The central nervous system (CNS) is an important pharmacological target, but it is very effectively protected by the blood–brain barrier (BBB), thereby impairing the efficacy of many potential active compounds as they are unable to cross this barrier. Among others, membranous efflux transporters like P-Glycoprotein are involved in the integrity of this barrier. In addition to these, however, uptake transporters have also been found to selectively uptake certain compounds into the CNS. These transporters are localized in the BBB as well as in neurons or in the choroid plexus. Among them, from a pharmacological point of view, representatives of the organic anion transporting polypeptides (OATPs) are of particular interest, as they mediate the cellular entry of a variety of different pharmaceutical compounds. Thus, OATPs in the BBB potentially offer the possibility of CNS targeting approaches. For these purposes, a profound understanding of the expression and localization of these transporters is crucial. This review therefore summarizes the current state of knowledge of the expression and localization of OATPs in the CNS, gives an overview of their possible physiological role, and outlines their possible pharmacological relevance using selected examples. Every living organism is in constant contact with its environment. One mechanism associated with this “contact” is the exchange realized in the selective uptake and release of molecules of versatile chemical properties. This is an important achievement of life, which is already realized on the cellular level. In more highly developed organisms, including humans, entire organ systems are specialized in selectively absorbing substances, while at the same time preventing or at least impeding the entry of potentially harmful molecules. This “sorting function” is realized in organs such as the intestine, the liver or the kidney, and involves different mechanisms. On the one hand, the body can selectively absorb substances, an example of which would be the absorption of amino acids or glucose in the intestine. In both cases, these are very polar molecules that do not cross the cell membrane by passive diffusion. The biological solution for their transit through membranes is the presence of selective pores/uptake transporters that allow facilitated, carrier-mediated diffusion. On the other hand, we are also exposed to toxins, which can cross the cell membrane by passive diffusion. A possible protective mechanism here is to throw these compounds right back out of the cell. This function is realized by membrane efflux transporters, which are capable of transporting their substrates against a concentration gradient in an energy-dependent manner. In addition to these transport-based systems, toxic substances can be subject to enzymatic conversion. This can neutralize them and/or make them accessible to transport processes. The interplay of transporters and enzymes is highly effective in protecting the body from environmental toxins and is additionally involved in the elimination of endogenous metabolites. However, it always represents a compromise between the necessary intake of essential nutrients and protection from potential toxins. Accordingly, it does not appear surprising that protection may not be complete. For this reason, structures requiring special protection are located behind further biological barriers. Examples are the blood–placenta barrier, the blood–testis barrier, and the blood–brain barrier (BBB). Here, again, membrane proteins including the efflux and uptake transporters play an essential role. At the molecular level, these transporters are, on the one hand, members of the ABC transporter family, which primarily govern cellular efflux, and, on the other hand, members of the solute carrier (SLC) superfamily, which facilitate cellular uptake processes.From a pharmacological point of view, the expression of drug transporters in the central nervous system (CNS) is of particular interest, especially at the BBB. This cellular structure is considered practically impermeable for most drugs. While considered advantageous for most drug development programs, the protective capacity of the BBB becomes a major challenge in the development of neurotherapeutics . In 2005SLC16A2 gene lead to severe defects in neurological development due to reduced thyroid hormone CNS exposure [On the other hand, the CNS depends highly on the selective entry of certain compounds. Uptake transporters, and possibly also efflux transporters like MRP1 (ABCC1) localized in the abluminal membrane of the endothelial cells , are assexposure ,11. Otheexposure ,13, or texposure , to nameSLCO nomenclature stands for the gene, while “OATP” denominates the respective protein. The aim of this review is to summarize the current state of knowledge regarding SLCO transporters in the CNS, especially at the blood–brain barrier, but also in the choroid plexus and neurons. Within this manuscript, we will address their expression and localization in these structures, but will also provide an overview of their endo- and exogenous substrates and discuss their known and potential physiological and pharmacological relevance.With the notion that facilitated BBB-endothelial entry could also realize central drug delivery, members of uptake and efflux transporter families recognizing pharmacological compounds as substrates are attracting interest in the field. Indeed, within the solute carrier (SLC) superfamily, pharmacologically important uptake transporters are found in the solute carrier organic anion (SLCO) family, among others. Members of this family are also known as organic anion transporting polypeptides (OATP). Accordingly, the In humans, 11 SLCO transporters exist, organized into six families and ten subfamilies based on sequence identity. OATPs are predicted to have 12 transmembrane domains and act in a sodium-independent manner, thereby mediating the cellular uptake of a wide variety of endo- as well as exogenous compounds, including several clinically important drugs. In general, OATP substrates are amphipathic organic anions with a molecular weight of more than 300 Da; however, neutral or even positively charged substrates  have also been described. OATPs are involved in the uptake of several physiological substrates including organic anions like bile acids, bilirubin, thyroid hormones and prostaglandins . MoreoveOverall, the function of OATPs is of interest from both a physiological and pharmacological perspective. However, while it is relatively straightforward to determine the function of a transporter such as OATP1B1, which is expressed in only one organ and for which there are also functionally relevant genetic variants available, this is significantly more difficult for other OATPs. Particularly for those assumed to be of relevance for the CNS. Moreover, due to significant species differences, animal models are of limited value. Nevertheless, there are several ways to address this question. Each of these is based on a precise determination of the expression and localization of the transporter in a specific organ or tissue.Within the following section, RNA expression data, antibody-based protein detection, which still plays an important role for localization, and modern mass spectrometry (MS)-based detection methods, are considered. Especially with regard to the latter method, it is important to emphasize that these methods not only target the whole brain, but also work with isolated microvessels or capillaries of the BBB and thus indirectly allow conclusions on localization.In 1998, in search of an uptake transporter for dehydroepiandrosterone-sulfate (DHEAS), the RNA of OATP1A2 was detected in the CNS of humans . Subsequ4) and reverse triiodothyronine (rT3)) transporter, OATP1C1, was described in multiple regions of the human brain [Already with their first description, the thyroid hormone , is responsible for the transmembrane transport of prostaglandins. As assumed for other OATPs ,40, OATPLike OATP1A2, OATP2B1 shows a broad tissue distribution, which also includes the CNS . ImmunohBesides these transporters, other OATPs have been found in the human brain. One example is OATP3A1, which is involved in the prostaglandine and thyroid hormone uptake ,50. OATPFinally, there are data on the thyroid hormone transporter OATP4A1 in the human brain . Here, mIn summary, several OATPs have been detected in the human brain compare . While O1S), pregnenolone-sulfate (PregS), and dehydroepiandrosterone sulfate (DHEAS) are examples of conjugated steroids that depend on transport mechanisms to cross cellular membranes due to their hydrophilic sulfate moiety. Particularly for DHEAS and PregS, there are reports showing their effects on processes in the central nervous system [It is generally accepted that OATPs are involved in the handling of sulfated steroid hormones. Estrone 3-sulfate  and the highly biologically active triiodothyronine (T3). The metabolism of thyroid hormones is rather complex; however, in the context of membrane transporters, it should be mentioned that one of the mechanisms contributing to the biological activity of thyroid hormones is the binding of T3 to respective intracellular receptors. T4 is linked to T3 by enzymes catalyzing the conversion by deiodination [4 and lower transport of T3 in OATP14-expressing HEK293 cells [X. laevis oocytes injected with human OATP1C1, Pizzagalli et al. did not observe enhanced uptake of T3, but they were able to validate transport of T4 and showed transport of the biologically inactive reverse triiodothyronine (rT3) [3. This conversion is catalyzed by enzymes such as the type II deiodinase, which has been shown to be expressed in the human brain [Another class of hormones that is dependent on transmembrane transport is the thyroid hormone family , which cdination . Independination . Even ifdination ,73, Sugi93 cells . Notablyne (rT3) . As mentan brain .X. laevis oocytes supporting that OATP1A2 mediates transport of both T3 and T4  [3 or T4 accumulation in OATP2B1 expressing X. laevis oocytes [4 and T3 interact with the transporter [While OATP1C1 transport function seems to be linked to the precursor thyroxine, there are data obtained in a similar experimental set-up of injected  1.7 μM) . For OAT oocytes , we provnsporter .Besides function, one has to consider localization when speculating on the role of a transporter in the handling of a molecular class. In this context, we want to mention that it is currently not elucidated whether OATP2B1 is localized in the luminal  or abluminal  membrane of endothelial cells. However, at the abluminal site of the endothelial cells, OATP2B1 might play a role in the salvage of centrally formed sulfated thyroid hormone metabolites. Importantly, without going into too much detail, thyroid hormones undergo a complex metabolism of which some metabolites contain hydrophilic moieties . For thoSLC16A2), a well characterized thyroid hormone transporter, which is highly expressed in neurons and mainly transports T3 [SLCO1C1 gene ). Subsequent in vitro data revealed a loss in T4 transport function of this particular variant [The importance of membrane transporters in the context of thyroid hormone homeostasis in the brain is exemplified by the Allan-Herndon-Dudley syndrome. This syndrome finds its genetic basis in mutations of the monocarboxylate transporter 8  were identified as OATP1A2 substrates [For OATP1A2, there have been in vitro experiments suggesting that this transporter may be—due to its expression in the BBB—involved in the brain entry of opioids. Briefly, using the bstrates . Anotherbstrates . It is bbstrates .One class of drugs that is widely accepted to be transported by multiple OATPs is the statins. These 3-Hydroxy-3-Methylglutaryl-Coenzyme-A-reductase inhibitors are used to treat dyslipidemia and to prevent cardiovascular events. The HMG-CoA reductase is the rate-limiting step in the mevalonate forming pathway. The end-products of this pathway are also involved in regulating biological activities in the central nervous system. As summarized by Fracassi and Marangoni et al., statins exhibit pleiotropic effects and have been shown to modulate a plethora of neuronal processes including the regulation of cognition and memory pathways . Indeed,In summary, several OATPs have been detected in the human brain so far. Based on our current knowledge, OATP2B1 and OATP1A2 appear to be the most relevant drug transporters, besides the physiologically important thyroid hormone transporters. Both OATP2B1 and OATP1A2 are found in the blood–brain barrier; however, information on their exact localization is still limited. Considering the situation in other tissues, one would rather expect a basal localization for OATP2B1, whereas an apical one would be conceivable for OATP1A2. Thus, in both cases, but especially assuming a luminal localization, these transporters would be of potential interest for concepts of CNS-targeted drug delivery. However, so far there are only very limited data available demonstrating a clinically relevant role of these transporters for drug distribution into the CNS. Although it is known from in vitro studies that respective drugs are transported by these transporters, clinical studies on this topic are missing. This is not necessarily surprising, as the corresponding effects are very difficult to detect. For example, in contrast to OATP1B1, there are no frequently occurring genetic variants for either transporter that are associated with a high functional impairment. Even if interaction studies would be an alternative, there are currently no transporter-specific inhibitors available. Another possibility for addressing this problem would be an appropriate animal model. However, it must be kept in mind that OATPs and especially OATP1A2 show major species-specific differences. Therefore, humanized animal models may be an interesting alternative. Indeed, an appropriate humanized OATP1A2 mouse model has recently been published, but the data presented to date concerning an effect on drug uptake into the CNS are rather disappointing . AnotherIn conclusion, the pharmacological relevance and the pharmacological possibilities of OATPs in the blood–brain barrier are of great interest, but in many respects, we are still at the beginning of understanding their relevance.


