Most delivery methods for these RNA-based therapeutics can be categorized as the addition of targeting moieties, the encapsulation of RNAs into lipid-based nanoparticles, and direct delivery into the target organ without extensive modification Fig. .Fig. 7De59, allowing recognition by the asialoglycoprotein receptor, a protein that is primarily associated with hepatocytes61, resulting in targeted delivery to the liver. The increased efficiency of this method has largely displaced the LNP-based delivery of small RNA drugs to the liver. In fact, the Alnylam company, which developed the first siRNA drug, patisiran, for the treatment of amyloidosis, designed this drug to use the LNP-based delivery method but is now testing their new siRNA drug, vutrisiran, which was engineered to use GalNAc-based delivery, for treating the same disease74. However, no other specific conjugates for cell-specific delivery have been evaluated or approved following the clinical trial. The success of GalNAc-based delivery supports the need for more research on conjugate-mediated cell-specific delivery.In the case of most GalNAc-conjugated siRNAs, such as givosiran and inclisiran, the GalNAc moiety is attached to the 3ʹ end of the passenger strand of the siRNA75. Nevertheless, the delivery of RNA-based drugs comprised of longer sequences, such as mRNA-based therapies, still relies on LNPs or other similar materials. Although the need to target a specific organ is relatively low when using a mRNA vaccine, delivery to the desired organ is important when using mRNA for the purpose of treating enzyme deficiencies, which can be made more difficult by the fact that LNPs are primarily transported to and eliminated in the liver or kidney76. This is why many of the current RNA-based therapies focus on diseases in these organs. Therefore, future work in this field should focus on expanding the delivery of these constructs to other organs and cell types.RNAs encapsulated in LNPs are significantly protected against nuclease-mediated degradation. However, the LNP itself can be toxic and may induce immunostimulatory side effectsFinally, many studies have evaluated the use of naked RNA delivery when the target is suitable for direct RNA delivery. Some examples of this include nusinersen, which is administered via intrathecal injection, and pegaptanib, which is administered via intravitreal injection Fig. . In bothIn summary, different delivery methods are used based on the available material and target. However, the drugs in the class of RNA-based therapies are significantly larger than those in other therapeutic classes, including small molecules, which makes their delivery more complex. This is exacerbated by the native charge on these compounds, which may inhibit their movement across the cell membrane. Therefore, more research on delivery methods for RNA-based drugs is needed.This review presents a solid case for the use of RNA-based drugs in the treatment of various diseases, especially those that are difficult to treat using other drug types. This is particularly clear considering the benefits of personalized RNA drugs, which may open up the possibility of high-efficiency treatment for very rare diseases for which conventional treatments have not been developed due to their cost. Moreover, RNA technology has mediated the rapid development of particularly efficient vaccines over the last couple of years, thus highlighting their utility in combating future pandemics and other infectious diseases. Although their delivery to target organs and their efficient introduction into cells remain challenging, the many advantages of RNA drugs  make the development of these technologies a worthwhile investment. Inspired by the development of various novel RNA drugs, including the novel COVID-19 vaccines in 2020, many researchers are making unprecedented efforts to develop new RNA-based drugs. Therefore, with the advancement of RNA therapy technology, the development of more diverse RNA-based drugs and drug-delivery methods is expected in the future.


Specifically, when making selections in isolation , people are less likely to choose the one whose race would increase group diversity than when making selections in collections . We set up eight experiments  in which participants make hiring or firing decisions among choices that are more white people than black people. We find that participants in the isolated choice group are less likely to choose black people, the smaller group, than those in the collective choice group. Our results show a potentially important contributing factor to the underrepresentation of black people in many organizations because hires are often made in isolation while layoffs are often made in collections, which provides a starting point for improving racial diversity in organizations by avoiding the isolated choice effect.With the view toward improving the racial diversity in organizations, this work seeks to uncover the reasons why larger groups have an advantage in terms of job opportunities. Based on people’s preference for diversity in commodity selection, we propose a potential feature that may exist in human resource management and call it the  Racial diversity in organizations has been society’s constant concern in the United States. For example, Title VIII of the Civil Rights Act protects individuals against racial discrimination in employment, and the majority of Fortune 500 companies emphasize workplace diversity on their company homepages . In spitNotably, inclusiveness and diversity become increasingly important for corporate strategy in a world of a large movement of migrations. A McKinsey study published in 2015 found that companies with a diverse ethnic workforce perform at least 35% better than comparable firms with a homogeneous workforce . Most ofisolated choice effect in the work of We are not the first to examine this phenomenon. Called In order to illustrate why many well-disposed organizations remain predominantly white though they espouse commitments to racial justice, we focus on racial diversity to examine whether the isolated choice effect is one of the contributing factors. Therefore, our work is an extension of that of n = 2,972), our hypotheses were largely supported and our work is expected to make important theoretical and practical contributions. This study provides a plausible explanation for low racial diversity in organizations, namely the isolated choice effect. Moreover, we show that the variety-seeking of consumer behavior theory can be applied to the implementation of personnel management in organizations.Across eight experiments  to prove our hypotheses in section “Materials and methods.” In the last section, we discuss our findings that show the important implications of the isolated choice effect and provide advice to increase organizational diversity.We organize our paper as follows. In the next section “Literature Review,” we review the studies related to the isolated choice effect and put forward our hypotheses. We then conduct eight experiments  and therefore consumers’ utility curves are normally convex toward the origin of coordinates, indicating that diversification provides consumers with higher utility. Much prior work also provide evidence that consumers prefer variety in their consumption bundles in order to meet their future needs . A consuOur study follows the insights of H1: Managers are less likely to hire applicants whose race would increase the diversity of the to-join list when making personnel selection choices in isolation than when making collections of choices.Based on the same logic, when a manager is asked to make firing decisions at a separate period (fire only one at a time), the set of people fired would be less diverse than when asking him or her to fire several people at a time. We thus hypothesize:H2: Managers are less likely to fire employees whose race would increase the diversity of the to-leave list when making personnel layoff decisions in isolation than when making collections of choices.n = 2,972). The experimental methods are questionnaire-based and are conducted by randomly distributing questionnaires on Amazon Mechanical Turk and the Wenjuanxing platform. The experiments ask participants to choose one person from several options according to the contextual setting, such as imagining