Objective biomarkers for the fatal neurodegenerative disease amyotrophic lateral sclerosis or motor neuron disease (ALS/MND) are critical for diagnosis, drug development, clinical trials, and insight into disease pathology. Key candidates for biomarkers present in biofluids include non-coding RNA (ncRNA) transcripts including microRNA, piwi-interacting RNA and transfer RNA. To determine if the central nervous system was the source of the dysregulated ncRNA biomarkers we previously observed in serum, we sought to identify dysregulated ncRNA candidates in cerebrospinal fluid (CSF) which may provide new insight into the disease pathology.n = 18), disease mimics (n = 8), and ALS patients (n = 40) in our Oxford Study for Biomarkers of ALS cohort, with RT-qPCR used to confirm their dysregulation.Small RNA sequencing (RNA-seq) was undertaken on CSF samples from healthy controls . Additionally, our previously identified serum ncRNA biomarker showed no change in CSF or correlation to serum.In this current study, we investigated the expression of non-coding RNA transcripts in the cerebrospinal fluid of ALS patients compared to healthy controls.RNA-seq identified dysregulated non-coding RNA transcripts, but these were not validated with RT-qPCR.We conclude that cerebrospinal fluid is not a suitable source of diagnostic biomarkers.This study suggests the CSF may not be the source of dysregulated ncRNA in the serum and highlights the difficulty in identifying ncRNA in CSF as biomarkers for ALS. TARDBP; TDP-43), chromosome 9 open reading frame 72 (C9orf72), and superoxide dismutase 1 (SOD1) [Motor neuron disease or amyotrophic lateral sclerosis (MND/ALS) is typically a fast-progressing fatal neurodegenerative condition with a lifetime risk of 1 in 300. It causes selective motor neuron loss, progressive muscle wasting, and ultimately death within two to five years after symptom onset, typically from ventilatory insufficiency [The development of biomarkers that are minimally invasive to obtain, and relatively simple and quick to analyse is key for clinical use, and those derived from biofluids such as blood are ideal. In recent years, the short non-coding RNA (ncRNA) species, microRNA (miRNA) have been targets of interest because of their small size, resistance to degradation factors both internally (RNase) and externally , and abundant expression ,4. As a Recently, we investigated ncRNA expression in the serum of ALS patients across multiple cohorts using RNA-sequencing (RNA-seq) and identified seven ncRNA that were differentially expressed and have potential as diagnostic biomarkers . The useThe origins of these ncRNA however are unknown. Their presence in the blood could reflect cellular contents released from dying muscle caused by ALS or could be reflective of their role in the paracrine system of communication between cells ,11. TheyPatients were recruited as part of the Oxford Study for Biomarkers in MND/ALS (BioMOx) from the Oxford MND Centre. Ethical approval for the collection and use of the biofluid samples and associated clinical data were obtained from South Central Oxford Ethics Committee B (08/H0605/85) and NRES Central Committee South Central – Berkshire (14/SC/0083). All participants provided written consent . The study included patients with ALS, patients with ALS mimic conditions ) and healthy control (HC) subjects . PatientClinical information collected included time from disease onset, diagnostic delay, mean disease duration, site of disease onset, presence of any ALS genetic markers, and clinical disability according to the ALS Functional Rating Scale-Revised (ALSFRS-R) scale. Patients were classified as having fast- or slow-progressing ALS based on their rate of change in the ALSFRS-R clinical rating , with a threshold of 0.6 points per month selected based on the median value seen clinically to divide the samples.CSF samples were obtained by lumbar puncture directly into polypropylene collection tubes and centrifuged at 3000 rpm for 10 min at 4 °C within 1 h of sampling and stored at −80 °C until extraction. Total RNA was isolated from 400 µL of CSF samples using the miRNeasy Micro kit (Qiagen) with a DNase I treatment (Qiagen).http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) before reads were analysed on the Qiagen GeneGlobe platform (https://geneglobe.qiagen.com/) to remove 5′ and 3′ adaptors using cutadapt [Healthy control, slow- and fast-progressing ALS (ALS-SP and ALS-FP respectively) samples were grouped into age- and sex-matched pools of four to five samples each and 7 µL of extracted RNA per sample were combined and concentrated to 8 µL using a SpeedVac at ambient temperature for 40 min. RNA-Seq libraries were then generated from the pooled RNA using the QIAseq miRNA library kit (Qiagen) following the recommended parameters for serum RNA. Libraries underwent 75 bp paired end sequencing on an Illumina NextSeq 500 machine at the University of Leeds, UK. Initial sequencing data were checked with FastQC  using fast cycling conditions on a QuantStudio 7 cycling machine. Quantitative cycle (Cq) values were averaged and normalised to the arithmetic average of hsa-miR-9-5p and hsa-miR-501-3p. An average for the healthy control samples was calculated and all samples were compared to this average for a ΔΔCq value.2 µL of RNA extracted from CSF was converted to cDNA using the TaqMan Advanced miRNA RT-qPCR chemistry following the manufacturer’s instructions. Candidate transcripts were profiled using pre-designed primers and master mixes . One-way ANOVA was carried out across the four groups with Tukey’s multiple comparison for parametric data and a Welch’s one-way ANOVA with Gomes-Howell multiple comparison when nonparametric according to a Shapiro-Wilks normality test. Correlations were analysed using Pearson’s rank correlation tests. Correlation analysis between matched serum and CSF samples utilised expression data for ncRNA expression that were collected as part of Joilin et al. [p < 0.05.Statistical analysis for RT-qPCR were conducted on ΔΔCq values for each sample with GraphPad Prism 8.0. Outliers were identified using the ROUT method . Using the Illumina NextSeq, we were able to generate an average of 38 million reads per sample pool. All sample pools as expected showed comparable quality control statistics, with the 75 bp reads having on average sequence and base quality scores of Q31.We sought to identify dysregulated ncRNAs in the CSF of ALS patients that could not only be used as potential biomarkers, but provide insight into the source of the ncRNA in serum and whether they were being trafficked between CSF and blood. To undertake an unbiased screen of dysregulated ncRNA, we undertook next generation RNA-seq to profile all small ncRNA in the CSF of healthy controls and ALS patients using our established protocols . ALS patSupplementary Tables 2 and 3). Despite using increased amounts of CSF and maximum input of extracted RNA from these samples, it appears that there is little intact ncRNA in the samples, which concurs with the difficulties experienced in quantifying the RNA concentration in these samples. These data would suggest the small number of aligned reads and the high number of reads annotated to rRNA may be the result of low RNA input and diversity, supported by an inability to directly quantify RNA concentration using A260 or intercalating fluorescence dyes, and limited analysis of the RNA-seq (Supplementary Table 5). Importantly, there was very little PCR bias introduced, as shown by a high level of correlation (pr > 0.99) before and after correction for unique molecular indices (UMI) which are additional sequences that index each original individual transcript.Using the analysis pipeline provided on the Qiagen GeneGlobe platform, removal of short and non-adapter reads resulted in 18 million reads per sample, of which we had an average alignment to the human genome (hg38) of 30.8%. Of those aligned, 51.73% were annotated . The majority of these reads were aligned to ribosomal RNA (rRNA) transcripts (59.5%) followed by miRNA (33.9%). The other forms of short ncRNA including tRNA and piRNA made up the remaining 6.6% (Supplementary Table 4). Interestingly, ALS-FP had a skew towards down-regulation (46 ncRNA transcripts down-regulated vs 15 ncRNA transcripts up-regulated) in comparison to the more balanced dysregulation in the ALS-SP groups , and between the ALS-SP and ALS-FP groups . Of note, only four ncRNA were significantly dysregulated in both the fast- and slow-progressing ALS groups compared to healthy controls .We next compared ncRNA expression in our slow- and fast-progressing ALS patients compared to our healthy controls (HC) in addition to between the two ALS groups. While the majority of transcripts did not show significant dysregulation, both groups did show both down- and up-regulation of ncRNA transcripts (pr (64) = 0.66, p < 0.0001).We next sought to confirm the dysregulation of candidate ncRNA in the CSF of both slow- and fast-progressing ALS samples. Using the TaqMan Advanced miRNA assay chemistry, we first aimed to identify potential normalisers that showed consistent and correlated expression using NormFinder  on our RA large number of ncRNA that showed large changes in the RNA-seq analysis also had low read counts across all sample pools, suggesting low expression that would be difficult to profile and have limited utility as biomarkers. Therefore, we limited our selection of candidate ncRNA to those that show significant dysregulation and sufficient expression levels  for further confirmation. These ncRNA included hsa-let-7c-5p, hsa-miR-9-3p, hsa-miR-196a-5p, hsa-miR-211-5p, hsa-miR-451a, and hsa-miR-6797-5p in our samples. Additionally, we also profiled the four ncRNA that were dysregulated in both the slow- and fast-progressing ALS patients .Supplementary Figure 1). Furthermore, the remaining miRNA showed little or no amplification with RT-qPCR in all or some samples, even with additional pre-amplification or cycling for particular ncRNA, suggesting that unlike serum, the expression of these ncRNA are not as ubiquitous across patient samples. Combined, these RT-qPCR data on individual samples did not confirm the RNA-seq analysis from pooled samples.For hsa-let-7c-5p, hsa-miR-9-3p, hsa-miR-196a-5p, and TRE-CTC1-2, we were able to show amplification across the majority of samples. However, there was no significant dysregulation of these targets identified between the groups, including the disease mimics which consisted of people with motor disorders similar to ALS . GroupinWe have previously identified seven ncRNA that may be candidates as biomarkers for ALS in the serum , but theSupplementary Figure 2). Lastly, some of the CSF samples from the BioMOx cohort had matching serum samples from the same patient, so using previously collected data from our previous study [Therefore, we profiled the expression of our seven ncRNA using RT-qPCR across our samples from healthy controls, disease mimics, and ALS patients . Firstlyus study , we undeUsing RNA-seq, we have observed that there are a range of ncRNA that are detectable in CSF and that there may be some dysregulation in their expression. However, they did not validate in RT-qPCR assays. As a result, we are unable to fully assess whether this dysregulation occurs in ALS patients and their potential utility as biomarkers for ALS. One reason for these difficulties may be due to the use of our pooling strategy. While pooling RNA from out samples together worked with serum in identifying biomarkers , this apThese observations suggest that for diagnostic biomarker profiling, serum may be more informative than CSF. It appears that ncRNA are lowly expressed in CSF and considering that it is less routinely obtained, the value in undertaking routine profiling of this biofluid for objective monitoring of the disease seems small. In contrast, serum collection from blood is more easily obtainable and routine, has abundant expression of ncRNA, and as described above could still reflect pathological processes such as ncRNA release from dying muscle. As such, from a clinical perspective, identifying ncRNA biomarkers in serum is the ideal proposition. However, identification of ncRNA in CSF could be done from unpooled samples if required, such as clinical trials affecting ncRNA, and if this provided validated targets may provide insight into pathology.We also observed that there was no correlation between the ncRNA we identified as dysregulated in serum with that of CSF. This suggests that dysregulation in the serum is not underpinned by the CSF and that the source of these ncRNA is not from the CNS, though this does not discount that ncRNA are not dysregulated in cells within the CNS in ALS patients, contributing to the pathology to the disease. Therefore, other tissues are more likely to be the source of the ncRNA present in the serum. A chief candidate for this includes muscle tissue, which wastes away during the disease and is likely to lead to the cellular contents being released into the extracellular space and taken up into the blood. This is supported by the up-regulation of hsa-miR-206, which is a muscle enriched miRNA linked to ALS and shown to be important for the maintenance of the neuromuscular junction , and hasClick here for additional data file.Click here for additional data file.