Computational methods to predict RNA 3D structure have more and more practical applications in molecular biology and medicine. Therefore, it is crucial to intensify efforts to improve the accuracy and quality of predicted three-dimensional structures. A significant role in this is played by the RNA-Puzzles initiative that collects, evaluates, and shares RNAs built computationally within currently nearly 30 challenges. RNA-Puzzles datasets, subjected to multi-criteria analysis, allow revealing the strengths and weaknesses of computer prediction methods. Here, we study the issue of entangled RNA fragments in the predicted RNA 3D structure models. By entanglement, we mean an arrangement of two structural elements such that one of them passes through the other. We propose the classification of entanglements driven by their topology and components. It distinguishes two general classes, interlaces and lassos, and subclasses characterized by element types—loops, dinucleotide steps, open single-stranded fragments—and puncture multiplicity. Our computational pipeline for entanglement detection, applied for 1,017 non-redundant models from RNA-Puzzles, has shown the frequency of different entanglements and allowed identifying 138 structures with intersected assemblies. Ribonucleic acids play vital roles in all living systems. They also constitute the genomes of most infectious viruses such as HIV, HCV, Ebola, influenza or SARS-CoV-2, fuelling recent worldwide pandemic. The discovery of numerous biological functions of these complex molecules is accompanied by the research into understanding their three-dimensional structure . And theIn silico modeling of RNA 3D structure has substantially developed in the recent decade  element. RNA structure elements are determined by the secondary structure of the molecule. Typically they include loops, stems, and single-stranded fragments. In our entanglement identification protocol, long double-stranded fragments are not treated holistically—each pair of the neighboring base pairs (i.e. dinucleotide step) defines a separate structure element. Loops and dinucleotide steps are referred to as closed structure elements, while single-stranded fragments are called open.The tight dependence on the secondary structure is what differs the problem studied here from the problem of entangled protein and chemical polymer chains - the latter one has no coincidence to base pairing . AlgoritBased on the entanglement’s topology, we distinguish two general classes: interlaces and lassos. The first type occurs when two closed structural elements form the Hopf link topology . Each inIn both classes, we distinguish subclasses defined by the types of entangled elements—each is assigned a label, which consists of structure element symbols  and the parent class symbol . Thus, for example, D&L denotes a dinucleotide step interlaced with a loop, while L(D) represents a dinucleotide step lassoed by a loop. Figure https://github.com/RNA-Puzzles and divided by challenges. The 233 of these RNA 3D models were predicted by web servers in the fully-automated processes (web server category). The remaining 795 structures were modeled in a hybrid approach, integrating automatic and manual methods (human category). In this dataset, we have found 33 redundant predictions and removed them, remaining with a collection of 1,017 RNAs for further analysis.The test set has been created from the RNA-Puzzles benchmark data . Initialn single-stranded fragments linked by n canonical base pairs. Closed structural elements include loops  and dinucleotide steps. In some applications, the latter ones are also classified as loops (named stacking loops)—e.g. in the energy models used by the RNA secondary structure prediction programs  class, occurs most frequently. Such entanglement has been found in the experimental structure targeted in Puzzle 18 . Recent We have examined RNA 3D models for the relationship between entanglement occurrence and the properties of structures to contain them. The tests prove no correlation between the occurrence of entanglements and structure size (cf. P of entanglement formation in pseudoknotted structures (P = 0.21) appears four times higher than in RNA models without pseudoknots (P = 0.05). 84% of entangled models have pseudoknots. We have learned that base pairs forming a pseudoknot often contribute to entanglement. Interestingly, L(S) lassos in our dataset occur only in the pseudoknotted RNA 3D models , model 8 submitted by Das group in Puzzle 13 (PZ13D8), and model 1 submitted by Adamiak group in Puzzle 5 (PZ05A1). The examples are representative of the analyzed dataset: each of them comes from a different round of RNA-Puzzles , was modeled using the other prediction method  . The natK7C . TheThe second example is taken from the set of 55 submissions to Puzzle 13 , challenging prediction of the ZTP riboswitch (PDB ID: 4XW7) with the 71-nucleotide sequence . Nine ofThe third example is selected from 24 non-redundant RNA 3D models submitted to Puzzle 5. The lariat-capping ribozyme (PDB ID: 4P9R) with a 188-nucleotide long sequence  was a taEscherichia coli (PDB ID: 1C2W), reconstructed by cryo-electron microscopy at 7.5 Å resolution (Finding an entanglement in the PZ18 target of RNA-Puzzles (PDB ID: 5TPY) motivated us to process with our algorithm some RNA 3D structures from the Protein Data Bank . For thesolution . This stsolution ,22. A knsolution ,22.Haloarcula marismortui (PDB ID: 1FFK). Its 2.4Å resolution structure was determined using X-ray diffraction (The second example is a 3044-nucleotides long 23S rRNA from fraction . Althougfraction .This work presents the problem of entangled elements occurring in RNA 3D structures. The entanglement is defined as the spatial arrangement of two structural elements, where at least one of them punctures the other. Such an arrangement can have an interlaced or lasso topology. An analysis of 1,017 non-redundant RNA 3D structure models from the RNA-Puzzles collection has shown that 14% of the predicted models and one experimentally determined RNA 3D structure included entanglements. Some instances had more than one issue. In total, we identified 201 entanglements—82% of them in the lasso and 18% in the interlaced topology.https://www.cs.put.poznan.pl/mantczak/spider.zip.In the predicted RNA 3D models, entanglements - the artifacts of computational procedures—come from the overlap of structural elements when assembling the structure from fragments or when transforming a coarse-grained to a full-atomic model. In the experimental structures, they might occur as a result of the model reconstruction from imprecise experimental data. The lack of suitable algorithms to identify entanglements has so far prevented the detection of such conformations and the sifting of entangled RNA 3D structures from the resulting set of models. Therefore, we provide the program developed for this study to enable easy identification and classification of entangled structure elements in RNA 3D models. We suggest the potential application of the program to validate both computationally generated and experimental structures. We hope it will contribute to improving the quality of RNA 3D structure prediction systems. The program, a component of the RNApolis group , is avaihttps://github.com/RNA-Puzzles/standardized_dataset.Computational tests described in the manuscript were performed on a benchmark set with RNA 3D structure models predicted within RNA-Puzzles, available at gkab716_Supplemental_FileClick here for additional data file.