themselves as an HR and choosing one from several candidates. All options are more white people, which is in line with most practical situations. The 2 × 2 × 2 groups of experiments are set up based on gender factors, two contexts of hiring and firing, and whether or not the decisions participants make are truly separate in time. To be specific for the last factor, in the first four experiments, we intentionally recruit participants in the isolated choice condition four times as many as in the inverse case (the collective choice condition). Each decision in the isolated choice condition was made by a different person, and thus they were not “time-split decisions” in the sense. In the last four experiments, the number of participants in the two conditions was equal because decisions made in the isolated choice condition were truly separated in time.To test our hypotheses, we designed eight experiments  would choose a lower proportion of black people than participants who are in charge of recruitment of multiple positions .In Study 1A, we set a hypothetical personnel selection scenario for investigating the influence of the isolated choice effect on the racial diversity of people on the to-join list. We predicted that participants who are in charge of single recruitment  — each of the 452 was responsible for choosing only one candidate. As a result, the numbers of selected candidates are balanced across conditions because the number of participants in the isolated choice condition was 452, which is four times as many as 113 in the collective choice condition.Participants were asked to think of that they were headhunters of a foreign trading company. The company was trying to hire five suitable candidates to fill five different positions: Purchasing Specialist, Accountant, Area Sales Representative, Quality Inspector, and Administrative Assistant . Participants were divided into two groups. Each of the 113 participants in the Firstly, participants were shown the statements of the job they are in charge of . After reading the job statements, they were asked to select one person among three candidates for each role  according to their pictures , years of experience, and ages. Except for the Area Sales Representative which included three whites, all the others contain two white males and one black male candidate. And we varied candidate quality such that the black man always had a moderate amount of experience and a moderate year of age.  In order to enable participants in the collective choice condition to make all the hiring decisions in a more simultaneous manner, we put the five options on a single screen. Please refer to our isolated choice condition was counted in the regression only once, while each decision made under the collective choice condition was counted four times. The regression result shows that hiring in isolated choice condition has a negative impact on the probability of choosing a black man , thus supporting H1 that managers in the isolated choice condition (who are responsible for hiring only one candidate) selected a lower proportion of black people than in the collective choice condition .The relevant variable that we are interested in was whether a black male was selected. Because there is a decision for obscuring study focus, we do not account it in our analysis. In the case of collective choice, black people were selected by 25.9% of all the selections made; however, in the case of isolated choice, black people were selected by only 14.8% of all hiring decisions. To predict whether a black male was added to the to-join list for each decision, we ran an ordinary least square regression. Since our analytical unit was a single selection, each decision made by participants in the Given gender differences may affect our findings, we used a similar recruitment scenario to Study 1A but reversed candidates’ gender in each option. This time, participants were responsible for jobs that were closer to our real life and experience: they were asked to select restaurant staff that are commonly seen in daily life.collective choice condition were paid $0.5. They were required to make five hiring decisions that took about 5 min to complete. Correspondingly, the rest 400 participants in the isolated choice condition were paid $0.1 because they made only one hiring decision that took about 1 min to complete.We recruited participants who met our criteria same as Study 1A through Amazon Mechanical Turk. 500 participants were left after excluding those who were trapped in our filtering questions. Among them, 100 participants who were assigned to the collective choice condition was responsible for hiring five candidates and each of the 400 in the isolated choice condition was responsible for hiring only one candidate. The number of participants in the latter was four times as many as that of the former such that the conditions were evenly balanced.In the experiment setting, participants were the owner of a newly opened restaurant. The restaurant was trying to hire five suitable staff to fill five different positions: Dishwasher, Kitchen Assistant, and Senior Chef , Waitress, Restaurant Receptionist. Again, participants were divided into two groups – each of the 100 in the collective choice condition were shown the five options with no page break. Please refer to our In the beginning, job descriptions of each position were shown to participants in charge . And then they were asked to choose one person for each role based on a joint assessment of the three candidates according to their pictures, years of experience, and ages. With the exception of three whites for Senior Chef, there were two white women and one black woman for each of the other four positions. And we varied candidate quality as the method mentioned in Study 1A. Participants in the bisolated−hiring−1B = −0.219, SE = 0.028, p < 0.001; 95%CI: −0.274, −0.163), thus providing further evidence for H1 that managers in the isolated choice condition (who are responsible for hiring only one candidate) selected a lower proportion of black people than in the collective choice condition .The relevant variable of interest in this study was whether a black female was selected. Because there is a decision for obscuring study focus, we do not account this in our analysis. In the case of collective choice, black people were selected by 24.5% of all hiring decisions; however, black people were selected by only 18.8% of all hiring decisions in the other case. We ran an ordinary least squares regression to predict whether a black female was chosen for each hiring decision. Our result indicates the negative effect of being in the isolated choice condition on people’s likelihood of hiring a black (see isolated choice condition) would choose a lower proportion of black people than participants responsible for firing several employees at a time . We use the word “choose” here because firing is an act of choosing someone to leave, just as hiring is an act of choosing someone to come in.Considering that personnel layoffs are also essential for employment, we therefore examined whether the isolated choice effect influences racial diversity of the to-leave list when people make firing decisions. Specifically, we predicted that participants responsible for firing a single employee . Each of the 100 participants assigned to the collective choice condition were shown the five options with no page break. For details of the surveys, please refer to our Participants were shown in advance the same job descriptions as Study 1A, along with the same employees’ photos and ages. We replaced “years of experience” with “years working in the company.” Except for the Area Sales Representative which included three whites, all the others contain two white males and one black male candidate. And we varied employee quality such that the black man always had a moderate amount of working years and a moderate years of age. After reading the information, participants were asked to choose one from three employees for each position to be downsized. Participants in the isolated choice condition, the effect on the likelihood of downsizing a black was significant , thus supporting H2 that managers in the isolated choice condition (who are responsible for firing only one employee) choose a lower proportion of black people than