CRISPR-Cas13 is a newly emerging RNA knockdown technology that is comparable to RNAi. Among all members of Cas13, CasRx degrades RNA in human cells with high precision and effectiveness. However, it remains unclear whether the efficiency of this technology can be further improved and applied to gene therapy.In this study, we fuse CasRx crRNA with an antisense ribozyme to construct a synthetic fusion guide RNA that can interact with both CasRx protein and ribozyme and tested the ability of this approach in RNA knockdown and cancer gene therapy. We show that the CasRx-crRNA-ribozyme system (CCRS) is more efficient for RNA knockdown of mRNAs and non-coding RNAs than conventional methods, including CasRx, shRNA, and ribozyme. In particular, CCRS is more effective than wild-type CasRx when targeting multiple transcripts simultaneously. We next use bladder cancer as a model to evaluate the anticancer effects of CCRS targeting multiple genes in vitro and in vivo. CCRS shows a higher anticancer effect than conventional methods, consistent with the gene knockdown results.Thus, our study demonstrates that CCRS expands the design ideas and RNA knockdown capabilities of Cas13 technology and has the potential to be used in disease treatment.The online version contains supplementary material available at 10.1186/s13059-023-02852-w. Gene silencing technology is critical for the functional analysis of genes , 2. WithIn recent years, CRISPR/Cas9 has emerged as a powerful and convenient DNA editing technology . In 2017Since CRISPR has the ability to specifically target DNA or RNA sequences, it can be used as a molecular scaffold to bind different functional molecules . ModifyiFusion of the Cas protein with a functional domain will increase the size of the transgene required for expression . Since mIn this study, we describe a new guide RNA engineering strategy for CasRx to enable additional cutting of target RNAs by fusing the guide RNA of CasRx to an antisense ribozyme and test the ability of this approach in RNA knockdown and cancer gene therapy. We show that the CasRx-crRNA-Ribozyme system (CCRS) has much better RNA knockdown efficiency on both mRNAs and non-coding RNAs than those of the traditional methods, including CasRx, shRNA, and ribozyme. We then applied this strategy to the targeted inhibition of tumor cells both in vitro and in vivo, which also showed a better therapeutic potential than existing strategies.′-end of the crRNA. In the antisense RNA-mediated ribozyme system, the guide RNA includes a hammerhead ribozyme with a 5′-antisense RNA and a 3′-antisense RNA. In other words, the arrangement of CasRx crRNA and ribozyme guide RNA in the core functional domain and RNA recognition domain is reversed. From these features, we constructed a fusion guide RNA by connecting the 3′-end of the crRNA of CasRx to the 5′-end of the ribozyme  and firefly luciferase (Fluc) driven by different promoters on a single vector. Rluc was chosen as the CCRS target by designing the fusion guide RNA and Fluc was treated as the internal control Fig. a. The CCSince CasRx has the ability to process poly crRNAs , we wantThese results suggest that CCRS can knock down foreign reporter genes more effectively than existing strategies.We next compared the ability of CCRS constructs to knockdown endogenous transcripts in HEK293 cells. We selected three coding RNAs  and three non-coding RNAs . The relative transcript level was measured at 48 hours post-transfection by qRT-PCR. We observed varying levels of knockdown, and found that CCRS targeted all mRNAs , linear lncRNAs , and circular RNA (circFAM120A) more efficiently than wild-type CasRx, shRNA, and antisense ribozyme  dramatically decreased CCRS knockdown efficiency Fig. . Any sinBecause CasRx is capable of processing its own CRISPR array, we next sought to extend the application of CCRS in a multiplexed targeting manner. We tested whether CCRS can be used to simultaneously target multiple transcripts  in the same cell for the delivery of multiple targeting fusion guide RNAs in a simple single-vector system Fig. b. IntereSince CCRS allows for multiple endogenous transcripts to be simultaneously regulated, this opens up new possibilities for cancer gene therapy by targeting multiple oncogenic genes at once Fig. a. AnotheNext, we tested the anticancer effects of AAV-mediated CCRS on bladder cancer and compared the efficiency of CCRS with the existing approaches including CasRx and shRNA. Cell proliferation was determined at various time points using the CCK-8 assay. We found that CCRS resulted in approximately 70% inhibition of T24 cell proliferation, while CasRx and shRNA had a moderate affect Fig. b. In theTo further explore the potential applications of AAV-mediated CCRS, we conducted in vivo experiments with subcutaneous tumor models, where AAVs were injected directly into tumors ten days after inoculation of T24 cells expressing luciferase. We found that the volume, weight and fluorescence intensity of xenografted tumors injected with AAVs were decreased compared with that of the control group Fig. d. HematoTogether, these results suggest that CCRS can be used to strongly inhibit the growth and metastasis of bladder cancer cells in vivo and achieves a higher efficiency than traditional technologies.Although RNAs, including mRNA, lncRNA, and circular RNAs are abundant and play important and diverse roles in mammalian cells, the molecular tools available to regulate RNA expression are limited . SeveralHere, we connected the 3′-end of the crRNA of CasRx to the 5′-end of the ribozyme, thus developing CCRS technology. Our results show that the RNA knockdown effect of CCRS is more effective than wild-type CasRx, ribozyme, and shRNA when targeting luciferase reporter genes and endogenous RNAs. Examining single base substitutions revealed that CCRS not only improves the RNA cleavage efficiency of CasRx but also maintains the targeting specificity, thereby indicating that CCRS is a reliable RNA knockdown strategy with more application potential than previous strategies.It is important to note that although CasRx can be used to simultaneously target multiple transcripts, its knockdown efficiency decreases with an increasing number of guides targeting different transcripts. This phenomenon may be related to the dilution effect, because the CasRx protein not only processes the precursor crRNA but also binds and cleaves a variety of different transcripts. Indeed, increasing the number of knockdown targets to 12 slightly reduced the effectiveness of CCRS.Our results show that the performance of CCRS in this case is more effective than that of wild-type CasRx, which may be attributed to the ribozyme activity and the longer complementary antisense sequence of CCRS that enhances the binding of CasRx to target RNA. However, the performance of CasRx was only slightly improved when we used crRNA with the same length as the antisense sequence of CCRS, suggesting that the ribozyme and antisense sequences may synergistically promote the binding of RNA targets. The knockdown efficiency of CCRS was significantly affected by using either the dead version of the ribozyme or dCasRx, which further suggest that CCRS should utilize both CasRx and ribozyme activities to cleave the targets.Next, we used bladder cancer as a model to evaluate the anticancer effects of CCRS targeting multiple genes both in vitro and in vivo. We selected several endogenous targets that are commonly used as cancer treatment targets. CCRS showed a higher anticancer effect than CasRx, ribozyme, and shRNA, consistent with our findings from the in vitro gene knockdown studies. Compared with the CasRx system, CCRS contains only one extra ribozyme and, therefore, can be easily loaded into AAV for gene therapy. This demonstrates the feasibility of CCRS for the treatment of cancers or other diseases.The antisense ribozyme can also be applied to guide RNAs of other Cas13 enzymes besides CasRx. Many Cas13 variants, such as Cas13bt, Cas13x, and Cas13y, have been shown to be very efficient. As Cas13bt, Cas13x, and Cas13y are compact Cas13 members, they may have advantages compared to CasRx , 41, 42.CCRS may also be used to regulate gene expression in bacteria, due to the high biological activity of CasRx nuclease and ribozyme in prokaryotes , 44. As Although our results suggested that CCRS is conceptually simpler than the independent CasRx/ribozyme system to design, the target sequences of CasRx and ribozyme in the CCRS are stringently associated and may be selected carefully. In order to optimize the effectiveness of the CCRS system, further research is needed to determine how to select the best targets.In conclusion, this study describes an alternative RNA targeting tool that can knock down gene expression with higher efficiency