Choice-consistency is considered a hallmark of rational value-based choice. However, because the cognitive apparatus supporting decision-making is imperfect, real decision-makers often show some degree of choice inconsistency. Cognitive models are necessary to complement idealized choice axioms with attention, perception and memory processes. Specifically, compelling theoretical work suggests that the (imperfect) retention of choice-relevant memories might be important for choice-consistency, but this hypothesis has not been tested directly. We used a novel multi-attribute visual choice paradigm to experimentally test the influence of memory retrieval of exemplars on choice-consistency. Our manipulation check confirmed that our retention interval manipulation successfully reduced memory representation strength. Given this, we found strong evidence against our hypothesis that choice-consistency decreases with increasing retention time. However, quality controls indicated that the choice-consistency of our participants was non-discernable from random behaviour. In addition, an exploratory analysis showed essentially no test–retest reliability of choice-consistency between two observations. Taken together, this suggests the presence of a floor effect in our data and, thus, low data quality for conclusively evaluating our hypotheses. Further exploration tentatively suggested a high difficulty of discriminating between the choice objects driving this floor effect. On the first day, shares of company A cost 200€ and shares of company B 150€. The stock trader buys 3 shares of company A and 0 shares of company B on the first day. On the second day, the share price of company A sinks to 150€ and the share price of company B rises to 200€. How should the stock trader respond to such a volatile stock market?money pump phenomenon). Such investment behaviour might, for example, arise from an inconsistent company value definition. By contrast, a consistent stock trader would base trading decisions on financial analysis, for example considering liquidity, book-to-market value, degree of state-ownership and past performance. This would result in a more robust value definition of company shares than the share price on a given day. Such a stock trader would, ideally, buy stocks at low prices and sell stocks for a profit, using the price volatility advantageously.A naïve (and inconsistent) stock trader might be tempted to prematurely sell the shares of company A and instead invest into company B. However, this would incur sensitive losses to the trader . More importantly, continuously selling shares cheaper than buying them will inevitably lead to the loss of all capital and being driven out of the market  paradigm is a better-suited paradigm to assess the influence of memory retrieval of goals on choice-consistency. MAVC describes the comparative judgement of visual objects that are characterized by multiple attributes, e.g. orientation, colour and shape. Further, we will argue how the revealed preference framework allows a broader evaluation of choice-consistency than traditional accuracy measures of perceptual decisions.. 1.1In our interpretation, the decision process as postulated by Query Theory  proposesFor example, when a stock trader decides whether to invest in shares of company A or company B, the trader compares them regarding liquidity, book-to-market value, degree of state-ownership, past performance etc. to a benchmark of what they consider a good investment. The investment option in the choice set which comes closest to the benchmark in memory, or so-called choice goals, is chosen (assuming that not investing is no option).This process is strikingly similar to the decision process proposed by the generalized context model (GCM) of categorization ,18 in seIn MAVC, participants have to choose one out of a set of objects subjectively most similar to a previously learnt exemplar. The choice set objects vary in their similarity to the exemplar regarding multiple attributes. Such choices are comparable to, although not identical with, delayed-match-to-sample tasks –24.An important advantage of MAVC tasks over value-based choice tasks is that we can experimentally induce and manipulate exemplar representations in MAVC, whereas goal representations are usually pre-existing, unknown and difficult to manipulate in value-based choice. We can, for example, experimentally manipulate memory representation strength of exemplars through changes of the retention interval (RI) between exemplar presentation and choice. These processes are well-studied and several off-the-shelf models for the relationship of memory representation strength and RI exist, e.g. exponential and power models .. 1.2Based on the predictions of Query Theory  and neurH1: As memory representations of exemplars are integral for MAVCs , we expePrevious research on the retention of information shows that forgetting curves are nonlinear . CCEI denotes the CCEI of a participant for one test block, and t denotes the assigned RI for that block (ranging from 0 to 30 s). σ accounts for random noise in the data. We assume all parameter values for σ to be equally likely a priori.with tμ denotes the expected choice-consistency given a RI and is specific to the model candidates.. 2.6.2.1The first candidate model assumes that choice-consistency decreases exponentially with retention time. This means that the decreasing rate of consistency is constant over retention time. Following Averell & Heathcote , the funa ∈  determines an asymptotical minimum level of choice-consistency after an infinite RI. The parameter b ∈  determines choice-consistency at t = 0, which allows for imperfect choice-consistency unconditional on time-dependent processes when b < 1. The parameter α ∈  determines the retention time-constant decreasing rate of consistency. We assume that all parameter values are equally likely a priori.The parameter . 2.6.2.2c ∈  determines the expected value of the choice-consistency. We assume that all parameter values for c are equally likely a priori:The second candidate model assumes that choice-consistency does not decrease as a function of the RI. The expected value of the choice-consistency distribution is, therefore, a constant:. 2.6.3et al. ) to guide our interpretation. The Bayesian model comparison using the complete dataset quantifies the evidence for or against each model in light of all data. We would use the same interpretation framework as before, which means that we consider a Bayes factor of BF ≥ 10 as conclusive evidence.Should we find conflicting evidence for H2 and H3, we would use the Bayes factor for the complete dataset  favouring a one-way ANOVA style model including the four-step RI factor over a null model. Inference was based on the replication Bayes factor fully using the evidence from our pilot experiment with BF10(origd) = 1000 [First, to ensure that our RI manipulation is effective, we tried to replicate the effect of the RI on absolute reconstruction error of exemplars from memory that we found in our pilot experiment (see pilot experiment) in our control task. Specifically, we wanted to find strong evidence (Bayes factor of at least ) = 1000 .We used the JAGS software  to analyFollowing Blaha , we thinFirst, we created a scatter plot of RI and choice-consistency in the training set together with histograms of the marginal distributions. This allowed for a visual inspection of the relationship of both variables as well as the marginal distributions. We did not want to see choice-consistency increase as a function of RI, as such pattern is not covered by our model space. The marginal distribution of the RI should, trivially, be uniform . The marginal distribution of the choice-consistency should, ideally, be a right-tailed Gaussian, meaning a tail for large consistency values. Second, we planned to create a plot that is overlaying scatter plots of RI and choice-consistency in the training set with the posterior predictive distributions of the exponential model and the null model . This would allow us to visually inspect how well the models can explain the data and, further, if there are any important qualitative differences between predictions and data. This is an important step to inform future modelling efforts and to identify systematic short comings of a model. Further, we would create the same plots for RI and choice-consistency in the test set overlaid with the out-of-sample posterior predictions of both models to qualitatively evaluate the generalizability of the models . . 2.9.1Should we find that choice-consistency is either near perfect or at very low levels across all RIs, this would indicate ceiling or floor effects respectively. While this is theoretically possible (e.g. in the case of an ineffective RI manipulation), we reduced the likelihood of finding such a pattern by using a continuous manipulation of the RI instead of a factorial design. Therefore, our design covered a wide range of RIs  instead of 2 to 3 RIs that a factorial design would cover. Still, it was not possible to entirely rule out the possibility of an ineffective RI manipulation on theoretical grounds only. Therefore, we conducted a pilot experiment to demonstrate the effectiveness of our manipulation using the control task from our main experiment (see below).. 2.9.2As one anonymous reviewer pointed out, our MAVC paradigm does not include a no-choice option. Intuitively, for some trials, it would be difficult for participants to make a similarity judgement. However, we decided not to include a no-choice option in our paradigm as one core assumption of revealed preference theory is that there is a well-defined preference structure , and thi. 2.10We conducted a pilot experiment to validate the effectiveness of our RI manipulation. Specifically, we wanted to demonstrate that our RI manipulation is sufficient to blur the memory representation of the exemplar. Furthermore, we explored the influence of presentation time of the exemplar on the memory representation strength.. 2.10.1. 2.10.1.1X- and Y-axis (see procedure of registered report) for either 1, 5, 10 or 30 s. Then, participants were presented with a mask of 10 randomly oriented cubes  for our pilot experiment. The sample size was not determined a priori. Instead we used a Bayesian stopping rule, recruiting further participants until we reached a Bayes factor of at least BF ≥ 10 for our hypothesis test. Importantly, the sample size is smaller than the minimal sample size we plan to recruit for the here described registered report. Participants were recruited from the same population we target for the here described registered report.We included a total of 25 participants  times more likely than the null model (including only the random subject intercept) given the data  times more evidence for the inclusion of the presentation time factor. We interpret this as definitive evidence. Inspection of the trend of means reveals that there is a negative relationship of presentation time and absolute error of reconstruction  times more evidence for the exclusion of the interaction term. We interpret this as definitive evidence. Note, however, that is was an explorative analysis.To quantify the evidence against an interaction effect of both factors, we compared the evidence of the most likely model with the evidence for a model including both main effects, the random subject intercept and an interaction term. We found that there was . 2.10.3We conducted a pilot experiment to validate the effectiveness of our RI manipulation. We showed that the precision of the orientation reconstruction of an exemplar from memory decreases with retention time over an interval of 30 s. Therefore, we are confident that the planned RI manipulation of the here described registered report is effective to weaken the memory representation strength of an exemplar. Further, we explored the influence of different presentation times on orientation reconstruction precision. We found that precision increases with presentation time. In the context of our registered report, it is important that the memory representation strength freely varies among different RIs for a given presentation time. Descriptively, the variance of the absolute error in reconstruction is highest for a presentation time of 1 s. However, also the mean absolute error is highest for a presentation time of 1 s. A presentation time of 5 s represents a compromise with the second highest variance and second highest mean of the absolute error in reconstruction.. 3. 3.1M = 22, range = 18−40; education: 72 completed high school, 5 completed a university degree) according to our inclusion criteria until reaching the preregistered stopping rule of our analysis plan.For our main experiment, we included 77 participants  = 32.894 ± 0.01%, BF10(fulld) = 46717770 ± 0.01%, ReplicationBF = 44623.3; To quantify the evidence for an effect of the RI factor on memory representation strength, we compared a one-way ANOVA style model including the 4-step RI factor to a null model. We found conclusive evidence for the RI model for the new and the full dataset (including our pilot data), as well as for the successful replication .Next, we tested in the training set whether choice-consistency as operationalized by the CCEI decreased with an increasing RI. Results showed conclusive evidence against our hypothesis . Results showed strong evidence against this, indicating a potential floor effect in our data (BF10 = 0.094).As apparent in figure 11. 3.3. 3.3.1r = −0.033). This was not driven by the difference in RIs of both measurements, or by low between-subject variability of the measure . A similar result showed for the money pump index (r = 0.023). However, interestingly, the Houtman–Maks index and the minimum cost index showed a much higher  test–retest reliability , which might be attributed to less vulnerability to outliers.In an attempt to further understand the quality of our data beyond our preregistered quality controls, we conducted a descriptive test–retest reliability analysis of the CCEI for the training and test set. As we reported recently elsewhere , there a measure a,b. A si. 3.3.2post hoc hypothesis that the generally low choice-consistency might be driven by too high a difficulty of discriminating the different X- and Y-orientations of the choice objects. To further explore this notion, we compared the mean absolute error in the reconstruction task, as an upper limit to the discriminatory performance, to the mean increment difference of orientation along the X- or Y-axis in the choice set, using bootstrapping. Results showed that the mean increment difference was generally lower than mean reconstruction error, tentatively suggesting a high difficulty of discriminating between the choice objects , it is unlikely that the low consistency level was driven by too long RIs.Generally, the lack and low reliability of choice-consistency indicates that our participants did not consistently integrate deviations in the X- and Y-orientations of the choice objects was too difficult. This was also, anecdotally, suggested in oral feedback of our participants during the data collection. To further explore this notion, we compared the mean absolute error in the reconstruction task, as an upper limit to the discriminatory performance, to the mean increment difference of orientation along the X- or Y-axis in the choice set, recovered from the task parameters, using bootstrapping. Results showed that the mean increment difference was generally lower than mean reconstruction error, tentatively suggesting a high difficulty of discriminating between the choice objects. As all choice-consistency indices quantify performance only relative to the increment orientation differences of choice objects, numerically low choice-consistency levels can correspond to only small inconsistencies in degree orientation.Another explanation for the overall low choice-consistency could be that the discrimination between the different . 4.2X- or Y-axis in the choice, or on an individual level by using an adaptive staircase procedure.Future studies investigating visual choice-consistency should, therefore, establish a sufficient level of choice-consistency at baseline. This could be achieved, for example, by pilot testing to adjust, in a group-wise fashion, the increment difference of orientation along the Another important consideration for the design of future studies is the low reliability of choice-consistency in the present study, but also, generally, in other task domains . While o. 5In this registered report, we set out to experimentally test the influence of memory retrieval of exemplars on choice-consistency in a novel visual choice paradigm. Due to unforeseen methodological pitfalls, our data are inconclusive to the preregistered hypotheses. However, our preregistered quality controls and additional exploratory analyses offer important insights for the design of future studies.Click here for additional data file.