in the collective choice condition  when downsizing.The dependent variable we were interested in was whether a black male was fired. We did not account the decision made for Area Sales Representative in our analysis. In the case of collective choice, black people were selected by 21.8% of all firing decisions; in the case of isolated choice, however, black people were selected by only 10.3% of all firing decisions. We ran an ordinary least squares regression to predict whether a black male was fired. The regression result shows that under the In Study 2B, we ran a conceptual replication of Study 2A to explore whether the isolated choice effect on personnel layoff works for females.collective choice condition were paid $0.5. They were required to make five firing decisions that took about 5 min to complete. And the rest 420 participants in the isolated choice condition were paid $0.1 becxause they made only one firing decision that took about 1 min to complete.Five hundred and twenty-five participants who passed our filtering question were qualified. Among them, 105 participants who were assigned to the collective choice condition was responsible for the decruitment of five people and each of the 420 in the isolated choice condition was responsible for the decruitment of only one person. The numbers of participants in the two groups were assigned such that the conditions were evenly balanced.Participants were asked to imagine that they were the owner of a restaurant. The restaurant was trying to reduce costs by cutting five employees — one person for each of five positions: Dishwasher, Kitchen Assistant, Senior Chef , Waitress, and Restaurant Receptionist. Participants were divided into two groups — each of the 105 in the collective choice condition were shown the five options with no page break. The surveys are available in our Job descriptions of each position identical to Study 1B were shown to participants in charge. There were three employees for each position. Participants were then asked to select and fire an employee based on a joint assessment of the three employees’ photos, years of service at the restaurant, and age. With the exception of three whites for Senior Chef, there were two white women and one black woman for each of the other four positions. And we varied employee quality as the method mentioned in Study 2A. Participants in the isolated choice condition has a negative impact on the probability of choosing a black woman , thus providing further evidence for H2 that managers in the isolated choice condition (who are responsible for firing only one employee) selected a lower proportion of black people than in the collective choice condition  when downsizing.The relevant variable that we are interested in was whether a black female was fired. Because there is a decision for obscuring study focus, we do not account it in our analysis. In the case of collective choice, black people were selected by 21.4% of all firing decisions; in the case of isolated choice, however, black people were selected by only 9% of all firing decisions. We ran an ordinary least squares regression to predict whether a black female was downsized for each decision. The regression result shows that firing in the isolated choice condition of the above experiment, each participant was only responsible for selecting one person. But in reality, personnel administrations are often executed by the same manager who has to experience several times of isolated choice conditions. Therefore, in Study 3A, we referred to the snack experiment Simonson did in 1990 [11]. In his study, Simonson provided students with snack options over a 3-week period. Students who randomly chose one snack each week were significantly more likely to choose the same snack each time than students who chose three snacks at a time. We predicted that participants responsible for hiring several candidates but only one at a time  would construct a collection with a lower proportion of black people than participants responsible for hiring several candidates simultaneously .In the collective choice condition and 102 for the isolated choice condition. Participants in the collective choice group were paid 2 yuan to fill out a survey that took about 5 min to complete. However, participants in the isolated choice group were paid 15 yuan to fill out three surveys that were distributed in separate periods. They were asked to think of themselves as the interviewers of a research institution that was looking for some male researchers. There were three first-round interviews, each with five male applicants, all of whom have a chance to enter the final round. Among the five applicants in each interview, two of them were black people with moderate quality.Because of the experiment setting, recruiting online participants requires much effort and resources, we conducted the following four experiments (Study 3A–Study 4B) in our community in China. We recruited 204 participants from our community, and evenly assigned them to two groups – 102 for the (a)Group 1 (collective choice condition)Participants were required to choose one person for each interview. They were given only 5 min (so as to make it as simultaneous as possible) to guess the candidates most likely to enter the final round based on their appearance, age, and work experience. As a result, each participant chose three out of 15 people at a time. All options for the three interviews were on the same page.(b)Group 2 (isolated choice condition)Different from Group 1, participants in this group had a 3-days interval between each selection (interview). That is, they chose one candidate every 3 days. Please refer to our isolated choice condition has a negative impact on the probability of choosing a black man , thus supporting H1 that managers in the isolated choice condition  selected a lower proportion of black people than in the collective choice condition (who made multiple hiring decisions simultaneously).The relevant variable we were interested in was whether a black male was selected. In the case of collective choice, black people were selected by 45.1% of all hiring decisions; in the other case, however, black people were selected by only 31% of all hiring decisions. We ran an ordinary least squares regression to predict whether a black male was hired. The result shows that hiring in the Considering that gender differences may affect our results, we used a similar recruitment scenario to Study 3A but reversed the gender.collective choice condition and 114 for the isolated choice condition. Participants in the collective choice group were paid 2 yuan to fill out a survey at a time. However, participants in the isolated choice group were paid 15 yuan to fill out three surveys that were distributed in separate periods. Participants were asked to think of themselves as the interviewers for a cram school that was looking for some female teachers. Again, there were three first-round interviews, each with five female applicants, all of whom have a chance to enter the final round. Among the five applicants in each interview, two of them were black people with moderate quality.We recruited 228 participants from our community and evenly assigned them to two groups – 114 for the (a)Group 1 (collective choice condition)Participants were required to choose one candidate for each interview. They were given only 5 min to guess the candidates most likely to enter the final round based on their appearance, age, and work experience. As a result, each participant chose three out of 15 people at a time. All options for the three interviews were on the same page.