than existing approaches. CCRS can be used not only for gene function research but can also be potentially used in gene therapy of diseases.2. Cell cultures were also confirmed to be free of Mycoplasma contamination by using 16S rRNA-based Mycoplasma group-specific polymerase chain reaction (PCR).The HEK-293, HEK-293T, and T24 cell lines used in this study were purchased from American Type Culture Collection (ATCC). Normal human primary fibroblasts derived from the epidermis were kindly provided by Dr. T. Chen . All cell lines were grown in DMEM medium supplemented with 10% fetal bovine serum  in the presence of 5% COE. coli cells. The desired expression clones were identified using PCR amplification and electrophoresis and then confirmed with Sanger sequencing. The sequence information of the related elements is shown in Additional file The backbone plasmid expressing human codon-optimized CasRx protein was purchased from Addgene (# 109049). The shRNA-expressing plasmids were purchased from Syngentech Co., Ltd. . crRNA sequences and crRNA-ribozymes that exactly match the corresponding shRNA sequences were designed. The cDNA sequences for crRNA, ribozyme, crRNA-ribozyme, and shRNA were then synthesized and inserted into corresponding backbone plasmids digested with restriction endonucleases. All vectors were transformed into One Shot TOP10 Chemically Competent HEK-293, HEK-293T, and T24 cells were cultured in plates until they reached 70% confluence. Cells were transiently transfected with the constructed plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol.Both the Renilla luciferase and firefly luciferase activities were measured in a 1.5-ml Eppendorf tube using the Promega Dual-Luciferase Reporter Assay kit (Promega E1980) according to the manufacturer’s protocol 48 h after DNA transfection. The relative luciferase activity was calculated as the Renilla luciferase value normalized to that of the firefly luciferase value. The assays were performed in triplicate and experiments were repeated five times.−△△ct method.Total RNAs were isolated from cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. The concentration and purity of total RNA were measured using UV spectrophotometric analysis at 260 nm. cDNAs were synthesized using the Superscript III First Strand Synthesis System (Invitrogen). Real-time quantitative reverse transcription PCR (qRT-PCR) was performed using a SYBR Green PCR Master Mix (Invitrogen). GAPDH was chosen as the endogenous control. PCR mixtures were prepared according to the manufacturer’s protocol and amplification was performed under the following PCR parameters on an ABI PRISM 7300 Fluorescent Quantitative PCR System : 40 cycles of 15 s at 95 °C, 20 s at 55 °C, and 30 s at 70 °C. All primer sequences are shown in Additional file Total RNAs were obtained using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. One gram of agarose was heated in 72 ml water until dissolved and then cooled to 60 °C. Ten milliliters of 10X MOPS running buffer and 18 ml 37% formaldehyde (12.3 M) were then added to prepare the gel. The gel was prerun for 10 min at 80 V, and 5 μg of total RNA was then loaded and separated until the bromophenol blue migrated 3 cm into the gel. The gel was visualized on a UV transilluminator.25 Dynabeads, and mRNA was purified according to the manufacturers’ instructions (Invitrogen). The purified mRNA was first fragmented by using divalent cations before being subjected to library preparation. Adapters were ligated to fragmented RNAs by using truncated T4 RNA ligase 2 (NEB) according to the manufacturer’s instructions, and RNA was reverse transcribed to DNA by using SuperScript® III  and circularized by using Circligase™ . Barcodes were added by PCR using Phusion® polymerase (Thermo Scientific). DNA libraries were sequenced on an Illumina HiSeq 2500 (Illumina). Reads were processed by Cufflinks v2.1.1 and fold changes were then calculated according to fragments per kilobase of transcript per million mapped reads (FPKM) values.Total RNA was isolated, mixed with oligo (dT)The pAAV packaging plasmid, pHelper plasmid, and pAAV plasmid were co-transfected into HEK-293T cells using Lipofectamine 3000. The culture supernatants were collected at 48 hours after plasmid transfection, concentrated, and used as virus stocks for subsequent AAV infection experiments. The AAV titer was calculated by qPCR using 2× EvaGreen Master Mix (Syngentech).Cell proliferation was assayed using the Cell Counting Kit-8 (CCK-8)  according to the manufacturer’s instructions. At various time points  post-transduction, 10 μl of CCK-8 reagent was added to each well of the 96-well plates, and the cells were incubated for an hour. Absorbance was read at a wavelength of 450 nm using a microplate reader . The assays were performed in triplicate and experiments were repeated five times.According to the manufacturer’s protocol, cell apoptosis was determined by the caspase-3/ELISA (enzyme-linked immunosorbent assay) assay . Caspase-3 is a marker for inflammation and apoptosis signaling. The experiment for each sample was repeated five times independently.Cell migration was determined by the wound healing assay. Briefly, cells were seeded into 12-well plates at equal density and cultured to 80% confluency. Artificial gaps were generated using a sterile pipette tip. Areas of wound were marked and photographed using a digital camera system. Cell migration distance (mm) was calculated using the software program HMIAS-2000. Each experiment was repeated five times.7) were hypodermically injected on the backs of BALB/c-Nude mice. Next, intratumoral injection of AAV  was performed. Tumor volumes were calculated using the formula: V = L × W2/2, where L is the length and W is the width of the tumor. The mice were sacrificed and tumors were removed at the end of the experiment.The mice were housed under standard laboratory conditions. BALB/c-Nude mice were randomly assigned into either the experimental group or control groups (five mice for each group). Bladder cancer T24 cells (5 × 107) were hypodermically injected on the backs of BALB/c -Nude mice, and then a single injection of AAV  via tail vein was conducted 10 days after inoculation. Four weeks later, mice were anaesthetized with isoflurane and injected with D-luciferin sodium salt (150 mg/kg) intraperitoneally. Subcutaneous tumor was then monitored by an in vivo imaging system, Xenogen IVIS .For in vivo imaging experiments, BALB/c-Nude mice were randomly assigned into either the experimental group or control groups (three mice for each group). Bladder cancer T24 cells (5 × 105) were suspended in 200 μL PBS and injected into the lateral tail veins of 5-week-old male B-NDG mice . Four weeks later, mice were anaesthetized with isoflurane, and D-luciferin sodium salt (150 mg/kg) was injected intraperitoneally. Bladder cancer cells were detected with an in vivo imaging system, Xenogen IVIS . The total flux in photons per second was calculated for the lung region using Living Image 4.3.1 .T24 bladder cancer cells stably expressing luciferase . Significance tests were performed using the SPSS version 20.0 software . Statistical significance was determined using Student’s Additional file 1: Figure S1. The expression level of Renilla luciferase (Rluc) mRNA in HEK293 cells. Figure S2. Detection of the potential influence of CasRx on the engineered crRNAs. Figure S3. Knockdown of Renilla luciferase (Rluc) using CCRS, CCRS-mutants and other related technologies. Figure S4. Comparison of knockdown effects of different versions of hammerhead ribozymes in the CCRS system. Figure S5. Targeting endogenous transcripts with CCRS in primary cultured cells. Figure S6. RNA-denaturing gel electrophoresis examining RNA integrity. Figure S7. Analysis of the specificity of CCRS-mediated knockdown. Figure S8. Evaluation of CCRS knockdown specificity on endogenous gene. Figure S9. Evaluation of CasRx knockdown specificity on luciferase gene. Figure S10. qRT-PCR analysis of relative target RNA knockdown by CasRx and shRNA. Figure S11. Targeting other endogenous transcripts with CCRS. Figure S12. qRT-PCR analysis of relative target RNA knockdown by AAV-CCRS and other existing technologies. Figure S13. The inhibition rate (%) of T24 cell proliferation. Figure S14. ELISA assay on Caspase-3 activity in T24 cells treated by AAV-CCRS and other existing technologies. Figure S15. AAV-CCRS efficiently inhibited T24 cell migration. Figure S16. AAV-CCRS efficiently inhibited in vivo tumor growth. Figure S17. Histopathological inspection of the mouse lungs treated with AAVs.Additional file 2: Table S1. The cDNA sequences of the engineered elements used in this study.Additional file 3: Table S2. Primer sequences used in real-time quantitative PCR.Additional file 4. Review history.