We further highlight the emerging applications of these useful toolkits in cell biology, disease and imaging. Finally, we discuss the potential limitations, such as off-target editing, low editing efficiency and limitation for AAV delivery, and provide possible optimization strategies.Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13 has drawn broad interest to control gene expression and cell fate at the RNA level in general. Apart from RNA interference mediated by its endonuclease activity, the nuclease-deactivated form of Cas13 further provides a versatile RNA-guided RNA-targeting platform for manipulating kinds of RNA modifications post-transcriptionally. Chemical modifications modulate various aspects of RNA fate, including translation efficiency, alternative splicing, RNA–protein affinity, RNA–RNA interaction, RNA stability and RNA translocation, which ultimately orchestrate cellular biologic activities. This review summarizes the history of the CRISPR-Cas13 system, fundamental components of RNA modifications and the related physiological and pathological functions. We focus on the development of epi-transcriptional editing toolkits based on catalytically inactive Cas13, including RNA Editing for Programmable A to I Replacement (REPAIR) and xABE (adenosine base editor) for adenosine deamination, RNA Editing for Specific C-to-U Exchange (RESCUE) and xCBE (cytidine base editor) for cytidine deamination and dm Prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) RNAs and CRISPR-associated (Cas) proteins perform adaptive immune functions that protect prokaryotic cells by degrading invading genetic material, such as bacteriophages and exogenous plasmids . Once thLeptotrichia shahii C2c2 (LshC2C2) is the first identified single-component effector protein. Leptotrichia wadei (LwaCas13a) does not rely on the PFS motif and exhibits more robust targeting efficiency than Leptotrichia shahii Cas13a (LshCas13a). They both perform a “collateral effect” of promiscuous RNase activity in bacteria upon CrRNA base-pairing with targeted transcripts. Due to this feature, LwaCas13a and other orthologs of LshCas13a have been elegantly programmed to platforms termed Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and SHERLOCKv2, which have been used to detect multiple strains of Zika and Dengue virus, identify bacterial pathogens, genotype human DNA, and detect mutations in cell-free DNA from cancer patients (Prevotella sp. P5-125 (PspCas13b) stood out because of its highest efficiency and accuracy. PspCas13b monitors RNA cleavage without PFS constraints but with high fidelity in mammalian cells, which can be used for further applications (Ruminococcus flavefaciens strain XPD3002 (CasRx) is small enough to be packaged in AAV delivery system with a median size of 930 amino acids and has one of the most robust endogenous knockdown efficiencies without PFS limitation and detectable off-target performance  , Cas13b, Cas13c, Cas13d, Cas13x, and Cas13y ; Table 1patients . To asseications . Cas13d ge >60%) . Recentlge >60%) . TherefoOne of the major concerns and ramifications of gene therapy applications based on CRISPR-Cas13 is that Cas13 does not cause permanent variation in the genetic code in contrast to CRISPR-Cas9. Therefore, consistently importing exogenous effectors are required to achieve or maintain therapeutic efficacy, which may trigger a severe immune response. However, they still exhibit several strengths. Firstly, as RNA manipulation is tunable in yield and reversible in time, Cas13 platforms might be substitutional options for DNA-correction strategies, which cause severe or even lethal side-effects . For exaN6-Methyladenosine (m6A), the methylation occurring at the N6 of adenosine, is one of the most abundant mRNA modifications in eukaryotic cells (6A is enriched in the 3′ untranslated region (3′UTR) and areas surrounding the coding sequence (CD) of mRNA with the typical consensus sequence DRACH   . A seriefication . Recentltability , 2015. Ytability . YTHDC2 tability . In additability . eIF3 prtability . Moreovenditions . In recenditions .6A is found in almost every type of RNA, including mRNA, tRNA, rRNA, miRNA, lncRNA, circular RNA (circRNA), and snoRNA, and is virtually linked to major bioprocesses and normal cell development. The long non-coding RNA X-inactive specific transcript possesses 78 m6A residues, some of which induce transcriptional silencing via tethering to YTHDC1  and contribute to the development of leukemia is probably related to the variation in function of m6A in specific regions of RNA. In other words, various readers may recognize different m6A in specific RNA and lead to diverse readouts. Therefore, it is urgently necessary to expand the toolkit to dissect individual m6A functions instead of ranges of modification.nd BCL-2 . Anothernd BCL-2 . The expficiency . All the in mice . R-2HG dpagation . Newly ipagation . The reaAnother prevalent RNA modification is adenosine-to-inosine editing, which is mediated by adenosine deaminase acting on the RNA (ADAR) family. Instead of pairing with uridine, inosine preferentially pairs with cytidine . There arAdar2 mRNA. As a result, frameshift mutations delete the RNA-binding and deaminase domains . Long and stable Alu acts as a platform for recruiting multiple ADARs, then ADARs edit at nearby short hairpins. Editing at reverse complementary sequences, which brackets a circRNA, melts the stem structure and ultimately represses circRNA formation   ppp (5′) Nm and m7G (5′) ppp (5′) Nm. 6Am was a reversible modification, as they found that FTO, an m6A eraser, had demethylation activity toward m6Am. They also illustrated that m6Am-initiated RNA in FTO KD cells had a longer lifetime owing to resistance to the decapping protein DCP2. Likewise, 6Am increased RNA stability, whose degradation relied on DCP2. Further study of the direct substrates of FTO comprehensively revealed that FTO targeted multiple RNA species besides messenger RNA, including small nuclear RNA (snRNA) and tRNA. However, substrate preference was found to be closely related to FTO location; FTO in cytoplasm prioritizes demethylating m6Am, while the nuclear version tends to affect m6A  and incorporated into the spliceosome through a set of processing events in which FTO is responsible for the transformation of two methylated states; m1 and m2  methylates m7GpppAm in an S-adenosylmethionine-dependent manner, in which MTase and the helical domain form a pocket to recognize the m7G cap. Given that the N-terminal WW domain of CAPAM has a high affinity for the Ser5-phosphorylated CTD of RNAPII, CAPAM introduces m6Am to a nascent mRNA chain co-transcriptionally  the stress and cell state conditions of these studies differ, thus, m6Am predominantly affects translation or RNA stability; (2) m6Am is a cell type-specific epi-transcriptomic marker; and (3) diverse sequence motifs and unidentified readers result in uncertain annotations.The di-methylated nucleoside mne (m7G) . Severalfect m6A . FTO alsn snRNAs . Furthermal ring . Mauer e1 and m2 . Severaltigation . Cap-spetionally . In contwith m6A . Of note process . SeveralN1-methyladenosine, which was identified in the 1960s, is more enriched in tRNA and rRNA than in mRNA , and bovine tRNAGlu, TRMT61B acts as a mitochondrion-specific methyltransferase at position 58 in the TΨC loop. TRMT61B also regulates m1A947 formation in mitochondrial 16s rRNA, which is critical for the mito-ribosome structure and translation of mtDNA-encoding proteins  and correction of disease-related mutations (35% correction of AVPR2 and 23% correction of FANCC) remained inefficient; even the specificity was subpar. To maintain the core structure of REPAIRv1, a mismatch in the editing site (C-A) of the base-pairing region was introduced, and substitution in the RNA-binding region of ADAR was generated to lower the affinity of ADAR and gain more specificity. Compared with REPAIRv1, REPAIRv2 (dCas13b-ADAR2 (E488Q/T375G) exhibited a lower off-target rate, but the editing efficiency slightly reduced (23.7% for KRAS and 13% for PPIB)  or ADAR2 (E488Q) were adapted to increase productivity . To optior PPIB) . Similaror PPIB) . Furtheror PPIB) . There alication . Leveraglication . As therEscherichia coli cytidine deaminase, Riemerella anatipestifer), ADAR2dd became the catalytic domain of RNA Editing for Specific C-to-U Exchange (RESCUE), a new generation epi-transcriptome editing platform   . Based oESCUE-S) . In addi6A-mediated RNA regulation at individual transcripts. By replacing ADAR2dd in the REPAIR platform with the N-terminal domain of YTHDF1 or the CCR4-NOT binding domain of YTHDF2, dCas13b can be used to study specific reader’s functions on single transcripts in an m6A-independent manner, which ignores the cooperation and antagonism of various readers and protein complexes. The reporter targeting assay implies that YTHDF1 and YTHDF2 are consistent with the reported characteristics of translational promotion and RNA decay mediation  with a low off-target possibility. In addition, there was no additive effect when using sets of CrRNA to target the same transcripts (6A in a transcript.The targeted RNA methylation (TRM) system utilizes the catalytic domain of methyl transferase METTL3 or fusion version with METTL14 linked to dCas13b, and the zinc finger of METTL3 in TRM is deleted to enhance the binding affinity between dCas13b and RNA . By fusicificity . In cont effects . Selectinscripts . Nonethe6A modification. Given that the light-sensitive protein CBIN and its adaptor CRY2 are tightly packed together under blue light exposure, dCas13 fused with CBIN and CRY2 linked to the full-length FTO (PAMEC1) or essential domain of the METTL3/METTL14 heterodimer (PAMEC2) will also be located close to each other and catalyze the substrate under blue light . Although GAPDH A690 modification increased, the RNA levels did not change. As previous studies did not test the protein level and other variations, the role of GAPDH m6A690 was uncertain. In addition, targeting BRD8 and ZNF638 by nucleus-located version dCas13-M3nls modulates alternative splicing of these pre-mRNAs and controls exon21 exclusion or inclusion  and protein level (around three-fold). m6A residues in the 5′UTR of CTNNB15 may downregulate RNA stability and translation capability, which contrasts with the previous finding that m6A in the 5′UTR promotes translation by interacting with the translation initiation factor. PAMECs also manipulate RNA fate. PAMEC-2 adds a methyl group to the 3′-UTR of TPT1, giving rise to RNA decay  gene in different cell types, the pathogenic factor of cystic fibrosis  signals and track ACTB transcripts with stress granules by visualizing the stress granule marker G3BP1  ; Table 24.5-fold ; Table 2nd FOXM1 . dm6ACRIficiency ; Table 2in vivo treatment (in vivo casual gene correction and markedly slow disease progression in various animal models (AAV is characterized by low pathogenicity, high specificity, and high safety, as it does not integrate into the genome and various serotypes, making it the preferred delivery strategy for animal experiments and reatment . Nonethereatment . CRISPR-reatment . With anreatment . Recentlreatment . A seriereatment . Base-edl models . Thus, p6A; although few studies have modulated m6Am, m1A and other RNA modifications are less abundant. In addition, some platforms are applied in disease models; RESCUE and REPAIR could correct many RNA sequence-related disorders and dm6ACRISPR can control disease processes via engineering modifications in oncogenes and tumor suppressor genes. Importantly, more attention should be paid to off-target possibilities, low editing efficiency and limitations in the AAV delivery system. Although there is much to be explored and addressed, we believe that both currently established and future CRISPR-based RNA editing systems have great potential to act as therapeutic strategies for various diseases.The CRISPR-Cas13 system and its derivatives provide platforms for precisely altering RNA sequences and epi-transcriptomes in a temporary and controllable manner without permanently changing genomic sequences. It is extremely important to modulate RNA modifications, as these residues control RNA readouts, including RNA stability, translation efficiency, alternative splicing, and nuclear-cytoplasm translocation, all of which ultimately determine cellular physiological and pathological processes. Meanwhile, these platforms are also conducive to subtly study the epi-transcriptome because modifications in different transcripts have various effects; even modifications in the individual RNA vary in function. To date, the development of RNA engineering platforms is at the initial stages, and the majority are applied to the deamination and demethylation of mTT wrote the manuscript. HH co-supervised the project. YH and YW contributed to editing the manuscript. PQ supervised the overall project and co-wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Butenolide derivatives have the potential to be effective and environmentally friendly antifouling agents. In the present study, a butenolide derivative was structurally modified into Boc-butenolide to increase its melting point and remove its foul smell. The structurally modified Boc-butenolide demonstrated similar antifouling capabilities to butenolide in larval settlement bioassays but with significantly lower toxicity at high concentrations. Release-rate measurements demonstrated that the antifouling compound Boc-butenolide could be released from polycaprolactone-polyurethane (PCL-PU)-based coatings to inhibit the attachment of foulers. The coating matrix was easily degraded in the marine environment. The performance of the Boc-butenolide antifouling coatings was further examined through a marine field test. The coverage of biofouler on the Boc-butenolide coatings was low after 2 months, indicating the antifouling potential of Boc-butenolide. Since the prohibition of tributyltin in 2008, many studies have attempted to discover novel antifouling compounds from marine natural products . Many bitert-butyl pentyl)carbamate (Boc-butenolide)  and tubeworms (Hydroides elegans) in the laboratory. The modified Boc-butenolide was further formulated into an antifouling paint using polymer matrix poly(ε-caprolactone) based polyurethane (PCL-PU). Release-rate measurements of Boc-butenolide and a marine field antifouling test were conducted to evaluate the feasibility of using Boc-butenolide as an active ingredient in antifouling coatings.The antifouling compound 5-octylfuran-2(5H)-one (butenolide) has a melting point at 23 °C, which causes it to change from solid form to liquid at high ambient temperatures. In the present study, a butenolide derivative was modified with a Boc-protecting-group at the terminal of the alkyl side chain, enolide) . This moenolide)  was furtA. amphitrite larvae treated with Boc-butenolide and butenolide. At nominal concentrations of 50 ppm to 100 ppm, both compounds inhibited the settlement of barnacle cyprids. Settlement rates between the two compounds began to differ at 25 ppm. For Boc-butenolide, the cyprids showed some settlement at a rate of approximately 3%. The settlement rate continuously increased, reaching approximately 70% at a concentration of 3.125 ppm. For butenolide, the attachment of cyprids was inhibited between the concentrations of 6.25 ppm and 100 ppm, and approximately only 10% of the larvae settled in the treatment of 3.125 ppm.A. amphitrite larvae treated with Boc-butenolide and butenolide. For both compounds, there was no significant difference in toxic effects compared with the control group between the nominal concentrations of 3.125 ppm and 12.5 ppm. A pronounced difference in toxic effects was observed at 25 ppm. A very low toxicity was observed for Boc-butenolide-treated larvae, i.e., the mortality rate was approximately 8%, compared with the obvious toxic effect observed for the butenolide-treated larvae, in which mortality rate was over 90% among the individuals at high concentrations. All cyprids died in both treatments with higher antifoulant concentrations (50 and 100 ppm), indicating the toxic effects of the antifouling compounds at high concentrations.H. elegans larval settlement bioassay results were similar to those of the A. amphitrite larval settlement bioassay. No settlement of larvae was observed after treatments with Boc-butenolide at 50 and 100 ppm  was much higher than that of butenolide (45 µg/cm2/day) for 10 wt% samples. A possible explanation is the hydrophilicity difference between Boc-butenolide and butenolide. A Boc-protecting-group was added to the side chain of butenolide. Thus, the Boc-butenolide was more hydrophilic with the added highly electronegative N and O atoms of the Boc group and easier to dissolve in seawater, thereby resulting in a high initial release rate. A huge decrease in the release of antifoulant from the coatings for both Boc-butenolide and butenolide was found after 1 month. Notably, the release of Boc-butenolide from the coatings for all four concentrations was much lower than that of butenolide after 1 month. This finding might be due to the fact that a steady release in the later stage cannot be supported after a huge initial release of Boc-butenolide.PCL-PU polymer is biodegradable and environmentally friendly, and was applied as the polymer to develop the antifouling coating system in the present work. In the previous study, a PCL-PU/butenolide antifouling coating system was developed and test results showed that the polymer coating could be degraded in the sea . In thisA field test was conducted to evaluate the performance of the coatings in the marine environment. In the second month’s results, the coatings with higher concentrations of Boc-butenolide or butenolide showed better antifouling abilities . PerformThe differences in effectiveness and toxicity between Boc-butenolide and butenolide are possibly due to their structural difference. Considering that a Boc group was added to the carbon side chain of butenolide in Boc-butenolide, its structure was large and bulky. Molecular weight and hydrophilicity can affect the pharmacokinetics of the compounds, especially regarding their absorption by organisms . GeneralFrom the release-rate results, an exponential decrease in Boc-butenolide release into the seawater throughout the test period was observed. This result may be due to the high hydrophilicity of Boc-butenolide, which allows it to dissolve more easily in the seawater and achieve a high release rate. The release rate can be improved through the structural modifications of Boc-butenolide. Changes in physical and chemical properties, such as the melting point and hydrophilicity, can be achieved by structural modification . For instert-butyl pentyl)carbamate (Boc-butenolide) with a purity of >99% were purchased from ChemPartner  and used as received. Acetonitrile and methanol used were of HPLC grade.All chemical reagents used in this study, unless otherwise specified, were purchased from Sigma Aldrich  and VWR chemicals . 5-Octylfuran-2(5H)-one (butenolide) and Seawater was collected using a pump at the Coastal Marine Laboratory of Hong Kong University of Science and Technology. Filtered seawater (FSW) was obtained by filtering seawater through a 0.22 μm filter membrane from Millipore . Artificial seawater (ASW) was prepared according to ASTM D114198 standards (2013) .v/v.The stock concentrations of butenolide and Boc-butenolide were made by dissolving 100 mg of butenolide or Boc-butenolide in 1 mL of DMSO to make a stock of 100 mg/mL, stored at −20 °C. The butenolide and Boc-butenolide samples used in the larval settlement and mortality experiments were prepared by serial dilution of stock concentrations of butenolide and Boc-butenolide using ASW as the diluent, and the DMSO content in final samples were lower than 0.5‰ A. amphitrite colonies were collected from Tso Wo Hang Pier (22°23′32.1′′ N 114°17′18.7′′ E), Hong Kong, from April 2018 to June 2018. The adults were kept in a water tank with running seawater at the Coastal Marine Laboratory (the Hong Kong University of Science and Technology) for no more than a week before experimental use. Adults were induced to hatch under light sources for 1 h; the larvae were obtained using a method described previously by Harder et al. [Chaetoceros gracilis Schütt. The seawater culture medium was replaced daily with fresh FSW and algae. The nauplii reached the competent stage, known as cyprid, after 4 d of incubation at approximately 28 °C.Adult H. elegans colonies were collected from a fish farm at Yung Shue O, Hong Kong  from March 2019 to April 2019. The adults were kept in a water tank with running seawater at the Coastal Marine Laboratory of Hong Kong University of Science and Technology for no more than 3 d before experimental use. The larvae were collected according to the methods described by Qian and Pechenik [Isochrysis galbana (Tahitian strain) after hatching. The seawater culture medium was replaced daily with fresh FSW and algae, and the trochophore-stage larvae reached the competent stage after 5 d of incubation at approximately 25 °C.Adult A. amphitrite and H. elegans cultivated in this study comprised C. gracilis and Isochrysis galbana, respectively. In the laboratory, the algae were cultured with Guillard’s f/2 medium. The f/2 medium was prepared by adding designated amounts of NaNO3, NaH2PO4 H2O, trace metal and vitamin solutions into autoclaved FSW [2SiO3 9H2O was added for the