(b)Group 2 (isolated choice condition)Different from Group 1, participants in this group had a three-day interval between each selection (interview). That is, they chose one candidate every three days. Our bisolated−hiring−3B = −0.269, SE = 0.040, p < 0.001; 95%, CI:−0.348,−0.190), thus providing further evidence for H1 that managers in the isolated choice condition  selected a lower proportion of black people than in the collective choice condition (who made multiple hiring decisions simultaneously).Our dependent variable of interest was whether a black female was selected. In the case of collective choice, black people were selected by 43.9% of all hiring decisions; in the other case, however, black people were selected by only 29.2% of all hiring decisions. We ran an ordinary least squares regression to predict whether a black female was hired for each selection. The result shows that under the isolated choice condition, the effect on the likelihood of hiring a black people was significant (see isolated choice condition) would construct a to-leave list with a lower proportion of black people than participants responsible for firing several candidates simultaneously .In Study 4A, we examined whether the isolated choice effect influences racial diversity if firing decisions are always made by the same people. We predicted that participants responsible for firing several candidates but only one at a time Group 1 (collective choice condition)Participants were required to fire one person for each group in 5 min based on their appearance, age, and work experience. As a result, each participant fired three out of 15 people at a time. All options for the three choices were in the same page.(b)Group 2 (isolated choice condition)Different from Group 1, participants in this group had a 3-day interval between each choice (downsizing). That is, they fired one person every 3 days. Please refer to our bisolated−firing−4A = −0.193, SE = 0.039, p < 0.001; 95%, CI: −0.271, −0.116), thus supporting H2 that managers in the isolated choice condition  fire a lower proportion of black people than in the collective choice condition (who made multiple firing decisions simultaneously).The variable we were interested in was whether a black male was fired. In the case of collective choice, black people were selected by 42.4% of all firing decisions; in the case of isolated choice, however, black people were selected by only 32.3% of all firing decisions. We ran an ordinary least squares regression to predict whether a black was selected for each firing decision. The regression result shows that under the isolated choice condition, the effect on the likelihood of downsizing a black employee was significant Group 1 (collective choice condition)Participants were required to fire one person for each group in 5 min based on their appearance, age, and work experience. As a result, each participant fired three out of 15 people at a time. All options for the three choices were on the same page.(a)Group 2 (isolated choice condition)Different from Group 1, participants in this group had a three-day interval between each choice (downsizing). That is, they fired one person every three days. For further details of the survey please refer to our isolated choice condition has a negative impact on the probability of choosing a black woman , thus providing further evidence for H2 that managers in the isolated choice condition  fire a lower proportion of black people than in the collective choice condition (who made multiple firing decisions simultaneously).The relevant variable we were interested in was whether a black female was fired. In the case of collective choice, black people were selected by 43% of all firing decisions; in the case of isolated choice, however, black people were selected by only 35% of all firing decisions. We ran an ordinary least squares regression to predict whether a black was selected for each firing decision. The regression result shows that firing in the We developed two hypotheses to understand the role of different choice conditions in influencing the racial diversity of organizations. Our hypotheses were largely supported and their close connection provides new insights into interpreting the low organizational racial diversity many companies face.n = 2,972), we find that people select less race-diverse groups when making isolated choices  than when making collective choices . Both Study 1A and Study 1B demonstrate that when candidates are all male or all female, people are less likely to choose black people when asked to choose only one person. In practice, companies often do isolated choices when hiring, and even if they are interested in hiring several people for the same position, the interviews are always one-on-one. In this case, they attend less to how their selected candidate will affect the racial diversity of the group. But if they select several people at a time, they realize that the organization has fewer black people, thus increasing the likelihood of selecting black people. Both Study 2A and Study 2B show that people choose a greater proportion of black people in the case of collective layoffs when the members are all male or all female. In practice, mass layoffs often occur, which are almost collective choice conditions for personnel managers. If there are more whites in the company, more white people are likely to be laid off. But that’s when they notice the diversity of the members being laid off, thus increasing the percentage of black people. The results of Study 3A and Study 3B are consistent with the results of the first two studies. The difference is that the participants in the isolated choice made decisions every three days, which is more in line with the condition we defined. And in reality, HR hires at longer intervals, sometimes up to several months. Thus, even if the same person makes the hiring decision, he may be unable to notice the characteristics of the person he previously chose and is less likely to choose black people who would increase diversity in the organization. Similarly, both Study 4A and Study 4B are complementary to the third and fourth experiments. All participants in the isolated choice group make a layoff decision at intervals, not by a different person. Both settings yield more black people laid off by managers in the collective condition.Across eight experiments . On the other hand, because of the large transaction costs involved in changing positions, organizations generally do not fire individuals unless that person is detrimental to the organization’s interests. Most of the layoffs are due to the economic crisis  or the replacement of humans with artificial intelligence . That is, personnel managers making firing decisions are always being placed in a collective choice condition that several leaving people are chosen simultaneously. In light of human’s ability to focus on whatever is different or unusual and to quickly and accurately recognize the salience of diversity  hunting for the same job. We thus suggest examining the isolated choice effect on group diversity with the interactions between gender and race.Second, due to resource limitations, the last four experiments were conducted in China, where indigenous people have less contact with black people and white people. Future research can replicate our study to find out whether the experimental results in China differ from those in Europe and the United States.collective choice condition), he may notice the diversity and choose a black person and thus construct a more diverse group. But we have not actually measured this mechanism. It’s valuable for future research to test whether racial diversity is more salient in collective choice conditions than in isolated choice conditions and whether the salience of diversity mediates people’s hiring and firing decisions. For example, asking participants to what extent they consider their choice would influence the group diversity when making decisions. A moderation study is also meaningful to investigate whether it makes difference when drawing participants’ attention to racial diversity. For example, using a 2 × 2  factorial design by telling the diversity-valued group that “the organization strongly values diversity.”Third, because our experiments are put in place in the same way as in the real world, there are always fewer black people than whites in the workplace. We therefore argue that in the case that all people are equally likely to be selected, there is a greater probability that the first person chosen by HR will be a white person. That is, black is always the race that would increase group diversity. If the HR continues to make the second choice (i.e., he or she is in a In addition, according to demographic characteristics, some participants in the experiment worked for less than 1 year. They may lack experience in organizational personnel management and thus make choices that do not correspond to reality. However, we did not exclude such participants from the questionnaire due to limited resources. Future studies may consider recruiting only participants with longer work experience or even those who have worked as human resources recruiters, which is closer to reality.Our work empirically suggests the important role of the isolated choice effect in influencing organizational racial diversity. Whether it’s hiring or firing, people build more race-diverse groups when they have to choose several people at a time, rather than just one. Our work not only provides an important explanation for why some well-intentioned organizations remain remarkably homogeneous but also identifies potential solutions to address the problem.The original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by the Academic Ethics Committee of Shenzhen University. The patients/participants provided their written informed consent to participate in this study.HL designed the study, analyzed the data, and reviewed and revised the manuscript. WZ proposed the research idea, designed the study, collected and analyzed the data, and wrote the manuscript. WS reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.