Vaccinia Capping System to synthesize and cap mRNA.We created an “integrated reaction buffer” that supportsboth capping enzymes for catalytic and in vitro transcription processes,enabling one-pot, two-step capped mRNA synthesis. Additionally, wereport a novel, one-step analytic platform for rapid, quantitative,capped mRNA analysis. The assay involves target mRNA segment protectionwith cheap DNA primers and RNase digest of non-hybridized or non-targetsequences before analysis by single nucleotide-resolving urea-polyacrylamidegel electrophoresis (PAGE). The integrated approach simplifies productionprocesses and saves costs. Moreover, this assay has potential applicationsfor mRNA analyses and post-transcriptional modification detectionin biological samples. Finally, we propose a strategy that may enableunparalleled sequence coverage in RNase mass mapping by adapting thedeveloped assay and replacing urea-PAGE with mass spectrometry.The existing platformfor large-scale mRNA production is fast,but consumable costs, process technicality, and complexity representkey bottlenecks limiting global mRNA biologics manufacturing. Anotherchallenge is the lack of a consolidated platform for mRNA productcharacterization and assays that meet regulatory requirements. Bridgingthese innovation gaps to simplify processes and reduce cost wouldimprove mRNA biologics manufacturability, especially in low-resourcesettings. This study develops a “cotranscriptional”capping strategy that utilizes T7 RNA polymerase, and the  A recent study has reported an efficient expressionand purification protocol that enables large-scale production of Vaccinia enzymes, allowing scaled production of cappedmRNA with 100% efficiency.21 The workflowin the Vaccinia strategy involves transcription,followed by DNA template decontamination and RNA purification beforethe capping process. Extra purification steps involved in standardIVT and capping strategy in capping increase production cost and time.A commercially available system, mScript, is reported to synthesizecapped mRNA using a cocktail of T7 RNA polymerase, a trifunctionalcapping enzyme, a 2′-O-methyltransferase,and a poly(A) polymerase18 but also requiresan RNA purification step. However, this system is expensive, and theadditional therapeutic licensing required limits scaled production,especially in poorer countries.Another strategy deploys post-transcriptionalcapping, where the 22 However, there arewell-established methods for mRNA analysis in biological samples.One of those methods includes an RNase protection assay that requiresa labeled RNA probe, purification steps, and autoradiography to detectmRNA.23 Detection and quantification ofmRNA post-transcriptional modifications, including 5′ cap,poly(A) tail, and epitranscriptomes are also essential aspects ofmRNA analytics. Traditional methods for 5′ cap detection relyon nuclease-mediated hydrolysis of mRNA to generate mononucleotidesand subsequent detection of the incorporated P32 label at the 5′terminal phosphate by polyacrylamide gel electrophoresis (PAGE) orhigh-performance liquid chromatography (HPLC).25 Another strategy is to cleave small sections of the 5′ endusing RNase H combined with radiolabel detection by urea-PAGE.26 These approaches rely on autoradiography, whichmay not be ideal for industrial settings. Moreover, most of them areonly suitable for detecting short RNA of about 50 nucleotide length.Additionally, a strong imperativeexists to develop simple, integrated,and cost-effective platforms for characterizing mRNA due to the lackof consolidated and transferable mRNA analytical tools that meet regulatoryagency requirements.27 Witha cleavage site only 4–5 nt in length, the risk of generatingadditional fragments is high, thus impacting the method’s reliability.Other recent methods include a reportedly improved Rnase H method,28 ribozymes cleavage-based assays,29 or biosensors to detect mRNA cap structures.30 Although these approaches yield valuable, semi-quantitativeor quantitative information and avoid radiolabeling, reagent or processcosts may limit their industry applications, especially in low-resourcesettings.Other strategies exist for label-free RNA analysis and capped detection.One method employs RNase H with a fused biotin-tagged RNA-DNA probe(to cleave the mRNA 5′ end), a purification step, and massspectrometry to detect capping.31With the increasing need for equitable, global, anddecentralizedaccess to biologics, there is a growing demand for simple, rapid,flexible, and scalable biomanufacturing and bioanalytic systems thatwill enable affordable, safe, and consistent production of biologics.Vaccinia capping enzymes, respectively. The system’s essential componentsare T7 polymerase, Vaccinia cappingenzyme (D1 and D2 subunits), nucleotide triphosphates (NTPs), S-adenosyl methionine (SAM), and an optimized buffer compatiblewith both enzymes. Enzymatic post-transcriptional capping eliminatesthe need for patented synthetic cap analogues, the ARCA system, orexpensive commercial capping alternatives. In addition, it circumventsthe need for multiple purifications following IVT and before mRNAcapping used in conventional mRNA platforms. We demonstrate that thissystem produces yield comparable to the commercial IVT kit and has100% capping efficiency.To address this demand for more straightforwardand affordablebiomanufacturing platforms for mRNA therapeutics, we developed anintegrated, single-pot platform that allows for simultaneous IVT andcapping of mRNA mediated by T7 polymerase and In addition, we develop a simple andrapid urea-PAGE method fordetecting and identifying synthesized mRNA products and quantitativedetection of capping. This method utilizes inexpensive DNA primersor probes of 16–25 nt length to protect the 5′ end ofmRNA before digestion by single-stranded RNA-specific RNases (RNaseA and RNase T1), followed by analysis by denaturing urea-PAGE. A novelfeature of our method that distinguishes it from other methods isthat it utilizes inexpensive DNA probes and a cheap sensitive dye.In contrast to the original RNase protection assay, which uses labeledRNA probes, our method employs unlabeled, inexpensive DNA probes tocreate DNA–RNA hybrids resistant to RNA cleavage. Moreover,this method requires no biotin tagging, autoradiography, or priorpurification of IVT material before analysis. We also propose thatthe developed assay could adapt HPLC–mass spectrometry analyticalapproaches to identify mRNA and post-transcriptional modifications.Vaccinia enzyme-mediated capping into one process,we investigated their reaction buffer compositions established inthe literature and assessed each component’s concentrationversus activity profile, essentiality, and compatibilities. Analysisof the data in the literature reveals that Vaccinia capping and IVT buffers have a combined total of 11 chemicals butonly share four components in common, Tris-HCl, MgCl2,DTT, and KCl (Table S1). The KCl appearsonce out of the 11 transcription buffers in the literature/manufacturers’manual. Some chemicals were essential for capping but not for IVTand vice versa. Therefore, to design an integrated buffer system compatiblewith IVT and capping reactions, it was necessary to consider the potentialimpacts of the chemical components on each reaction buffer. For theintegrated buffer composition, we selected the final chemicals andconcentrations by subjecting them to the following formulated rules:(1) include the chemical if it was essential for one reaction typeeven if it is not present in the other. It is essential when a chemicaloccurs at 100% frequency in the buffer composition found in the literature.(2) exclude if a chemical is not essential and not found in the otherreaction. (3) Rule of integration: Use the highest concentration valuefor chemicals that occur in both unless it is detrimental to the otherbuffer or if evidence exists that the lowest quantity has the sameeffect as the highest value, in which case use the lowest value.To integrate the IVT and Vaccinia capping enzymes is unknown in the literatureand is possible that it is not particularly essential to T7 polymeraseactivity. A study has shown that elevated NaCl concentrations aredetrimental to T7 polymerase,32 but inhibitoryconcentration for Vaccinia enzymesis unknown. Moreover, the storage buffer of T7 RNA polymerase and Vaccinia capping enzymes have a high concentrationof NaCl (100 mM), so IVT will still benefit from residual salt fromthese.On this basis, we eliminated NaCl, Triton X-100, and Tween-20. Theremoval of these chemicals was precautionary as their effects on 2, 7.4 mM DTT, 1.8 spermidine, and 2.0 mM NTPs for T7 polymerase(Table S1). For Vaccinia capping enzymes, all buffers found in the literature contain identicalconcentrations of chemicals (Table S1).We then applied rule 3 to generate chemical concentrations for thepredicted integrated buffer (see Table S2).Further analysis of literature data to predict optimalchemicalconcentrations reveal a median of 41.1 mM for Tris-HCL, 9.9 mM MgClVaccinia Capping System do not inhibit but support IVT.Following the rational design of an integratedbuffer, it was necessary to experimentally test the performance ofthe integrated buffer and how it affects mRNA yield. First, we wantedto know the effect of the capping substrates  and enzymes on the IVT. Therefore, we set up an IVT reactionin the integrated buffer with or without capping enzymes. We transcribeda DNA template encoding an 86 nt RNA in these buffers. Following theIVT reaction, an optimized method using DNase I treatment in conjunctionwith solid phase extraction using a silica membrane spin column topurify further the RNA product was performed before agarose gel electrophoresisand nanodrop spectrophotometer analysis. The agarose gel analysisshows (We then tested the synthesis ofa longer mRNA (2191 nt) in formulatedintegrated buffers (IB1 and IB2) and other varied conditions, includingintroducing a capping enzyme at different reaction stages. The generatedproducts appear heterogeneous as two RNA bands appear on the agarosegel. The shorter RNA may be due to incomplete transcription drivenby sequence composition. Although it is hard to say without sequencing,the polyA tail or some secondary structure present in this 2191 ntRNA may be impacting the processivity of the T7 polymerase resultingin a fraction of the transcripts being shorter. Initial analysis ofthe RNA concentration shows that reaction performed in integratedbuffer 1 (IB1) containing capping enzymes appears to have a similaror higher RNA titer (2020 ng/μL) 2C. IB2sVaccinia capping enzyme system.A statistical comparison would enable us to determine if the presenceof the capping enzyme in the integrated buffer significantly reducesmRNA yield. Our analysis shows no significant difference in yield,confirming that the capping enzyme in the formulated buffer does notsubstantially affect mRNA yield (Tables S3–S5).Due to the costsof enzymes, it was not possible in this studyto perform a comprehensive experiment that probes the significanceof the RNA yields obtained from these highlighted reaction scenarios.However, to provide insight, we performed reactions in four replicatesto compare the 2191 nt mRNA yield of formulated reaction buffers ±the 33 and, by extension,the RNA strand on an RNA–DNA hybrid under this condition. However,as we have observed in experiments, it does not cleave dsRNA in asolution with excess salt. Moreover, DNA–RNA hybrids are morestable than DNA–DNA.34 