cultivation of C. gracilis. Algal stocks were then added into the culture medium in a 2 L Erlenmyer flask and subcultured bi-weekly. The cultures were bubbled and illuminated under 14 h/10 h light/dark cycle at 23 °C for incubation.The diet for aved FSW . Na2SiO3A. amphitrite cyprids were placed into each well of the 24-well polystyrene culture plate containing 2 mL of FSW and were subjected to different treatments. For all treatments and controls, three replicates were performed. The plates were then incubated at 25 °C in darkness. After 48 h, the number of settled and swimming larvae were counted using a Leica MZ6 microscope, and possible toxic effects were noted.The test compounds were dissolved with a small amount of DMSO. The test compounds were used in six concentrations from 100 ppm to 3.125 ppm with 2-fold serial dilution. The same amount of DMSO was used as the negative control for all testing concentrations. Approximately 20 ± 2 individual H. elegans larvae were placed into each well of a 24-well polystyrene culture plate that contained 2 mL of FSW with different concentrations of the test solution. Approximately 10−4 molarity of 3-isobutyl-l-methylxanthine was added into each well as an inducer for the settlement of H. elegans larvae [The test compounds were dissolved with a small amount of DMSO. The test compounds were used at six different concentrations with a 2-fold serial dilution from 100 ppm to 3.125 ppm. The same amount of DMSO was used as the negative control for all testing concentrations. Approximately 10 ± 2 individual s larvae . The plav/v. The stock concentrations of butenolide and Boc-butenolide were made by dissolving 500 mg of butenolide or Boc-butenolide in 1 mL of DMSO to make a stock of 500 mg/mL and stored at −20 °C. The working concentrations of butenolide and Boc-butenolide samples used in settlement bioassay were measured by reverse-phase HPLC using a Waters 2695 separation module coupled to a Waters 2669 photo-diode array (PDA) detector according to the peak area at 210 nm . Identification of butenolide and Boc-butenolide was determined based on their retention times . The samples were tested with a 20 min gradient of 50–99% aqueous acetonitrile (ACN) containing 0.05% v/v trifluoroacetic acid (TFA) at a flow of 1 mL/min. The working concentrations of butenolide and Boc-butenolide were calculated according to their standard curves using peak areas plotted against known quantities of standards. The recoveries for butenolide and Boc-butenolide were 90.9% and 99.5%, respectively.The measurement and analysis of butenolide and Boc-butenolide were performed according to previous reports ,21,22. TThe stability of Boc-butenolide was measured by the concentration changes of Boc-butenolide in ASW throughout 3 months. The starting nominal concentration of Boc-butenolide was 200 ppm. At every time point, 5 mL of the solution was drawn and mixed with 10 mL dichloromethane (DCM). The DCM fraction with the analyte was dried under nitrogen gas, redissolved reconcentrated in 1 mL of methanol and subjected to above HPLC analysis.The polymer/antifoulant coatings were prepared using the solution casting method described by Ding et al. . The coaThe release rate of butenolide was determined by HPLC for quantification. The polymer/antifoulant coatings were prepared on an epoxy panel (25 mm × 75 mm) according to the above procedure. The coated panels were then placed into ASW. At certain time points , the panels were transferred into separate containers, which filled with 100 mL of fresh ASW. After 24 h immersion, the analyte in ASW was extracted with equal portion of dichloromethane (DCM). The DCM fraction with the analyte was dried under nitrogen gas, reconcentrated in 200 μL of methanol and subjected to HPLC analysis  using a reversed-phase system with a C18 column (Phenomenex Luna C18(2), 250 × 4.6 mm, 5 microns, Torrance, CA, USA) and a photodiode array detector  operated at 210 nm ,21,22.p-value lower than 0.05.The field tests were conducted in a fish farm in Yung Shue O, Hong Kong  from January 2018 to March 2018. PVC panels (53 mm × 125 mm) covered with coatings were immersed in seawater at a depth of 1 m from the surface. The panels were retrieved once monthly, the dirt on the panels was removed by washing the panel gently with seawater before being photographed. The panels were placed back into the sea for monitoring. The antifouling potential of different panels were compared to determine the efficiency of the coatings. The estimation of the panel fouling coverage was achieved by ImageJ  [A. amphitrite and H. elagans larval settlement bioassay results indicated that Boc-butenolide has similar antifouling ability against macrofoulers but with lower toxicity at high concentration. Boc-butenolide was released from the coatings and demonstrated antifouling ability for at least 2 months (as long as Boc-butenolide was released from coating). The release rate decreased with the increase in concentration of Boc-butenolide in the coatings. Our experiment demonstrated that Boc-butenolide exhibited good antifouling ability and could be a substitute compound for antifouling paints, and future efforts should focus on developing Boc-butenolide as a non-toxic antifouling compound with improved controlled release in the marine environment.The structure of butenolide was modified to Boc-butenolide to solve the problems of low melting point and smelliness of butenolide.


Illumina RNA-seq analysis was used to characterize the whole transcriptomes of peripheral blood mononuclear cells (PBMCs) from patients with congenital generalized lipodystrophy. RNA-seq information for seven patients with type 2 congenital generalized lipodystrophy  was obtained and compared with similar information for seven age- and sex-matched healthy control subjects. All seven CGL2 patients carried biallelic pathogenic mutations affecting the BSCL2 gene and had clinical symptoms of varying severity. The findings provide the whole-transcriptome signatures of PBMCs of CGL2 patients, allowing further exploration of gene expression patterns/signatures associated with the various clinical symptoms of patients with this disease. Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.15022521 Thus, RNA-seq allows for a more precise identification of the gene expression differences of cells when normal and abnormal tissues are compared. Indeed, RNA-seq has become a ubiquitous tool for the study of human diseases, including metabolic disorders, cancers, and infectious diseases2. Overall, RNA-seq-identified changes in the transcriptome that are associated with a disease can reveal dysregulation affecting associated biological pathways. The approach also aids in the identification of molecular markers reflecting disease progression.RNA sequencing (RNA-seq) is a term that describes the use of next-generation sequencing (NGS)-based methodology to target whole transcriptomes. This approach has gained wide popularity in biomedical research, in which profiling of the gene expression levels in samples can be very useful. Compared with other high-throughput transcriptome profiling technologies, such as DNA microarrays, the RNA-seq approach has the advantage of a wider dynamic range of measurement, which enables more sensitive detection of the global gene expression signatures of a given cell population or tissueAGPAT2, BSCL2, CAV1, or CAVIN18. Metabolic disorders in CGL patients are the main factors causing morbidity and mortality. Deficiency in adipose mass results in leptin deficiency9, leading to the main clinical features of these diseases, including hepatomegaly, muscular hypertrophy, cardiomyopathy, insulin resistance, and hypertriglyceridemia3. Furthermore, leptin deficiency might be associated with the lowered immunity frequently observed in CGL patients, as leptin is an important modulator of both the innate and adaptive immune systems. For example, leptin activates polymorphonuclear neutrophils (PMNs), exerts proliferative and antiapoptotic activities on T lymphocytes, affects cytokine production and regulates activation of monocytes and macrophages12. To date, approximately 500 patients with CGL have been reported in the literature. In Taiwan, type 2 disease (CGL2), which is caused by BSCL2 mutation, is most common based on surveillance across Asia (73% to 100%)17.Congenital generalized lipodystrophy  is a group of rare autosomal recessive disorders characterized by defects in the biogenesis of lipid droplets; individuals with these genetic diseases lack adipose tissue from early in life due to mutations in 18. Hypertrophic cardiomyopathy (25% of CGL2 patients) often leads to death in the third decade of life. In addition, early onset of liver cirrhosis, renal failure, and recurrent bacterial infection are potential lethal complications. Diagnosis of CGL is based on the patient’s clinical and biochemical phenotype and is usually confirmed by molecular testing19. Therapeutic strategies include restriction of total fat intake , treatment with fibric acid derivatives, prescription of n-3 polyunsaturated fatty acids to control hypertriglyceridemia, and standard glucose-lowering treatments20. In addition, leptin replacement therapy has been approved since 2014 by the Food and Drug Administration (FDA) for the treatment of severe metabolic abnormalities that result in generalized forms of lipodystrophy21.Compared to CGL1, CGL2 presents with a more severe phenotype, including extensive fat loss, cardiomyopathy, intellectual impairment (50% to 78%), and premature death (15%)Clinically, we have observed that some CGL2 patients develop hyperactivity and seizures in late childhood but that others develop metabolic syndrome that is difficult to control with glucose-lowering drugs. To elucidate the processes underlying these different outcomes, the aim in this study was to explore differences in RNA signatures between CGL2 patients and matched control subjects.This study investigated differentially expressed genes that might be associated with various subcategories of CGL2 patients and aimed to identify distinct regulatory pathways involved in these various phenotypes. The overall workflow is presented in Fig. BSCL2 mutation by Sanger sequencing after their initial diagnosis of CGL. Each was further categorized into various subgroups based on age, symptoms, and individual biochemical profiles, namely, child or young adult, normal blood lipid or hyperlipidemia, intellectual disability or not, and diabetes mellitus (DM) or not. Additionally, patients with severe DM or hyperlipidemia, namely, patients 1, 4, 6, and 7, were assigned together into another subgroup, the metabolic syndrome subgroup (presence of insulin resistance or a diagnosis of DM); patients 2, 3, and 5 comprised the “nonmetabolic syndrome” subgroup. Peripheral blood samples were collected after obtaining written informed consent from the participants themselves or one of their parents if the individual was younger than 18 years old. The study protocol was approved by the Institutional Review Board of National Yang-Ming University (IRB number: YM108113E) and the Institutional Review Board of National Taiwan University Hospital (IRB number: 201901028RINB). Informed consent for these data to be openly shared was obtained from all subjects or their guardians, and the participants were warned that RNA-seq data carry some inherent risk of reidentification. To reduce variability, each blood sample was drawn in the morning before the participant broke their fast. Each blood sample was collected into a BD Vacutainer® EDTA tube, and soon after collection, an aliquot from each sample was used for RNA-seq transcriptomic profiling of peripheral blood mononuclear cells (PBMCs).Seven patients  who had been diagnosed with CGL2 Table  and seveEach whole-blood sample was heparinized, and the samples were used to isolate PBMCs by density gradient centrifugation using LymphoprepTM medium according to the manufacturer’s instructions. Specifically, a sample was diluted with phosphate-buffered saline  to double its volume, layered on top of 5 mL of Ficoll-Paque Plus  and centrifuged for 30 minutes at 400 × g. The PBMC layer was aspirated and centrifuged for collection. The PBMC pellet was treated for 15 minutes with RBC lysis buffer in the absence of light and then centrifuged again followed by washing twice with PBS. Total RNA was extracted using a miRNeasy Kit (Qiagen Cat. No. 74004), followed by DNase I treatment to avoid contamination with genomic DNA. All procedures were according to the manufacturer’s instructions. The fourteen samples containing total RNA from the subject’s PBMCs were submitted to The Genomics Center for Clinical and Biotechnological Applications of National Core Facility for Biopharmaceuticals, which is located at National Yang-Ming University, Taipei. At this facility, RNA quality assessment, RNA integrity assessment, and whole-transcriptome sequencing were carried out. The sequencing library was prepared using a TruSeq Stranded Total RNA Library Ribo-Zero™ Globin Kit . As the amount of input RNA for the preparation of sequencing libraries was ~1 μg, which is approximately the highest level suggested by the manufacturer’s manual, no positive control, such as SMARTer® Ultra™ Low RNA Kit for Illumina® Sequencing , was used. Paired-end RNA-seq (2 × 100 bases) was carried out using the Illumina HiSeq. 2500 platform.RNA-seq data analysis was performed to identify differentially expressed genes (DEGs) by assessing the number of reads mapped to the individual gene present in the human reference genome. The workflow of the RNA-seq data analysis was as follows: (1) preprocessing of raw RNA-seq reads and quality validation; and (2) read mapping and normalization.22 and MultiQC23, providing per-base and per-sequence assessments of the sample’s read quality, overall GC contents, and presence of adaptors, overrepresented k-mers and duplicated reads. In addition, Trimmomatic was used to discard low-quality reads, to trim adaptor sequences and to eliminate poor-quality bases24. The quality control steps and read trimming were iteratively performed to ensure that all low-quality reads and adaptor sequences were removed as much as possible because they would seriously interfere with the read-mapping step. The quality of the cleaned reads was further assessed by generating per-base box plots using FastQC. Next, MultiQC was applied to create a visualization of the output across the various different samples  using the splice-aware alignment tool STAR27 using the TMM  normalization method to calculate various effective library sizes. Boxplots and a multidimensional scaling (MDS) plot were generated to determine whether any unusual expression-pattern similarities between the samples due to batch effects might be present28. The boxplots suggested that the variation in the distributions of normalized gene expression levels across different samples was small and that their median values and IQRs (interquartile ranges) were highly similar  when CGL2 patients were compared to age- and sex-matched controls29. The raw read count dataset was deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE15933730 (Table 31.The FASTQ files for the raw RNA-seq reads in this study have been deposited in NCBI Sequence Read Archive (SRA) under the study accession SRP281919Total RNA was quantified using a Nanodrop ND-1000 spectrophotometer, and its quality was then assessed using an Agilent 2100 Bioanalyzer according to the manufacturer’s instructions. Acceptable quality values are in the range of 1.8−2.2 for A260/A280 ratios and with an RNA integrity number (RIN) of >7.0.22, which ensured that the adaptors were removed from the raw reads. This program also verified that the quality of the cleaned RNA-seq reads was suitable for downstream analyses (Fig. The quality of the raw and cleaned RNA-seq reads was evaluated using FastQC


Our aim was to explore the biological function and clinical significance of the m6A methyltransferase METTL3 in intrahepatic cholangiocarcinoma (ICC). In this study, we revealed that METTL3 was upregulated and predicted poor prognosis of patients with ICC. Multivariate regression analysis demonstrated that METTL3 expression was an independent predictor for overall survival in patients with ICC. Moreover, METTL3 knockdown inhibited ICC progression, while METTL3 overexpression showed the opposite effect. METTL3 inhibitor STM2457 also showed anti-tumor effect in ICC. Mechanistically, METTL3 transcription was driven by H3K4me3 activation. Upregulation of METTL3 mediated m6A modification of IFIT2 mRNA and accelerated IFIT2 mRNA decay in a YTHDF2-dependent manner, which promoted the development of ICC and lead to poorer prognosis. In summary, our findings revealed that H3K4me3 activation-driven METTL3 transcription promotes ICC progression by YTHDF2-mediated IFIT2 mRNA degradation, suggesting that METTL3 may serve as a potential target for human ICC therapy.N6-methyladenosine (m Intrahepatic cholangiocarcinoma (ICC) is the second most common primary malignant liver cancer, which accounts for ~10% of all such cancers . The pro6A) modification is the most prevalent mRNA modification in eukaryote  and Alkylation repair homolog protein 5 [ALKBH5]). In addition, specific “reader” proteins  can recognize m6A sites and affect RNA process including mRNA stability, decay, splicing, and translation  No. 382).4-week-old female BALB/c nude mice were used for HuCC-T1 tumor xenograft models and 4-week-old female B-NDGn = 56), and IHC score ≤3 was defined as low METTL3 expression (n = 40). For IHC staining of IFIT2, IHC score >3 was defined as high IFIT2 (n = 34), and IHC score ≤3 was defined as low METTL3 expression (n = 62).This staining assay was performed as described previously . The IHCICC cells were crosslinked in 1% formaldehyde  for 10 min at room temperature and followed by 250 mM Glycine quenching. Cell lysates were processed using the SimpleChIP Enzymatic Chromatin IP Kit  according to manufacturer’s instructions. The H3K4me3 antibody was used for immunoprecipitation at a dilution factor of 1:1000. ChIP DNA was purified, and subsequent qPCR was conducted using the 2 × ChamQ Universal SYBR qPCR Master Mix (Vazyme). A flank region with no signal was used as a negative control.METTL3-1 and shMETTL3-2, were harvested at 48 h post-infection, followed by RNA extraction using TRIzol solution (Life Technologies). The cDNA library was prepared by Novogene . The paired-end reads were generated by the Illumina® HiSeq 2500 platform supplied by Novogene. An R package, DESeq, was used to quantify transcription levels and identify differentially expressed genes, using a cut-off of P < 0.05.HuCC-T1 cells, infected with lentiviruses expressing sh6A antibody was used for immunoprecipitated. Both input and immunoprecipitation RNA samples were then subjected to the sequencing libraries preparation using NEBNext Ultra RNA Library Prep Kit for Illumina and submitted for sequencing on Illumina HiSeq 2500 or MeRIP-qPCR analysis.Total RNA was isolated from indicated HuCC-T1 and HCCC-9810 cells, and the mRNA was further purified using Dynabeads mRNA Purification Kit . After fragmentation with RNA fragmentation reagent , the anti-mIn short, we induced the covalent cross-link between the protein and the directly bound RNA by ultraviolet irradiation. In this way, RNA-protein crosslinking is achieved. After cross-linking, the RNA is partially hydrolyzed to reduce the bound RNA fragments to a “footprint” size , which can be cloned by RNA adaptor ligation and reverse transcription (RT)-PCR amplification. Then sequence these PCR products on a high-throughput platform, which was completed on Illumina Hi-Seq. The RNA-IP performed as described previously .6 cells/ml. The cells were harvested in lysis buffer. The anti-YTHDF2 antibody was used for immunoprecipitated in a dilution of 1:100. The RNA of input and immunoprecipitated samples were isolated with the TRIzol reagent and subjected to sequencing using Illumina HiSeq 2500 platform supplied by Novogene or qPCR analysis. For qPCR analysis, a flank region with no signal was used as a negative control.HuCC-T1 and HCCC-9810 Cells were seeded in a 150-mm dish at a density of 1 × 10t test, Wilcoxon test, or Chi square test. DFS and OS were measured by Kaplan–Meier method, and the differences between groups were evaluated by log rank test. The independent predictive factors were determined by the cox proportional hazard model. All the statistical analyses, p < 0.05 were considered statistically significant.The statistical analysis of the data was carried out in SPSS 22.0 software and GraphPad Prism 9 software. The data were expressed as mean ± standard deviation and compared by Supplemental