Over the past decades, RNA viruses have been threatened people’s health and led to global health emergencies. Significant progress has been made in diagnostic methods and antiviral therapeutics for combating RNA viruses. ELISA and RT-qPCR are reliable methods to detect RNA viruses, but they suffer from time-consuming procedures and limited sensitivities. Vaccines are effective to prevent virus infection and drugs are useful for antiviral treatment, while both need a relatively long research and development cycle. In recent years, CRISPR-based gene editing and modifying tools have been expanded rapidly. In particular, the CRISPR-Cas13 system stands out from the CRISPR-Cas family due to its accurate RNA-targeting ability, which makes it a promising tool for RNA virus diagnosis and therapy. Here, we review the current applications of the CRISPR-Cas13 system against RNA viruses, from diagnostics to therapeutics, and use some medically important RNA viruses such as SARS-CoV-2, dengue virus, and HIV-1 as examples to demonstrate the great potential of the CRISPR-Cas13 system. In addition, many other RNA viruses threaten the worldwide public health and economic burden, such as dengue virus, which infects 96 million annually across more than 100 countries [https://www.hiv.gov/hiv-basics/overview/data-and-trends/global-statistics (accessed on 31 May 2022)) [Throughout the history of human suffering and struggling against infectious diseases induced by viruses, the majority have been RNA viruses. This is exemplified by the outbreak of coronavirus disease in 2019 (COVID-19) induced by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) ,4. As ofountries , and humy 2022)) ,8.The Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR-Cas) systems, known as the bacterial immune systems against invading viruses, provide adaptative immunity to the hosts against invading nucleic acids . DespiteIn this review, we highlight the use of the CRISPR-Cas13 system for diagnosis and therapeutics of some representative and important RNA viruses such as SARS-CoV-2, dengue virus, and HIV-1. Several reviews have focused on the CRISPR-Cas systems’ emerging applications for infectious diseases diagnosis and antiviral therapy ,15. HoweThe CRISPR-Cas13 system, which is made up of dual components—a corresponding CRISPR RNA (crRNA) and a single Cas13 nuclease—belongs to the Class 2 Type VI CRISPR-Cas systems that contrast with Class 1 CRISPR-Cas systems, in which the nuclease complex consists of multiple subunits . AccordiCas13a, also named C2C2, was first introduced in 2015 by Zhang’s group using a computational pipeline strategy , and wasIn addition, depending on a computational-sequence database-mining approach, Zhang et al. introduced Cas13b in 2017 , includiCas13c was also discovered in 2017 when Zhang et al. sought to identify a more robust RNA-targeting CRISPR system . Cas13c Cas13d is a recently discovered type VI CRISPR-Cas variant with a smaller size of about 930 amino acids than Cas13a (1250aa) . The crRDuring the burst of Cas protein variants, Cas13X and Cas13Y were the latest to be found that held the smallest size of 775–803 amino acids , which cThe conventional diagnostic tests for RNA-based viruses can be divided into two main types, the immunological and molecular methods . The enzIn 2017, Zhang’s group came up with a rapid, inexpensive, and sensitive nucleic acid detection method with CRISPR-Cas13a, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) ,32. WithFocusing on the time needed for detection, Arizti-Sanz and colleagues developed an extraction-free, rapid, and sensitive diagnostic tool for SARS-CoV-2 RNA, termed Streamlined Highlighting of Infections to Navigate Epidemics (SHINE) . Aiming However, to achieve high sensitivity, both SHERLOCK and SHINE used the preamplification process to lower the analytic limit of detection, which was considered time-consuming and to easily cause false-negative or false-positive results due to amplification errors. In an attempt to avoid that, Fozouni et al. developed an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 . By actiIn addition, as another target amplification-free method, the Fast Integrated Nuclease Detection in Tandem (FIND-IT) approach was developed by Liu and her colleagues . By smarDespite the creative methods based on the CRIPSR-Cas13 system that have been described above, a rapid, scalable, and easily deployable diagnostic tool for SARS-CoV-2 detection is still in great demand. As an extension of SHERLOCK, Ackerman et al. in 2020 described an attempt to enable scalable detection of many samples while simultaneously testing for many pathogens, SARS-CoV-2 included, named Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN) . By combHowever, CARMEN required custom-made chips and readout hardware, which hampered its application in a clinical setting. Lately, Nicole et al. upgraded CARMEN to microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic Acids (mCARMEN) to scale up its diagnostic capability . By replIn another study, Manning et al. presented a high-throughput SHERLOCK SARS-CoV-2 test that showed that all processes were automation-compatible in a single 384-well plate [Despite Cas13a being used in RNA virus detection, Cas13d was also developed into a SARS-CoV-2 diagnostic assay, termed the Sensitive Enzymatic Nucleic-acid Sequence Reporter (SENSR) . SENSR rHaving established a general detection method for RNA viruses based on SHERLOCK, Zhang’s group immediately set out to detect dengue viruses that were responsible for dengue hemorrhagic fever and dengue shock syndrome using this CRISPR-Cas13 system . By testTo optimize the unnecessary viral nucleic acid extraction step in both SHERLOCK and SHERLOCKv2, Myhrvold et al. also adopted HUDSON to pair with the CRISPR-Cas13a system to detect dengue virus . With thIn addition to that, a sensitive electrochemical detection method was used to detect dengue virus with the aid of CRISPR-Cas13a-assisted catalytic hairpin assembly . Once th5 copies/mL, which made the method sensitive and easily able to handle detection of HIV-1.HIV, the cause of AIDS, has an urgent demand for diagnosis as early as possible because infected patients have a relatively aggressive clinical course with the onset of immunodeficiency . Therefo3 copies/mL. CARMEN has also been applied to differentiate 169 human-associated viruses, including RNA viruses such as Zika virus, hepatitis C virus, influenza A virus, etc. In 2019, Liu and his colleagues utilized the CRISPR-Cas13a nanomachine for avian influenza A (H7N9) on site detection by targeting the H7N9 HA gene [In addition to SARS-CoV-2, dengue virus, and HIV, many other RNA viruses threatening human health could be detected in a rapid way by the CRISPR-Cas13 system. SHERLOCK was the first test used to detect Zika virus in clinical isolates as low as 2 × 10 HA gene . Visual  HA gene . Ebola v HA gene . Moreove HA gene , as showConventional RNA virus therapeutics place a focus on vaccination and antiviral drugs. As the primary strategy to prevent viral infection in hosts, vaccines have been widely used in combating viral diseases and have made a huge difference, but only a few viruses have FDA-approved vaccines. Even worse, the