Therefore,we reasoned that the DNA–RNA hybrid would remain intact andunaffected by the RNase A treatment under high saline conditions.In a strategy to develop an inexpensive integrated platformfor the synthesis and analysis of mRNA, it was desirable to createa method that can rapidly identify mRNA and detect capped structureswithout the need for purification of the IVT product. Our strategyexploits RNase A’s specificity for single-stranded RNA (ssRNA)species under specific conditions. RNase A cleaves dsRNA species inlow-saline solutionConsequently, oligonucleotides designed to anneal to any segmentof a single-stranded RNA molecule will protect that region; conversely,mRNA sequence regions not hybridized to the oligonucleotide will becleaved by RNase A. Therefore, when cleaved with RNase A, bands correspondingto the DNA probe and RNA fragment will be prominent on a denaturingPAGE. It is possible to resolve the RNA–DNA hybrid oligos onthe PAGE because, first, under denaturing conditions, such as existingon the urea-PAGE, the oligo strands will separate. Second, we haveobserved in experiments that RNA and DNA oligos of the same lengthhave different electrophoretic mobilities, with RNA migrating sloweron a polyacrylamide gel. Therefore, DNA and RNA fragment of the samesize can be separated. Lastly, we hypothesize if the RNA target isnot present in a sample or the designed probe is not complementaryto the target mRNA then only the DNA probe band will be observed.We propose that, on this basis, this assay can be used to confirmthe identity of an mRNA. The general scheme of the Rnase A-based assayis illustrated in 3A.To test our hypotheses, we designed a 24 and 26 nt DNA oligonucleotide(£2.5 per 50 nmoles) that anneals to the 5′ end of thetarget RNAs. This oligonucleotide was annealed to the RNA under salineconditions, and the reaction product was analyzed on 8% urea 8–20%polyacrylamide gel (urea-PAGE), as described in the Similarly, the 26 nt DNAprobe assay generated two prominent bandsand a single band for treatment and control (26 nt DNA probe) samples,respectively  with the RNase A/T1 mixtureunder saline conditions (700 mM NaCl) and in the presence of a 20nt probe. The urea-PAGE analysis of the digested samples reveals twoprominent bands see 3C corresThe developed mRNA detectionapproach is flexible and enables thedesign of DNA oligonucleotides to target unique mRNA sequences andregions of any length. As a result, nuclease digest could generateunique RNA fragments and bands on PAGE. Moreover, targeting the 5′end or the 3′ end of mRNA with an appropriate complementaryDNA probe can detect capped mRNA and poly(A) tail, respectively.Vaccinia capping enzyme, IVT, and capping components.First, we performed the Vaccinia enzymesystem-based “co-transcription” capping reaction inthe integrated buffer  using a 2191 bp DNA template. The negative controlhas the same composition except for Vaccinia capping enzymes. Following the IVT-capping reaction, we treatedthe IVT product with a 20 nt DNA probe designed to protect the mRNA’s5′ end, digested with RNase A, and analyzed by urea-PAGE analysis.The result shows two bands corresponding to the capped and uncappedmRNA  used in this experiment have identical 5′ end sequencesand are targeted by the same 20 nt DNA probe. Similar to the assayperformed for 2191 nt mRNA, the reaction product was digested withRNase A in the presence of the designed 20 nt DNA probe and analyzedon the urea-PAGE. The result shows two bands corresponding to cappedand uncapped mRNA. The result for the 86 nt RNA is consistent withthat obtained for the 2191 nt mRNA, demonstrating that the lengthof RNA does not significantly impact transcription and capping inthis integrated system.Although urea-PAGE analysis shows thata capping reaction occurred,it also revealed that the capping efficiency is not 100%. Visual inspectionof the urea-PAGE estimated that about 70% 2191 nt and 50% 86 nt RNAwere capped 4A. It waVaccinia cappingenzyme loses efficiency with longer incubation time. A previous studyhas shown that longer incubation increases capping, but the enzymeloses activity for hours,21 which is consistentwith our assumption. The IVT/capping reaction was performed over 3h, and we reasoned that much of the earlier transcripts would havebeen effectively capped (within 1–2 h) than those synthesizedmuch later in the reaction. In addition, we also speculated that theamount of capping enzyme used might not be sufficient. It is alsopossible that buffer conditions or the release of by-products, suchas pyrophosphates, during the reaction may have impacted the cappingenzyme activity.To optimize capping efficiency in this system,we assumed that the We first tested the efficiency of capping the86 nt transcript in this experiment with a control experiment (reactionwithout capping enzyme). We performed the reaction as before to testour hypothesis that insufficient enzyme concentration and loss ofenzyme activity may be responsible. However, we supplemented the reactionwith more capping enzymes in the last hour. A comparison of thesetwo, following urea-PAGE analysis, reveals a mobility shift, witha fragment from the capping reaction 4A, more Vaccinia cappingenzyme into the one-buffer system.The result demonstrates thesuccessful integration of IVT and cappingprocesses mediated by the Figure S2). First, we transcribed them in IB1,IB2, and commercial VCE + IVT reaction buffer mix (1:1 mix) beforeagarose gel electrophoresis. To test if the developed buffer generatesspurious products, no template control was performed in the IB1 bufferwith or without VCE. The result shows that transcription occurredacross all tested conditions except for the no-control template  and S1 (1152 bp) sequences see S2. FirstFollowing transcription, the S1 and N15 RNA samples generatedunderdifferent conditions were subjected to the DNA-probe RNase protectionassay before UREA PAGE analysis. From experience in our lab, we haveobserved optimal single-nucleotide resolution in the range of 16–24nt DNA oligos on our urea-PAGE. We reasoned that RNA fragments withinthis range would be optimally resolved in our system. Therefore, ourchoice of oligonucleotides ranging from 16 to 20 nt in length wasto ensure optimal single-nucleotide resolution while demonstratingprobe design flexibility for cap analysis in our system. We anticipatethat single-nucleotide resolution of shorter or longer oligonucleotidefragments is also possible with optimal electrophoresis run time andurea-PAGE composition. Consistent with previous results, expectedRNA fragments corresponding to capped and uncapped sequences wereobserved see 5B,C. TakVaccinia enzyme-mediated capping. This approach eliminatesthe extra unit operation and attendant costs of performing two-stepsynthesis and purification steps in the traditional enzymatic methodwhile retaining high capping efficiency. We have decreased the complexityby reducing the synthetic steps, thereby enhancing scalability andmanufacturability. We show that the system produces mRNA yields similarto those of commercial IVT kits.This studypresents a less expensive single-step method that combinesthe advantages of both co-transcriptional and enzymatic capped mRNAsynthesis. We developed this novel one-step platform for capped mRNAsynthesis by rationally designing and optimizing an “integratedreaction buffer” that supports both IVT and Additionally, we report a simple,novel method for rapidly andquantitatively detecting mRNA and the capped structure. This approachinvolves protecting a short target sequence within mRNA with a complementaryDNA probe, followed by RNase digest and one-nucleotide resolving urea-PAGE.This platform integrates a novel one-step synthesis of capped mRNAwith a one-step assay that, in conjunction with PAGE, identifies mRNAand structural RNA modifications. The assay uses inexpensive reagents:DNA primers (£2 per 100 μM per 300 assays) and susceptibledyes . It does notrequire costly labeled RNA probes or any tedious purification steps.However, adequate precaution is necessary for handling RNase duringthe protection assay to avoid accidental contamination of samples.We speculate that this method could be deployed for mass spectrometryanalysis of mRNA by designing different oligos targeting differentregions spanning the length of the mRNA. It may also find applicationsin isolating and detecting post-transcriptional modifications in biologicalsamples by designing oligonucleotide primers targeting the modifiedsequence in the mRNA in conjunction with mass spectrometry analysis.We predict that, if implemented, this would achieve higher sequencecoverage than is currently possible using traditional RNase-mass mappingapproaches.O-methyltransferase activities. In the future, we hope totest this and further optimize reaction conditions to improve RNAand capping yield.The limitation of this study is that only cap 0capping efficiencywas tested in the developed reaction buffer. Cap 0 is currently beingphased out due to potential immunogenicity concerns. We speculatethat the developed buffer will also support Cap 1 capping since thesame reaction buffer supports commercial VCE and mRNA Cap 2′-Vaccinia CappingSystem (New England Biolab), and synthetic gene from GeneArt GeneSynthesis (Invitrogen Life technologies). Two synthetic genes encoding86 nt RNA and 2191 nt mRNA were used for IVT . The DNA template sequence architecture, primersequences, and probes used in the DNA primer-based protection assayare depicted in Figure S1. Four DNA primerspurchased from Thermo Fisher Scientific were used in the protectionassay. The NTP set, 100 mM solution (ThermoFisher Scientific), andthe HiScribe T7 High Yield RNA Synthesis Kit were used for IVT. 50×TAEand 10×TBE buffers from Thermo Fisher Scientific were used toperform agarose and urea-PAGE, respectively. Other chemicals usedinclude urea pellets (Sigma-Aldrich), molecular grade agarose (Bioline),acrylamide/bis-acrylamide (Sigma-Aldrich), and nuclease-free water(Thermo Fisher Scientific). Thiazole orange (Sigma-Aldrich) was usedas a dye for urea-PAGE. Midori green direct (Nippon genetics) wasused as a stain for the RNA agarose gel electrophoresis and analysis.A Fastgene 470 nm blue LED transilluminator (Nippon Genetics) wasused to image all gels.Q5 High-Fidelity DNA polymerase,dNTPs, and designed primers fromMWG Eurofins were used for PCR and IVT performed using T7 RNA polymerase(New England Biolabs), 4041424344 and manufacturers’manuals .Their chemical component concentrations were compared and analyzedto obtain median concentrations. Similarly, a search identified thecomponents of Vaccinia capping bufferfrom the literature46 and manufacturing sources  and analyzedthem. The final components and concentration of the integrated bufferswere selected by applying three rationalized rules to the data: (1)include a chemical if it is essential for one reaction type even ifit is not present in the other. It is essential when a chemical occursat 100% frequency in the buffer composition found in the literature.(2) exclude if a chemical is not essential and not found in the otherreaction. (3) Rule of integration: use the highest concentration valuefor chemicals that occur in both unless it is detrimental to the otherbuffer or evidence exists that the lowest quantity has the same effectas the highest value, in which case use the lowest value*. Essentialwas defined as when a chemical occurs at 100% frequency in a buffer.By applying these rules, the chemical components and the concentrationof the predicted integrated buffer were established. 