As well, the application of CRISPR and base editors for screening shows promise in revealing the phenotypic outcomes from m6A modification, many of which are linked to physiological, and pathological effects. Thus, the therapeutic potential of CRISPR/Cas and base editors for not only m6A related, but other RNA and DNA related disease has also garnered insight. In this review, we summarize/discuss the recent findings on RNA editing with CRISPR, base editors and non-CRISPR related tools and offer a perspective regarding future applications for basic and clinical research.One key advantage of RNA over genomic editing is its temporary effects. Aside from current use of DNA-targeting CRISPR-Cas9, the more recently discovered CRISPR-Cas13 has been explored as a means of editing due to its RNA-targeting capabilities. Specifically, there has been a recent interest in identifying and functionally characterizing biochemical RNA modifications, which has spurred a new field of research known as “epitranscriptomics”. As one of the most frequently occurring transcriptome modifications, N6-methyladenosine (m6A) has generated much interest. The presence of m6A modifications is under the tight control of a series of regulators, and the ability of fusing these proteins or demethylases to catalytically inactive CRISPR proteins have resulted in a new wave of programmable RNA methylation tools. In addition, studies have been conducted to develop different CRISPR/Cas and base editor systems capable of more efficient editing, and some have explored the effects of  Genome editing and modification technologies such as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) originated from earlier nuclease technologies and other chemical techniques. However, these earlier methods were limited in terms of editing specificity and riddle with off-target side effects . When thBase editing is a method currently used to introduce single nucleotide variants into DNA or RNA . DiffereWhen CRISPR systems emerged, the advantages over TALENs and ZFNs were observable. CRISPR-Cas9 is capable of editing with higher efficiency and precision on DNA at multiple loci simultaneously. It is able to target a given genome sequence through modifying the guide RNA sequence, whereas TALENs and ZFNs require the re-coding of proteins for each new target site . ImportaHowever, CRISPR-Cas9 was still prone to relatively major off-target effects, even with the help of protospacers to increase specificity . This beDD complexes by more than 900-fold. A further subclass of Cas13b proteins known as Cas13bt has recently been constructed into variant REPAIR and RESCUE editors for transcript knockdown. Cas13bt′s smaller size permits packaging of the editor into an adeno-associated virus for delivery (Type VI (Cas13) systems are used in prokaryotes to target and ultimately cleave RNA. Systems such as Cas13a and Cas13b, which have different cleavage preferences, and guide CRISPR RNA (crRNA) structures , have bedelivery .DD in order to generate various RNA base editors, namely A to I (xABEs) and C–U (xCBEs) base editors, which were capable of editing at various mammalian loci. Subsequently, truncations of dCas13X.1 generating mxABEs and mxCBEs overcame the size limitations other Cas13 systems faced for in vivo delivery. The xABE and mxABE editors were found to perform A–I conversions more efficiently than the REPAIR systems when paired with a crRNA guide, and the mxCBE systems were found to outperform the RESCUE systems for C–U editing, demonstrating high transcriptome fidelity and reducing off-target edits  before undergoing translation. To date, m6A modifications are observed to be the most abundant type of endogenous mRNA modification in eukaryotes . Many m6dCas13 effectors are not the only CRISPR systems capable of m6A modification. CRISPR-Cas9 conjugates were coupled with single-chain methyltransferase and ALKBH5/FTO in order to form writers and erasers, respectively. The target site specificity of these Cas9 editors was programmable through guide RNA . Specifiin vivo manipulation of oncogenic targets on EGFR and MYC transcripts for controlling cell proliferation, while limiting the number of off-target edits. Demethylation efficiency was increased by calibrating the distance between the target sites and methylated sites to 100–300 nucleotides  when combined with dCas13 and targeted with an sgRNA in order to form a dm6ACRISPR system . This coleotides . Howeverleotides , more releotides . In addileotides . We antiThe development of high-throughput technologies and genome-editing has revolutionized the field of functional genomics, which attempts to assess the function and interaction of genes in a systematic approach. Screening tools such as short interfering RNAs (siRNAs) were effective and consistent at silencing gene expression for genetic screens, however, both the cost, the short life of the siRNAs, and the lack of efficient delivery into primary cell cultures put limits on its application . On the PguCas13b and PspCas13b—that have been identified, however, CasRx was shown to be consistently more effective at target RNA knockdown, even more so when fused with an NLS (. We envision similar approaches will utilize CRISPR/Cas programmable RNA modification tools (as discussed earlier and below) to study the epitranscriptome through pooled screening, which will serve as powerful tools to assess all types of RNA modifications.Through the use of CRISPR-Cas13d in combination with improved designs of sgRNAs for circRNA back-splicing junction (BSJ) sites, circRNA silencing specificity is increased, indicating its effectiveness for high-throughput screening of functional circRNAs . In a sih an NLS . CasRx-Bh an NLS . We enviDD was embedded into dCasRx’s flexible loop instead of being linked at its terminal . Grünewald and colleagues  were ablin vitro studies were undertaken for optimizing base editor efficiency, there have been steps taken for using base editing in vivo, specifically in non-human primates and mice  is a gRNA independent system derived from human proteins . It is aThe principles of CRISPR-Cas systems have also been applied to the design of CIRTS (CRISPR/Cas inspired RNA targeting system). These endogenous transcriptome editing tools separate the main required functions  that Cas13 holds in one protein domain into a complex of several different proteins, each one responsible for a single function . There aAnother practical method is the use of antisense oligonucleotide (ASO), which can modulate RNA expression, and have been under development as therapeutic tools for years. In systems such as recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing (RESTORE), only delivery of the oligonucleotide is required. The RESTORE system has been found to achieve higher efficiency than its Cas13 counterparts with limited off-targeting . On the Due to the results and successes of base editor approaches, more recent studies have begun to utilize base editing technology to study m6A modifications. In order to observe the effects of m6A modifications on miRNA and long non-coding RNA, an adenine base editor system (ABE7.10) was used to induce single site base change. After the targeted mutation of an m6A site (T-A conversion) upstream of miR-675 in the H19 locus in HEK293T cells, miR-675, and H19 expression were observed to be suppressed, resulting in an increase in apoptosis . It was in vivo, despite the usual risk of DNA damage and small sample sizes  systems do not utilize double-strand breaks for gene knockouts . Insteadle sizes . Currentm6A has been shown to be associated with many physiological and pathological processes. However, it is only one of many different types of modifications which can be targeted. As such, the possible use of CRISPR and base editors as tools for therapeutic processes has been explored and is currently under promising development.One of the fields of research is sickle cell disease. A customized base editor—ABE8e-NRCH - was developed in order to convert the sickle cell disease allele into a non-pathogenic form (Makassar beta-globin) . Transplin vivo gene editing is by no means an inferior method. LNP delivery of Cas9 endonuclease mRNA and transthyretin-targeting gRNA (NTLA-2001)   was propLA-2001) . The lipLA-2001) . Cas9 of RNA-targeting is the more current and popular methodology for phenotypic observation and novel therapeutic approaches, genome-targeting itself is still a very viable option. Recently, delivery of Cas9 mRNA with herpes simplex virus type 1 (HSV-1) erasing lentiviral particles  stopped HSV-1 viral proliferation, as well as eradicated any latent viral reserves . An addeFor industrial purposes, DNA and RNA targeting systems will likely play a prominent role in agricultural production. Like humans, m6A modifications are critical in plants. In early trials, it was shown that demethylation of m6A by FTO demethylase resulted in increased levels of yield and biomass for rice and potato crops . What m6in vitro m6A detection of pathogenic transcripts will help preclinical research studies and serve as a blueprint for further extended research into other RNA modifications. We expect CRISPR-based approaches will allow the study of the modifications endogenously, as opposed to using conventional exogenous approaches that rely on luciferase reporter assays, which are currently used to measure the role of RNA modifications on protein stability. It is foreseeable that further exploration will bring us to the stage where we are eventually understanding the functions of individual m6A sites, by introducing a variety of writer and eraser fusions that assist with various biological studies, and optimizing CRISPR complexes for high quality pooled screens.It is known that m6A modifications can affect RNA stability, and therefore increase or decrease the amount of translation that occurs as well as the longevity of the molecule itself. Therefore, introducing m6A modifications on individual RNA species could serve as an approach to fine-tune the stability of RNAs, which could have applications for RNA vaccine and RNAi therapeutics. In addition, engineering TRM systems for Costwise, current base editors, CRISPR, and techniques such as CIRTS and RESTORE are more economical than their predecessors. While base editors in general can be delivered in a variety of ways and provide an opportunity for the pursuit of personalized medicine, toxicity is still a concerning obstacle especially for systems such as BARBEKO that rely on sgRNA delivery. Overall, the major transition to Cas systems—especially CRISPR/Cas13—in search for a solution for programmable RNA editing was a fruitful process. Editing efficiency was improved and off-target effects were generally reduced, although they currently remain higher than desired. As more CRISPR proteins are being discovered, we expect that ones with smaller size and lower off-target efficiency will be available to address these shortcomings. With the right coupling, both Cas9 and Cas13 conjugates can target methylation sites for writing/erasing, and the same applies to PUF systems as well. PUF complexes are capable of targeting multiple subcellular compartments, although off-target effects need to be improved by modifying the PUF domain. On the other hand, off-target effects are less of a pressing issue for ASO systems. By adjusting the ADAR domain of ASO systems, the editing efficiency can increase. However, the risk of off-target editing also increases, which is an area that requires further optimization.