high evolutionary rates of RNA viruses often lead to vaccine failure. Following viral infections, small molecular antiviral drug treatments have become the only clinical therapy to resist viruses. However, the antiviral drug screenings rely highly on a biological or mechanistic understanding of virus infection, which takes much investigation time before a drug comes onto the market. Besides that, drug resistance and the performance of pathogen mutations should still be considered. Therefore, developing adaptable antiviral therapeutic platforms has always been kept in urgent demand due to the threat of RNA viruses. When applying CRISPR-Cas systems to RNA virus treatment, CRISPR-Cas9 and CRISPR-Cas12 are only appropriate for the certain RNA virus class that relies on its reverse-transcription ability to pass on the genetic material. However, CRISPR-Cas13 can be used in inhibiting any virus of which the genetic material is RNA. The CRISPR-Cas13 system that holds a specific RNA targeting ability offers the potential to combat RNA viruses in a “virus against virus” way .In 2020, Abbott and his colleagues employed a prophylactic antiviral CRISPR in human cells (PAC-MAN) strategy to genetically intervene in SARS-CoV-2 infection . ThroughBy targeting the SARS-CoV-2 spike protein with CRISPR-Cas13a, Wang et al. also successfully eliminated viruses in human HepG2 and AT2 cells . Given tWith the help of the smallest Cas protein, Cas13x, Xu et al. performed a bioinformatics analysis and selected crRNAs targeting RNA sites coding for RdRP and E proteins of SARS-CoV-2 to test Cas13X’s antiviral ability . After cDengue virus, which is the most common arthropod-borne virus, has no specific therapy so far. As a single-stranded positive-sense RNA virus, dengue virus infection has been attempted to be treated using the CRISPR-Cas13 system. Li et al. discovered in 2019 that CRPSIR-Cas13a cleavage of the dengue virus NS3 gene could efficiently inhibit viral replication . By screIn another work, Singsuksawat et al. carried out a novel strategy by using Cas13b RNP with a short spacer that enhanced knockdown activity . It has In 2020, Yin et al. repurposed the CRISPR-Cas13 system to inhibit HIV-1 infection by targeting HIV-1 LTR, gag, tat, and rev regions . By tranBesides Cas13a, the small Cas13d was used in HIV-1 treatment. Kulkarni’s group designed multiple crRNAs targeting the conserved regions of HIV-1 gag, pol, and cPPT genes . Data shIt should be noted that many other RNA virus infections could be treated by the CRIPR-Cas13 system as well. For example, Tng et al. found that a strong suppression of chikungunya virus RNA in infected insect cells was induced by Cas13b with suitable crRNAs . Cui et RNA viruses have historically caused occasional epidemics and pandemics of highly contagious diseases. Mainly focusing on the three typical RNA viruses—SARS-CoV-2, dengue virus, and HIV-1—we showed the potential of the CRISPR/Cas13 system against RNA viruses from diagnostics to therapeutics. Given that the CRISPR-Cas13 system can promiscuously cleave ssRNA after target recognition in vitro, a variety of nucleic acid detection platforms have been developed. To improve the sensitivity of the CRISPR-Cas13-based detection method on RNA viruses, amplification techniques such as PCR, RPA, LAMP, transcription amplification, and so on have been elaborately incorporated into the detection system. Amplification-free detection platforms have also been explored using the combination of crRNAs or cooperation of Cas proteins to prevent wasted time and amplification errors. Moreover, rapid virus evolution and mutation has been considered as the major contribution factors to the misdiagnoses of RNA viruses, and serological antigen tests have been developed as a useful complement to improve the diagnostic accuracy ,60,61. INevertheless, not only diagnostic tools but also antiviral uses should be developed for the CRISPR-Cas13 system to help people combat RNA viruses. Always starting with the genome of a specific RNA virus, the potential crRNAs are designed to correspond to the most conserved gene, and then are combined with the Cas13 protein to perform their antiviral function. With high efficiency of target silencing, CRISPR-Cas13a, -Cas13b, and -Cas13d have been used in combating different RNA viruses, such as SARS-CoV-2, dengue virus, and HIV. However, it should be noted that the delivery methods and safety issues of the CRISPR-Cas13 system used in vivo are still immature, just like other CRISPR-Cas systems. There is great room for CRISPR-Cas13 system improvement in further applications. Upon use of CRISPR-Cas13a, C2C2 also was discovered, and researchers found its collateral activity could be directly observed biochemically in vitro and indirectly observed through growth suppression in bacteria. However, researchers have implemented the multiplexed leave-one-out and RNA-sequencing (RNA-seq) analyses, suggesting a lack of collateral RNA degradation in mammalian cells . That ma


The aim of this study is to investigate in depth diabetes mellitus associated with immune checkpoint inhibitors (DM-ICIs) by analysing a case series. We also evaluated the clinical impact of flash glucose monitoring (FGM) systems in the management of this entity.  We conducted an observational cohort study of DM-ICIs diagnosed in two hospitals in Seville (Spain). Patients with a new diagnosis of diabetes mellitus (DM) or with sudden worsening of preexisting DM after starting treatment with ICIs, with a random 5 hour-postprandial C-peptide value of <0.6 nmol/L and without possibility of subsequent withdrawal of insulin treatment, were included. n = 6; 85.7%), with a mean age of 64.9 years. The mean glycated hemoglobin (HbA1c) upon diagnosis was 8.1%, with diabetic ketoacidosis (DKA) observed in 6 cases (85.7%). Subcutaneous flash glucose monitoring (FGM) systems were used in six cases, with a mean follow-up period of 42.7 weeks. During the first 90 days of use, mean average glucose was 167.5 mg/dL, with a coefficient of variation (CV) of 34.6%. The mean time in the range 70-180 mg/dL (TIR) was 59.7%, with a mean time above range (TAR) 181-250 mg/dL of 27.8% and a mean TAR > 250 mg/dL of 10.2%. The mean time below range (TBR) 54-69 mg/dL was 2%, while the mean TBR < 54 mg/dL was 0.3%. The mean glucose management indicator (GMI) was 7.3%. No significant differences were observed in FGM values for the following 90 days of follow-up. A progressive improvement in all parameters of glycaemic control was observed between the first month of FGM use and the sixth month of FGM use. Of note, there was a decrease in mean CV , mean TAR 181-250 , mean TAR > 250 mg/dL , mean TBR 54-69 mg/dL , and mean TBR < 54 mg/dL , along with an increase in mean values of TIR 70-180 mg/dL . The lack of statistical significance in the differences observed in the mean FGM values over the follow-up period may be related to the small sample size.  A total of 7 cases were identified, mostly males ( DM-ICI is recognised by a state of sudden-onset insulinopenia, often associated with DKA. The use of FGM systems may be a valid option for the effective management of DM-ICIs and for the prevention of severe hyperglycaemic and hypoglycaemic episodes in this condition. In the last few years, immune checkpoint inhibitors (ICIs) have positioned themselves as one of the therapeutic options with the greatest benefits in the treatment of different types of cancer. Their favourable effects have been proven in a wide variety of clinical scenarios, including an increase in survival in advanced metastatic tumours such as advanced melanoma , 2. ConsFrom a biomolecular point of view, ICIs act by blocking inhibitory pathways in T lymphocytes (T cells) mediated by CTLA-4 (cytotoxic T-lymphocyte antigen 4), PD-1 (programmed cell death protein-1), and PD-L1 (programmed death ligand 1), with the aim of avoiding immune tolerance and increasing the T-cell ability to identify and respond to tumour cells . As a coβ-cells, as a consequence of overactivation of T cells (mostly CD8+) in the context of treatment with ICIs [Diabetes mellitus associated with immune checkpoint inhibitors (DM-ICIs) is a rare condition in clinical practice. Its incidence is estimated to be around 1-2% among patients receiving treatment with ICIs, although an upward trend has been