10× Integratedbuffers were prepared by adding each component at the right concentrationdeduced from the analysis of the concentration data obtained fromthe literature. The buffer composition can be broadly classified asIVT-CAP core components  and capping substrates (SAM and GTP). Two versionsof the 10× integrated buffer (IB) were prepared: (1) Integratedbuffer 1 (IB1) is composed of 500 mM Tris, 99 mM MgCl2,10 mM DTT, 18 mM spermidine, 20 mM NTPs, 1 mM SAM, and 5 mM GTP . (2) Integrated buffer 2 (IB2) of 500 mM Tris, 99mM MgCl2, 10 mM DTT, 18 mM spermidine, and 20 mM NTPs.1× solution, when required, is obtained by dilution with nuclease-freewater. Stock solutions of all the required components for an integratedbuffer were prepared in sterile 15 mL polystyrene falcon tubes (brandname) using nuclease-free water (Thermo Fisher Scientific). Thesestock solutions were filtered with single-use 0.2 μm PES syringefilters to minimize adventitious contamination and stored at the appropriatetemperature recommended by manufacturers.IVTbuffers were extracted from eleven literature refs –40414243An 86 and 2161 bpDNA encoding mRNA was amplified from the 86th and 2161st regions ofa plasmid (termed pSpmep2) and used as the template for the in vitroreaction. PCR was performed using primers flanking the dsRNA geneunder the following conditions. 0.02 U/μL Q5 High-Fidelity DNAPolymerase, 200 μM dNTPs, 0.5 μM each of forward and reverseprimers, and 10 ng of DNA template. The following PCR parameters wereused: the initial denaturation was 1 cycle of 30 s at 97 °C,followed by 30 cycles of  and a final extension at 72 °C for 2min. IVT was performed using T7 RNA polymerase (New England Biolabs).For IVT using CB ,a 20 μL of reaction was set up and contained 10 mM NTPs, 1×reaction buffer, 1 μg of DNA template, and 2 μL of T7RNA polymerase in 20 μL of RNase-free water. IVT reactions usinga formulated “integrated” buffer were prepared as follows:20 μL of reaction was set up by mixing 1 μg of the DNAtemplate (dissolved in 10 μL of nuclease-free water) with 2μL of 10× integrated buffer. Then, an appropriate volumeof nuclease-free water and 2 μL of T7 polymerase (NEB) was addedto make the final 1× reaction solution.Vaccinia CappingSystem (NEB), were used to performa capping reaction in the “integrated” buffer and benchmarkedagainst the reaction in the Vaccinia capping buffer (NEB). Four distinct reactions were performed: (1)20 μL capping reaction using a commercial kit: heat 10 μgof RNA (purified 86 or 2191 nt) in 15.0 μL of nuclease-freewater at 65 °C for 5 min. Place the tube on ice for 5 min andadd the following: 2 μL of 10× capping buffer, 1 μLof GTP (10 mM), 1 μL of SAM ,and 1.0 μL of Vaccinia cappingenzyme (NEB). (2) Standalone capping reactions in “integrated”buffers 1 and 2: 5 μL of IVT product (containing an estimated70–90 μg of RNA) in 1× integrated buffer was combinedwith 13 μL integrated buffer and 2 μL of Vaccinia capping enzymes (NEB). (3) Integrated (single-step)IVT and capping reaction: 1 μg of the DNA template (dissolvedin 15 μL nuclease-free water was mixed with 2 μL of T7polymerase (NEB) and 2 μL of Vaccinia capping enzymes (NEB). (4) Integrated (single-step) IVT and cappingreaction with supplementary capping enzyme: 1 μg of the DNAtemplate (dissolved in 15 μL of nuclease-free water) was mixedwith 2 μL of T7 polymerase (NEB) and 2 μL of Vaccinia capping enzymes (NEB). All reactions wereincubated at 37 °C for 2 or 3 h; reaction time depends on theexperimental design. For reactions incubated for 3 h, the reactionwas supplemented with 2 μL of Vaccinia capping enzyme (NEB) added after 2 h  or not(control for treatment) before incubation for another 1 h.Enzymes from the kit, IVT-generated RNA was made with 50μL of nuclease-free water, treated with 2 μL of DNaseI, and incubated at 37 °C for 20 min 80 μL of 5 M NaCland 150 μL of isopropanol were added to this mixture beforetransfer to a silica spin column (Geneflow Limited). The spin columnwas centrifuged at 13,000 rpm for 30 s, and the flowthrough was discarded.500 μL 70% ethanol was added, and the spin column was centrifugedat 13,000 rpm for 30 s. The supernatant was discarded, followed byrepeated centrifugation for 30 s to remove residual ethanol. The columnwas eluted with 100 μL of nuclease-free water. The RNA concentrationand quality were determined using a NanoDrop 2000c spectrophotometer(Thermo Scientific).1 μg (for 86 nt RNA) or 5 μg(>1000 nt RNA) of purified and un-purified IVT-generated mRNA wasmixed with 800 ng of either 20, 24, or 26 nucleotide DNA primer targetingthe 5′ end of RNA. For cap analysis, DNA oligos of 16–20nt length are designed to enable single-nucleotide resolution withoutprolonged run time. Following annealing, 10 μL of 700 mM NaClsolution was added. A non-target 27 nucleotide DNA was used in thecontrol assay. 2 μg of RNase A or a mixture of RNase A (2 μg)and T1 (2 U) (Thermo Fisher Scientific) was added and incubated for15 min. After the RNase digest, the sample was loaded on 8–20%urea-PAGE (10.1 × 8.2 cm × 1 mm gel) and run for 2 h at200 V. The gel was stained with thiazole orange (10 ng/mL) solutionin 1× TBE buffer. The gel was imaged using a 470 nm blue LEDtransilluminator (Fastgene).1% agarosegel was usedfor gel electrophoresis. RNA loading dye 2× (NEB) was added tothe RNA sample and loaded on the gel. 1× TAE buffer (40 mM Tris(pH 7.6), 20 mM acetic acid, and 1 mM EDTA) was used to perform electrophoresisat 100 V for 45 min. The gel was imaged using a 470 nm blue LED transilluminator(Fastgene).The experimentsubjected to statisticalanalysis was conducted in quadruplicate. Statistical analysis wasaccomplished using Graphpad Prism 9. One-way analysis of variance(ANOVA) and Tukey’s multiple comparison analysis were performedto determine if there were significant differences between the differentconditions tested.


Our manuscript presents a paradigm for carrying out distributed science. We have harnessed an online RNA design game, Eterna, to challenge a large community of RNA designers to create diverse RNA sensors. RNA is an attractive, biocompatible substrate for the design and implementation of molecular sensors. We tasked the diverse Eterna community, comprising a global network of molecular design enthusiasts, to submit thousands to tens of thousands of “solutions” to these RNA sensor design challenges. Crucially, community designs were synthesized and tested experimentally in the real world using high-throughput methods for biochemical assays built on repurposed DNA sequencers. The best player-generated designs for RNA sensors approached the thermodynamic optimum. Internet-based scientific communities promise a means to apply distributed, diverse human intelligence toward previously intractable scientific problems. However, current implementations have not allowed communities to propose experiments to test all emerging hypotheses at scale or to modify hypotheses in response to experiments. We report high-throughput methods for molecular characterization of nucleic acids that enable the large-scale video game–based crowdsourcing of RNA sensor design, followed by high-throughput functional characterization. Iterative design testing of thousands of crowdsourced RNA sensor designs produced near–thermodynamically optimal and reversible RNA switches that act as self-contained molecular sensors and couple five distinct small molecule inputs to three distinct protein binding and fluorogenic outputs. This work suggests a paradigm for widely distributed experimental bioscience. Several recent platforms have demonstrated the engagement, via the internet, of vast untapped human cognitive potential, transforming the scale by which scientific questions can be approached with human intelligence. However, previous work has only allowed “citizen scientists” to process data already collected , 2, to i47RNA is an attractive substrate for diagnostics , cellulaR2 = 0.94)  (AR) of the RNA switch as the fold change in the observed D,obsK between the off state (weak reporter ligand binding) and the on state (strong reporter ligand binding). In the limit of low reporter (output ligand) concentrations, this fold change in D,obsK is equal to the ratio of measured intensities commonly reported for switches in literature. In contrast to measuring signals at single reporter concentrations, however, we determined changes in the signal from the full range of on-state reporter affinities, which allows for maximized absolute signal changes and for quantities to be directly related to biophysical models  –23. ResuFig. S1) A. In thil models .ARmax) expected for this challenge, as determined by the dissociation constant of the FMN aptamer and the experimental trigger concentration of input ligand, was 132 ± 33 . Over seven consecutive rounds of design, synthesis, characterization, and community-wide discussion, 149 members of the Eterna community submitted over 30,000 designs . Of these, 30 designs from five distinct individuals fell within error of the thermodynamic maximum .As a first test, we challenged players to couple input and output RNA aptamers that were individually well characterized but had not been previously combined into an RNA switch: an 11-nt aptamer for the cellular metabolite flavin mononucleotide   and a 19132 ± 33  . This game update allowed the community to autonomously explore a wide variety of such element orderings  in remaining rounds  the relaxation of puzzle design constraints (enabled through updates of the Eterna source code) that allowed for exploration of a much larger design space and 2) the public dissemination of annotated experimental data that let players iterate and improve upon any past design. Designs exhibited striking improvements over iterative rounds; in the first round, the AR values, often approaching the thermodynamic ARmax of 132 .Changes to design constraints and iterative refinement of promising solutions C yieldedar in AR E. The reSI Appendix, Fig. S4). To further test the general usability of these sensors in different experimental contexts, we shifted our readout from RNA-MaP to cytometric analysis of RNA switches displayed on micromagnetic particles  and of “miniaturizing” designs by promoting stable base pairings between the 5′ and 3′ regions or nonswitching stems and hairpins that sequester nonswitching nucleotides B and F. articles . Using tarticles H.ARs than the final player designs after iterative refinement .The high performance of player-designed switches encouraged us to compare a concurrently developed, physics-based computational algorithm for RNA switch design—Ribologic —against finement A. To comAR values above 10 and in several cases approaching the thermodynamically allowed maximum . We note that Eterna performance was benchmarked against a single competing algorithm, which is a limitation of this study. We also note that in general, there is decoupling between the performance of RNA switches based on our in silico energy calculations and the measured performance of these switches  , 29 and We next asked if player guidance could improve the automated design of RNA switches. We used player solutions to seed the designs for player–Ribologic hybrid designs. To do this, we fixed the location of the included aptamer motifs and allowed Ribologic to converge on new designs seeded with player-submitted solutions. In two examples, coupling player-guided aptamer placement with the Ribologic design infrastructure yielded switches surpassing the best player designs, providing an existence proof of a system wherein experience-guided aptamer and reporter placement can enable semiautomated design of highly functional molecular sensors and suggesting routes to approach next-generation RNA switch design algorithms B.SI Appendix, Fig. S5).An important and previously undemonstrated goal for RNA design is the reusability of designed RNA switches to enable energy-neutral applications. In our final experiments, we therefore tested the reversibility of switches by exposing 482 FMN–MS2 Eterna designs to 52 buffer exchanges (cycling between 0 and 200 μM FMN) over 29 h. In all switches, we observed consistent toggling of the fluorescence output A and B, To understand how top Eterna players achieved success in designing efficient stand-alone riboswitches, the community engaged in “all-hands” online discussions, presentations during annual Eterna conferences, and compilation of puzzle-solving strategies in game forum posts and widely shared Google documents. As expected, players approached new puzzles in a highly varied manner, but several recurring principles were articulated.First, no single puzzle architecture was universally suitable for all given input–output combinations. Rather, the testing of various puzzle architectures that differed in the relative placement of the input and output aptamers was required for identifying high-efficiency switches. For the first FMN–MS2 on-switch puzzles , many suSecond, in initial FMN–MS2 challenges that allowed for iterative refinement of solutions, players commonly modified the most successful designs in every round in order to improve performance in subsequent rounds, leading to design “families” sharing high sequence similarity B and C. AR of the design. Players generally sought a high predicted x ratio, and this feature was incorporated into a player-designed “arcplot” online tool that showed pairing probabilities of all bases in the absence and presence of the ligand in a single chart. Players also disseminated screenshots of arcplots with striking features  (SI Appendix).Finally, several players spearheaded the development and dissemination of tools that granted insights into individual switch puzzles and/or solutions. During light-up challenges, players developed visualization tools for simultaneously tracking both states of the sensor and tabulating metrics that were not provided in the Eterna interface. For example, one metric was a solution’s “x ratio.” This is the ratio of predicted pairing probabilities in the absence vs. presence of the input ligand of the outermost base pairs of the split aptamer, which approximately predicts the actual This study demonstrates that an iterative, massive open laboratory approach—internet-scale crowdsourcing of experimental hypotheses in combination with a high-throughput, quantitative experimental pipeline for hypothesis testing—enables design of near–optimally efficient, reversible, self-contained RNA switches that couple diverse input molecules to diverse output fluorescence modalities, opening the door to a variety of applications. We believe that other citizen science projects would benefit by enabling their communities to define and test all incoming hypotheses . More sphttps://eternagame.org). For RNA-MaP high-throughput functional assays, Eterna participants’ solution sequences were collected in a series of rounds, and oligonucleotides were synthesized corresponding to these solutions, bottlenecked, and then sequenced. Sequencing chips were recovered and mounted on a custom automated fluorescent microscope, and they were quantified for reporter binding across multiple ligand concentrations. Fluorescence images were aligned to the sequencing data; individual cluster intensities were quantified with a custom analysis pipeline, normalized, and fit to a binding curve, and AR values were calculated. Detailed materials and methods, including descriptions of AR quantification and ancillary quantification methods, are in SI Appendix, Materials and Methods. Data are also archived on the Eterna website (https://eternagame.org). Control and analysis software is available at https://github.com/GreenleafLab.RNA molecules were designed in Eterna (Supplementary FileSupplementary File


The increased affinity of Mg2+-PPi to the RMP-added primer/template (P/T) RNA duplex complex introduces a new hydrogen bond of a substituted cyano group in RMP with the K593 sidechain. The new interactions disrupt a switching mechanism of a hydrogen bond network that is essential for translocation of the P/T duplex product and for opening of a vacant NTP-binding site necessary for next primer extension. Furthermore, steric interactions between the sidechain of S861 and the 1′-cyano group of RMP at position i+3 hinders translocation of RMP to the i + 4 position, where i labels the insertion site. These findings are particularly valuable to guide the design of more effective inhibitors of SARS-CoV-2 RNA polymerase.The mechanism of remdesivir incorporation into the RNA primer by the RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains to be fully established at the molecular level. Here, we compare molecular dynamics (MD) simulations after incorporation of either remdesivir monophosphate (RMP) or adenosine monophosphate (AMP). We find that the Mg Remdesivir is an antiviral drug that inhibits viral replication after it is converted into the triphosphate form and bound to the active site of RNA-dependent RNA polymerase (RdRp) for its incorporation into the RNA primer . HoweverEbola virus and more recently against SARS-CoV-2 (The replication-transcription complex (RTC) of SARS-CoV-2 is composed of RNA-dependent RNA polymerase (RdRp or nsp12), nsp7, nsp8, nsp13, and the template/primer (P/T) RNA duplex. RdRp is the enzyme that catalyzes the replication and transcription of viral RNAs essential for viral replication . It is tRS-CoV-2 . CompareRS-CoV-2 . Its modRS-CoV-2 .i, i + 1, and i + 2 can be extended efficiently, where i labels the insertion site while i + 1 and i + 2 indicate sites after one- and two-base pair translocations of the RNA duplex, respectively  complex controls the transcriptional pause, translocation, pyrophosphorylysis, and eventually, possible cleavage by exoribonuclease. A common efficient translocation mechanism of P/T duplex is often powered by the release of pyrophosphate   simulations. RNA polymerases often use pauses as mechanisms of transcriptional regulation . Elementhosphate . This iseaction)  and in slymerase , the rellymerase . For othlymerase . In many2+ ions bound in the pol active site, MgA and MgB. We studied dynamic properties of the RTC using MD simulations shortly before and immediately after polymerization and/or translocation (but not the translocation process itself since that would require longer MD simulations than presented in this study). Our study differs from other recent similar MD simulations that studied different aspects of RNA synthesis  and PPi were added to the model. In addition, two Zn2+ ions were included in the structure as part of the Zn-Cys motif important for the stability of the nsp12. One reference set of MD simulations was carried out with three AMPs at position i + 1, i + 2, and i + 3 of the RNA duplex, followed up by three sets of MD simulations with one RMP replaced at each of the three positions  was used as a starting point for our MD simulations after removing nsp13 helicase . Two Mg2ositions . MD simu Maestro  was used package . MD simu package . The sys+ ions were added as counterions to neutralize the whole system. The remdesivir parameters were generated by optimizing the molecule with B3LYP/6-31G* first, and then calculating the electrostatic potential for partial atomic charges  maps derived from MD trajectories were calculated for the complex structure using CCP4 as previously described , from whi + 1, i + 2, and i + 3. PPi interacts with the Mg2+ ions mainly through electrostatic interactions in all three structures, although the strength of the interactions of PPi varies in the three complexes. The H-bond is stronger when RMP is in position i + 3 (the i + 3 complex). The H-bond interaction analysis shows that the PPi forms H-bonds with three surrounding amino acid residues R553, R555, and K621  for the PPi to each of its interaction partners are sharp with peaks around 3 Å when RMP is at position i + 3 whereas for AMP at position i + 3, the corresponding distributions are broader with some peak positions shifted by more than 2.0 Å . Results of this analysis are consistent with the calculated iso-probability densities  from the average structure shows that the RMSF value for the PPi is 0.7 (0.3) Å and 1.3 (0.4) Å (numbers in parenthesis is uncertainty or one standard deviation of RMSF) in the ensities , showingi + 3 RMP complex, the RMSF analysis shows that the RMSFs for the three nucleotides of the primer strand at positions i to i + 2 are 0.8 (0.3) Å, 0.7 (0.2) Å, and 0.7 (0.2) Å, respectively. In contrast, the corresponding values for AMP complexes are 1.0 (0.3) Å, 1.2 (0.3) Å, and 0.8 (0.2) Å. In addition, the RMSFs for single RMP nucleotides at positions i + 1 to i + 3 are 0.8 (0.2) Å, 0.8 (0.3) Å and 0.8 (0.3) Å. In contrast, the corresponding values for corresponding single AMP nucleotides increase to 1.2 (0.3) Å, 1.0 (0.4) Å and 0.9 (0.3) Å. Clearly, the incorporated RMP increases the rigidity of the RNA duplex. Again, these results are consistent with the iso-probability density analysis. Therefore, incorporation of RMP stabilizes the nucleic duplex relative to the system without RMP.For the i + 3 complexes, the difference MD-derived ED maps show that the largest difference in the entire map is observed on the PPi . For the i + 1 and i + 2 complexes, the larger differences are on the cyano group, while differences near the PPi are relatively very small . All these observations support our hypothesis that the PPi binds more tightly when RMP is added to the primer strand and occupies position i + 3, which is consistent with the paused translocation.For the  the PPi , and thei + 3 and the PPi bound at a distance of over 15 Å apart to determine how the RMP at i + 3 stabilizes the PPi , K593 interacts solely with the sidechain of Q815 which in turn interacts along a H-bonding network with the backbone carbonyl group of C813. These interactions lead to a reduction of H-bonding strength between the backbone amide of C813 and the sidechain of D761, so D761 coordinates more strongly with MgB, weakening the MgB-PPi interaction. The carboxyl group of D761 repels that of D618 and changes its position. As a result, D618 interacts less with K798 and in turn K798 interacts more strongly with PPi, pulling PPi away from three other positively charged residues . Therefore, we find that weakening the interactions of PPi with R553, R555, and K621 after switching PPi to a new interaction with K798 may be essential for releasing PPi in this polymerase. This step is equivalent to the reopening of the Fingers domain of RB69 DNA polymerase .i + 3, the RdRp complex remains in the pre-translocated state, which lacks the vacant NTP-binding site observed in the post-translocation state. Therefore, it prevents binding of the next incoming NTP. When RMP is replaced by AMP, the RdRp complex rapidly advances to the post-translocation state and the NTP binding site becomes vacant (i + 4. Therefore, mutation of S861 reduces the stalling effect (i + 3 to stall translocation.The mechanism for stalling primer extension by RMP has been suggested to be associated with a free energy barrier for translocation . Howevers vacant . Stallins vacant , which hg effect . In addii + 3, it induces a transcriptional pause under the reduced NTP concentrations. Our computational analysis points to a tighter binding of PPi as the physical basis of the pause. When RMP is in position i + 3, the cyano group of RMP forms hydrogen bonds with K593 and prevents a switch of a H-bonding network established by protein residues that are essential for primer/template translocation. This increases the binding stability of the PPi product so that PPi blocks the translocation of RNA duplex and slows primer extension.RMP acts as a delayed inhibitor and slows down the primer extension. During the first pass of viral RNA synthesis by the SARS-CoV-2 RNA-dependent RNA polymerase. When RMP is translocated to position