observed in recent years in relation to the increased presence of these drugs in cancer treatment regimens , 7. Frommostly CD+ in the mostly CD+ in the ith ICIs . Interesith ICIs , 11.Currently, knowledge about DM-ICIs is scarce, given the low number of available cases due to the exceptional nature of its onset. The identification of risk factors for its development or its association with a favourable tumour response has been the subject of recent research , 13. HowIn addition, the potential severity associated with this condition and the expected increase in its incidence as a consequence of the increasing indications for ICIs in cancer treatment regimens raise the need for the development of specific strategies for prevention and management of DM-ICIs .In this sense, the implementation of new technological tools such as flash glucose monitoring (FGM) tools could have a positive impact on patients with DM-ICIs, especially in the prevention of hypoglycaemia and severe hyperglycaemic crisis. In the International Time in Range Consensus, Battelino et al. have proposed glycaemic control targets based on metrics from interstitial glucose monitoring systems, such as a glycaemic variability target (as defined by a coefficient of variation (CV) ≤ 36%) or different percentages of time in glycaemic ranges, for 14-day periods of use with collection of more than 70% of the data . HoweverIn this paper, we report a series of seven cases of DM-ICIs diagnosed in two hospitals located in Spain, with special emphasis on the benefits of the use of FGM systems for the management of this condition.We conducted a retrospective observational study of DM-ICI cases referred during the 2018-2021 period to the Endocrinology and Nutrition Departments of University Hospital Virgen Macarena and University Hospital Virgen del Rocío in Seville (Spain).β-cell destruction as evidenced by a result < 0.6 nmol/L in a random 5 h postprandial C-peptide measurement (as it has been suggested by the ADA-EASD consensus report for the management of T1DM) [The following criteria were used to diagnose cases of DM-ICIs: (1) diagnosis of diabetes after receiving the first course of treatment with ICIs, (2) onset with an episode of acute hyperglycaemia ≥ 200 mg/dL or glycated hemoglobin (HbA1c) ≥ 6.5% , (3) demonstration of of T1DM) , 16.Patients with a history of type 2 diabetes (T2DM) or prediabetes who had an episode of acute deterioration of glucose control after starting treatment with ICIs, with evidence of newly developed insulinopenia associated with the need for insulin therapy, were also included .The state of glucose control prior to the development of DM-ICIs was assessed by baseline glycaemia levels recorded in routine monthly blood tests requested by the Oncology Center, together with HbA1c levels (determined by high-performance liquid chromatographic method) in case these where available. After diagnosis of DM-ICIs, islet autoantibodies (anti-GAD65 and anti-IA-2 autoantibodies) were measured by electrochemiluminescence immunoassay . Where available, human leukocyte antigen- (HLA-) II-DQ genotyping was determined by polymerase chain reaction and sequence specific oligonucleotide probe (PCR-SSOP), using the INNO-LIPA HLA-DQ model .Initially, follow-up of glycaemic control after diagnosis of DM-ICIs was performed by self-monitoring of blood glucose (SMBG) through glucometers in all cases. Subsequently, subcutaneous flash glucose monitoring (FGM) systems  were implemented in those cases in which it was technically possible, analysing glucose data from 30-day and 90-day ambulatory glucose profile (AGP) reports.p value < 0.05, considering p values < 0.1 as close to statistical significance. The statistical analysis software SPSS v.26 (IBM Statistics) was used to perform the statistical analysis.Quantitative data were expressed as mean, standard deviation (SD), median, and range (minimum-maximum). For the assessment of the evolution of AGP data, a repeated measures general linear model with Bonferroni correction was developed. The significance level was defined by a This study was conducted in accordance with the ethical standards proposed in the Declaration of Helsinki (1964) and in its subsequent modifications. Data were collected retrospectively as part of routine patient care, in accordance with the guidelines of the hospitals' Ethics Committees.A total of seven cases were identified. The overall characteristics of the study cohort are summarised in n = 6), and the overall mean age was 64.9 years . The mean body mass index was 26.11 kg/m2 . The cancer diagnosis was melanoma in 42.9% of cases , lung adenocarcinoma in 28.55% (n = 2), and urothelial carcinoma in 28.55% (n = 2). One case (14.3%) received treatment with Ipilimumab (anti-CTLA-4)/nivolumab (anti-PD1), while five cases (71.4%) received monotherapy with nivolumab, pembrolizumab, cetrelimab (anti-PD1 drugs), and one case (14.3%) with atezolizumab (anti-PD-L1). A personal history of T2DM was recorded in one case (14.3%) and prediabetes in another case (14.3%), while the remaining patients  had no history of diabetes.85.7% of the cases were male . The mean number of ICI treatment cycles administered prior to diagnosis was 4.3 .Out of the 7 cases, six had DKA at diagnosis, while one had isolated hyperglycaemia . Mean glBlood tests performed prior to the diagnosis of DM-ICIs as part of the usual cancer follow-up revealed an abnormal increase in fasting plasma glucose (between 106 and 148 mg/dL) in 4 out of 7 cases with respect to what these patients had shown in previous follow-up visits Figures . In the Mean HbA1c value at debut was 8.1% (65 mmol/mol), measured at a mean of 20 days after diagnosis of DM-ICIs. The C-peptide level was below 0.6 nmol/L in all cases, with a mean of 43.5 days comprised between DM-ICIs diagnosis and C-peptide measurement. C-peptide measurement was a random measurement performed within 5 hours postmeal, coinciding with blood glucose values between 100 and 200 mg/dL in all cases. In this regard, case 7 initially showed a detectable C-peptide level  which subsequently decreased to insulinopenic levels (0.03 nmol/L at 28 weeks after DM-ICIs diagnosis) .All cases required subcutaneous basal-bolus insulin regimen for the management of DM-ICIs. The overall mean insulin requirement amounted to 0.6 IU/Kg body weight/24 hours (SD: 0.13), remaining constant throughout the follow-up period, with no decrease observed during the first weeks after the onset of DM-ICIs . In two n = 2 anti-GAD65+, n = 3 anti-IA2+), while the other two cases (28.6%) were islet autoantibody-negative. The HLA-II DQ genotype was explored in n = 3 cases, with two of them showing the DQA1∗05 : 01/DQB1∗02 : 01 haplotype  showed single islet autoantibody positivity  .In 6 out of 7 cases patients 1-6; 85.7%), FGM systems were used as part of the glycaemic control optimisation strategy, activating audible and vibrating alarms for hypoglycaemia (<70 mg/dL) and hyperglycaemia (>250 mg/dL). Initiation of use of these devices occurred after a mean of 28.7 weeks from diagnosis of DM-ICIs SD: 35.7 weeks) and continued for a mean of 42.7 weeks thereafter (SD: 20.3 weeks) , along with TAR > 250 mg/dL .Analysing the FGM data through the monthly AGP reports, we observed a progressive improvement in all parameters of glycaemic control from the first month of use of the FGM devices . Thus, Cp value = 0.09) as well as a decrease in TBR 54-69 mg/dL  and TBR < 54 mg/dL   .The percentage of time FGM sensor was active was higher than 80% in all cases at all timepoints, thus supporting a correct interpretation of FGM data in our cohort. Indeed, according to the International Consensus on Time in Range , the perFive patients (71.4%) discontinued ICI treatment after diagnosis of DM-ICIs, while 2 (28.6%) patients continued the scheduled treatment regimen. Of the five patients who discontinued treatment, one did so permanently. Four patients resumed ICI treatment after a mean of 14 weeks , and no deterioration in glucose control was observed in any of the cases (who remained on insulin therapy).Once the diagnosis was made, coordinated clinical visits were scheduled between Oncology and Endocrinology Centers for close clinical follow-up. The possibility of an unscheduled clinical assessment at the Diabetes Day Hospital of the Endocrinology Department was made