Flow velocities within coral reefs are greatly reduced relative to those at the water surface. The in-reef flow controls key processes that flush heat, cycle nutrients and transport sediment from the reef to adjacent beaches, all key considerations in assessments of reef resilience and restoration interventions. An analytical framework is proposed and tested with a suite of high-resolution numerical experiments. We demonstrate a single parameter that describes the total coral frontal area explains variation of horizontally averaged velocity within a reef canopy across morphologies, densities, and flow depths. With the integration of existing data of coral cover and geometry, this framework is a practical step towards the prediction of near-bed flows in diverse reef environments. The hard coral colonies that form coral reef benthos are irregular in form and vary on spatial scales that range from centimeters to kilometers. This irregular and complex form directly modifies the in-reef flow that occurs between the seabed and the top of the coral colonies , 2. WhilAcropora hyacinthus [Porites lobata [Pocillopora edyouxi [Despite the wide range of coral species globally, coral colonies can be broadly classified from a physical perspective based on their morphological form . For exaacinthus  are chars lobata  form mon edyouxi  create a edyouxi  and inde edyouxi  throughoMany reef systems are current-dominated systems, such as those located in macro-tidal regions  and reefIn this study, we present a practical framework for predicting the ratio of horizontally-averaged in-reef and near-surface velocities for the three coral archetypes in β) of the horizontally- and depth-averaged in-reef velocity :ΔU is the difference between U∞ and Uc.The analytical framework presented here builds on previous work by others, such as that by Lowe et al . It predcity Uc,  to the vFD) to the depth‐averaged in-reef flow velocity (Uc) and the area of the coral colony projected into the flow :ρ is the density of water, CD is a drag coefficient and ϕ is the porosity of the reef colony, which was calculated by treating the canopy as a box with the upper limit defined at the top of the coral morphology (z = h); the porosity is then equal to one minus the ratio of coral solid volume to box volume. We discuss the limitations of this approach to some morphologies such as plate corals later in the discussion. While complex forms such as branched corals  have been suggested to act as bluff bodies [Af is not simply the total coral area projected into the flow; rather, it is the sum of the projected areas of all coral surfaces . What this means is that, for example, for a branching coral each branch along the flow direction axis contributes to increase Af. We note this is not trivial for practical applications and we propose an approach to address this later. In this study, we evaluate the area projected into the incoming flow of each individual element  of each morphology and define Af as the sum of these projected areas.The attenuation of the in-reef flow occurs due to the substantial drag forces imposed by coral reef colonies. For a given coral colony, this drag is typically described through a quadratic law relating the drag force  is the total benthos frontal area per unit reef surface area. Below we show that λf is a critically-important determinant of the in-reef flow. While evaluation of CD for a complex coral reef colony is not straightforward, measured values tend to lie in the range 0.5–2 [CD = 1 for all coral morphologies. This assumption is borne out of practical necessity, as utilization of the framework presented here requires assumption of a coral drag coefficient value (which is otherwise not easily predicted). Later we discuss the implications of our choice and the need for a practical strategy to define CD, particularly for ‘non-front-facing’ morphologies.When this is extrapolated to the reef scale, the drag force exerted per unit reef area and unit fluid density is:ge 0.5–2 –39. RathH) and for a given water surface slope (S) requires a balance of the driving hydraulic gradient , conservation of momentum over the entire depth (τc) can also be deduced from a momentum balance, such that the shear velocity at the top of the benthos (u*) is given by:g is the gravitational acceleration constant and h is the average height of the reef benthos. In flows over rough colonies, the shear velocity increases linearly with the velocity difference across the benthos (ΔU):k is a coefficient of proportionality [β → 1) in the limits of both vanishing benthos frontal area (λf→0) and emergence of the benthos from the water (h/H → 1). Later in the discussion we propose how this framework can be applied in practice.The shear stress at the top of the reef benthos  simulations were conducted to obtain detailed information about the vertical and horizontal structure of the 3D in-reef flow across a wide range of system variables  and corahorizontally-averaged in-reef velocity, we believe that this simplification is both practically necessary and appropriate.One coral colony was placed in the horizontal center of the computational domain  treated as a non-zero-velocity, zero-flux slip boundary. Four mesh sizes were tested to determine the point of mesh insensitivity .z = h/2. After the initial spin up, each simulation was continued for a minimum of 10 flow-through periods, during which the velocity data were obtained. While LES provides time-dependent flow output, only the steady-state velocities are presented here. Critically, model results were validated against data from laboratory flume experiments.All simulations were initially run for 60 s to ensure the flow reached quasi‐steady state, determined from time series of the horizontally averaged velocity at -1) and water depth (0.16 m) were maintained. To determine the impact of additional system variables on flow attenuation, we maintained a coral cover of 20% and varied (i) the water depth (0.06–0.50 m) for a massive morphology, (ii) the angle of the flow (0-180°) relative to the orientation for a massive morphology, and (iii) the depth-averaged inflow velocity (0.05–0.50 m s-1) for the branched morphology. Given that our framework is physically-based, a computationally expensive factorial study was not required here. Rather, it was more important that the key variables controlling the in-reef flow were firstly identified, and then varied over realistic field ranges.We conducted 21 simulations across the three archetype coral colony morphologies with variable coral cover 5–50%, . For theUc and U∞) were taken as depth-averaged values of horizontally-averaged velocities in a 3-cm-tall profile: from z = 0.5–3.5 cm (spanning 0.1 ≲ z/h ≲ 1) for the in-reef velocity and from z = 7.0–10.0 cm (spanning 1.3 ≲ z/h ≲ 3) for the near-surface velocity. This provided mean in-reef and above reef velocities with standard errors that were, on average, 10% and 1% respectively. The measured spatially-averaged flow velocities above and within the coral benthos agreed with model results to within an average of 4%  open-channel flow flume . Experimge of 4% . This deu>(z)) are shown in z/h < 1), and a uniform high-velocity region well above the colonies. These regions are separated by a strong shear layer [U∞) as that 0.01 m below the water surface, and Uc as the mean velocity in the region 0 < z < h across the entire horizontal extent of the domain  the velocity is 0.048 ± 0.055 m/s  with increasing coral cover , which are indicative of field values, are sufficiently high that β is independent of Reynolds number (and thus velocity itself).There is negligible variation of velocity . This imβ─1) will increase with α1/2, whereAssuming an invariant drag coefficient, the analytical framework presented in β and α . The line of best fit in this figure represents the relationship β─1 ≈ α1/2. The agreement of the data across morphologies as well as the agreement of the functional forms of the line of best fit and Indeed, across all the numerical simulations conducted here and across all the dimensions of variation considered in β on α . While CD is expected to vary based on the flow and morphology, this variation is somewhat small relative to the natural variation of other components that make up the predictor variable (α). Furthermore, the scale at which inhomogeneity of coral morphologies affects drag warrants further investigation. While studies of cylinder arrays suggest that the average drag is similar for both homogeneous and inhomogeneous arrays, at an individual colony scale the drag can be expected to be different [The agreement of the data with the curve of β on α  is a funβ on α  are likeal cover . This deal cover . Consistifferent . Notwithh) of the reef benthos, (3) the total water depth (H) and (4) the surface velocity (U∞). We recognize that not all of this data will always be available, however we believe that it can be practically obtained. Spatial maps of reef benthos are routinely assembled by diver [λf/ϕ . This requires quantitative allometric relationships between λf/ϕ and the more easily-obtained measure of coral cover. For the three archetypes employed here, indicative forms of such relationships are presented in λf/ϕ-coral cover relationship, a table coral minimizes frontal area per unit biomass while branches serve to maximize it. Such curves can be developed for other morphological forms with relative ease using techniques such as CT scanning [To apply this framework, four key data are required: (1) a spatial map of reef benthos species , (2) the height (by diver , 50, droby diver , aerial by diver  or satelby diver , 54 methscanning  of colonin situ hydrodynamic measurements with wave or tide gauges [The three remaining pieces of data are less difficult to determine. The geometric height of the benthos can be obtained in a number of different ways including from representative spot measurements or vessel mounted echo-sounders , as welle gauges . Finallye gauges  or by tie gauges , 51, 62 λf/ϕ in An obvious consideration is how this framework can be applied to a reef composed of a range of coral species. The nature of this framework is such that mixed benthos can be characterized as the sum of the frontal areas of its constituent parts, contributing to a ‘total’ value of Traditional approaches used to describe the hydrodynamic impact of coral reef benthos is through a friction coefficient that describes the impact of reef on the overlying flow . This ap