available in the case there were alterations in glycaemic control requiring preferential or urgent clinical assessment.n = 2; n = 3 cases (42.8%), stable disease in n = 2 cases (28.6%), partial response in n = 1 case (14.3%), and disease progression (with exitus) in n = 1 case  had DKA at diagnosis.DM-ICIs is characterised by a state of insulinopenia with a sudden onset, leading to a high percentage of cases developing DKA at the time of clinical presentation (more than 67%) , 21. In Based on this mechanism, a C-peptide measurement below 0.6 nmol/L has been proposed as a diagnostic criterion for DM-ICIs by most authors , 12–14. β-cell destruction in DM-ICIs. In addition, some studies have suggested that DM-ICIs may be characterised by dysregulations of pancreatic α-cells and incretin system, with implications for α-cell glucagon production and/or incretin secretion  [Data from the FGM devices show that suboptimal control with a tendency to hyperglycemic episodes was maintained throughout the follow-up period, without a decrease in daily insulin requirements, which supports the theory of massive and persistent pancreatic cretion) , 25. Thicretion) , 26.In DM-ICIs, HbA1c levels are often not markedly high as compared to the observed glucose values that sometimes are extremely high at the time of diagnosis . This fiThe development of DM-ICIs in patients with a previous history of T2DM constitutes an entity with particular characteristics, with some cases reported in the current literature . HoweverIn our cohort, in one case (patient 7), C-peptide measurement during the first 24 hours after DM-ICIs diagnosis showed a normal value (0.9 nmol/L), which fell into the insulinopenic range in a subsequent measurement. In this regard, some authors propose a second confirmatory C-peptide measurement at least 15 days after DM-ICI diagnosis, or measurement under glucagon stimulation or with a mixed meal tolerance test in case of doubt . The decn = 5) were positive for islet autoantibodies, a percentage that is slightly higher than that reported in the main reports on DM-ICIs, which place it at around 50% [In our research, 71.4% of cases  within the genetic risk assessment for the development of autoimmune diabetes [∗03 binding imbalances  and, like other HLA class II polymorphisms such as DR4-DQ8 (DRB1∗04-DQA1∗03-DQB1∗03 : 02), has been associated with a strong risk for T1DM development [In the present case series, two cases (out of three studied) had the haplotype DQA1diabetes . This haelopment , 18, 35.elopment , 36. Howelopment . This poelopment , 20, 37.α cells), together with impaired exocrine pancreatic function [Some authors have suggested the existence of different subtypes of DM-ICIs depending on the clinical and biochemical profile at the onset . Thus, tfunction , 26. In function , 25. Thefunction .In line with this insight, in our series, 4 cases (57.1%) showed features compatible with a course similar to that of AFD, while 2 cases showed mixed features (negative islet autoantibodies) and 1 case showed a slower development time, with an onset in the form of isolated hyperglycaemia without DKA. Interestingly, the AGP reports extracted from the FGM devices showed how the patient diagnosed following an episode of isolated hyperglycaemia (patient 4) had a better glucose control compared to the other patients during postonset follow-up, with the highest TIR 70-180 mg/dL and the lowest CV (as compared to the other cases) and with a TBR < 70 mg/dL of less than 1.5%. Assuming the limitations derived from the analysis of a single case, this fact would support the theory of the existence of different onset forms of DM-ICIs, including one that shares clinical features comparable to those of typical T1DM.Different strategies have been proposed for the early identification of DM-ICIs. Some of them are providing health education notions focused on the identification of warning symptoms or the design of care circuits for specific assessment. In those cases, in the presence of a history of established T2DM or prediabetes, it is recommended to optimise glucose control –11, 19. Periodic blood tests performed at the same time of the treatment cycle administration may be useful to identify discrete variations in glycaemia levels that precede the debut of DM-ICIs . In our On the other hand, Fujiwara et al. have described changes in insulin secretion dynamics after initiation of treatment with ICIs in patients who subsequently developed DM-ICIs , so thatIn the event of a hyperglycaemic crisis in a patient being treated with ICIs, a differential diagnosis with similar conditions should be considered . The preIn terms of initial therapeutic management, the use of corticosteroids or other immunomodulators has not been shown to be effective in the treatment of DM-ICIs due to the frequent irreversibility of insulin secretion dysfunction, unlike other nonendocrine irAEs , 42. A cOn the other hand, given the difficulty of managing this condition, close and coordinated follow-up with other specialties could have added advantages in optimising the metabolic control of patients, allowing complications to be resolved at an early stage and progressively increasing the degree of autonomy in the management of diabetes. In this sense, multidisciplinary collaboration between different specialties (including oncologists and endocrinologists) plays a key role .p value > 0.05), probably due to the low number of cases in our cohort as a consequence of the rarity of this diabetes type. Yet, the increase in TIR and the decrease in TAR > 250 were close to statistical significance , suggesting that with a larger sample size, these differences could become statistically significant.The complexity of clinical management and adjustment of insulin therapy in DM-ICIs makes early implantation of continuous glucose monitoring systems a very useful tool in patients with this condition. In our case series, the benefits associated with FGM resulted in a progressive and generalised improvement in glucose parameters between the first and subsequent months of FGM use. In this regard, the CGM metrics observed decrease in CV and TBR < 70 mg/dL and TBR < 54 mg/dL was noteworthy, which is remarkable given the risk factors for the development of hypoglycaemia associated with this condition . We also observed a progressive increase in TIR 70-180 mg/dL, which overall was higher than 54% during the entire follow-up; also, we observed a decrease in TAR > 250 mg/dL from 16.3% in the first month to 7.7% in the sixth month. These differences failed to reach statistical significance  may limit the interpretation of our study conclusions, requiring comparison of our results with other similar cohorts. Second, the lack of measurement of C-peptide and islet autoantibodies at baseline limits the interpretation of the process through which DM-ICIs develop. The measurement of zinc transporter 8 autoantibodies  was not available in our setting, nor was the measurement of the HLA-DR haplotypes. HLA-DQ haplotypes could only be determined in three patients due to factors external to the investigation. On the other hand, use of FGM systems was relatively delayed after the disease onset; therefore, FGM use may not entirely reflect the exact patterns of blood glucose derangements in our cohort. In addition, longer-term follow-up of patients with DM-ICIs  would confirm the theory of a persistent insulin secretion dysfunction that cannot be reversed.In conclusion, DM-ICI is a condition characterised by a high complexity in terms of management, in which an early diagnosis allows the prompt initiation of insulin treatment and the use of technological systems (such as FGM devices) that represent strategies leading to significant clinical benefits, including improvement of glucose control, prevention of hypoglycemic episodes, and reduction of hospital readmissions. In this regard, the presence of an endocrinologist in the multidisciplinary team around the cancer patients is a crucial aspect since it facilitates the implementation of coordinated strategies during the follow-up.Therefore, large prospective studies using continuous glucose monitoring systems are needed to better investigate the patterns of glucose derangements in patients with DM-ICIs and to understand the most effective therapeutic interventions in this population.