Stringent control of [H+] is required to maintain the homeostatic acid-base balance in coral cells and is achieved through the regulation of intracellular pH (pHi). This task is especially challenging for reef-building corals that share an endosymbiotic relationship with photosynthetic dinoflagellates (family Symbiodinaceae), which significantly affect the pHi of coral cells. Despite their importance, the pH regulatory proteins involved in the homeostatic acid-base balance have been scarcely investigated in corals. Here, we report in the coral Stylophora pistillata a full characterization of the genomic structure, domain topology and phylogeny of three major H+ transporter families that are known to play a role in the intracellular pH regulation of animal cells; we investigated their tissue-specific expression patterns and assessed the effect of seawater acidification on their expression levels.Reef-building corals regularly experience changes in intra- and extracellular H+/H+ exchanger (SLC9), vacuolar-type electrogenic H+-ATP hydrolase (V-ATPase) and voltage-gated proton channel (HvCN) families in the genome and transcriptome of S. pistillata. In addition, we identified a novel member of the HvCN gene family in the cnidarian subclass Hexacorallia that has not been previously described in any species. We also identified key residues that contribute to H+ transporter substrate specificity, protein function and regulation. Last, we demonstrated that some of these proteins have different tissue expression patterns, and most are unaffected by exposure to seawater acidification.We identified members of the Na+ transporters that might contribute to the homeostatic acid-base balance in coral cells. This work will enrich the knowledge of the basic aspects of coral biology and has important implications for our understanding of how corals regulate their intracellular environment.In this study, we provide the first characterization of HThe online version contains supplementary material available at 10.1186/s12860-021-00353-x. Coral reefs are among the most valuable ecosystems on earth, harbouring more than one-third of ocean biodiversity and providing economic benefits to tropical coastal nations worldwide . Sclerace) due to physiological  and environmental  factors. For example, symbiont photosynthesis and respiration of both the host and symbiont drive wide pHe variations (from pH 8.5 to 6) in the internal fluid of the coelenteron [e in the extracellular calcifying medium (ECM) [e variations can also have an impact on the regulation of intracellular pH (pHi). For example, decreases in the pHe of the ECM (from 8.3 to 7.8) under acidified seawater conditions affect the pHi of calicoblastic cells. Despite these variations, coral cells are able to maintain their pHi within narrow limits (7.1–7.4) [i regulatory mechanisms that account for this stability.Corals are diploblastic animals, which means that they are made of two cell layers, an ectoderm and an endoderm, separated by a layer of mesoglea. Both ectoderm and endoderm are present in the oral and aboral tissue located on either side of the gastrovascular cavity (coelenteron) . Each tilenteron –18, and um (ECM) , where c7.1–7.4) , showingi stress, an appropriate response depends on the ability to sense acid-base disturbances and react through acid-base transport mechanisms [i regulation, fall into two groups, namely, HCO3− (solute carrier-SLC transporter family 4 and 26) and H+ membrane transporters [+ extrusion, H+ membrane transporters can be further divided into transporters, pumps and channels. Transporters can move H+ against their electrochemical gradient by coupling H+ transport with pre-existing ion gradients as an energy source. This group includes the SLC9 family, also known as Na+/H+ exchangers, which harness the electrochemical gradient of Na+ maintained by Na+/K+-ATPase [+ membrane transporters in charge of pHi regulation includes H+ pumps that allow H+ to move against its concentration gradient by coupling H+ transport to ATP hydrolysis. Vacuolar-type electrogenic H+-ATP hydrolases (V-ATPases), which transport H+ via V0 V-ATPase subunit-a [+ 2-ATPase (PMCA), which extrudes Ca+ 2 in exchange for H+ [i regulation is not the primary role of these H+ pumps in most mammalian cells [+ channels, allow H+ to passively diffuse down its electrochemical gradient whenever the regulatory gate is open and includes voltage-gated proton channels (HvCN) [Under pHchanisms . Over thchanisms –22. Acidsporters . Based o+-ATPase . The SLC+-ATPase . The sece for H+ , 27, 28,an cells , others an cells –32. Membs (HvCN) , 34.i regulation, the investigation of these H+ transporters is incomplete and includes only a limited number of marine species. For example, SLC9 and V-ATPase proteins have been identified in the transcriptome of calcifying primary mesenchymal cells of the sea urchin [Litopenaeus vannamei), squid (Sepioteuthis lessoniana), and sea anemone (Anemonia viridis), SLC9 has been shown to play a role in the response to low-pH stress [i regulation, only HCO3− transporters have been characterized [+ membrane transporters is limited to Ca+ 2-ATPase [1 V-ATPase subunit B [Despite their fundamental importance in pHa urchin  and in ta urchin , 37. In H stress –40. In ccterized , whereas2-ATPase  and V1 Vubunit B .+ transporters that might be involved in the intracellular pH regulation and maintenance of cellular physiological homeostasis in the symbiotic coral Stylophora pistillata. These transporters include Na+/H+ exchangers, which are ion transporters that concurrently transport Na+ into the cell and H+ out of the cell; voltage-gated H+ channels (HvCN), which represent a specific subset of proton channels that have voltage- and time-dependent gating and thus only open to extrude H+ from the cell; and V0 V-ATPase subunit-a, which connects the two portions (V0 and V1) of the multi-subunit enzyme V-ATPase and is crucial for proton transport [+ transporters to determine whether they are differentially expressed in coral tissues, and we discussed their potential physiological roles. In addition, we investigated the molecular response of S. pistillata to ocean acidification by assessing the effect of external seawater acidification on the levels of H+ transporter expression after 1 week and 1 year of exposure.In the present study, we provide a full characterization of the genomic structure, domain topology and phylogeny of the principal Hransport , 43–45. SLC9 in the genome and transcriptome of S. pistillata [S. pistillata SLC9 proteins (spiSLC9) with functionally characterized SLC9 in humans allowed us to group the corresponding S. pistillata genes within three subfamilies, namely, subfamilies A, B and C , one (spiSLC9A1) is a plasma membrane homolog, two (spiSLC9A6 and spiSLC9A7) are A6/7 organelle homologs; one (spiSLC9A8) is an A8 organelle homolog; two (spiSLC9B1 and spiSLC9B2) belong to subfamily B (the NHA subfamily); and one (spiSLC9C) belongs to subfamily C . The gene and transcript information of SLC9 family members is given in Additional files We identified genes homologous to human stillata , 47. Phyd C Fig. . Members spiSLC9A is a pla+/H+ exchanger activity, such as F161, P167–168, R440 and G455–456, for members of subfamily A    are conserved between spiHvCNs and hHvCN1, whereas the residues D112, D123, D185 and E119 are conserved only between spiHvCN1.1 and hHvCN1. Regarding post-translational modifications, several phosphorylation sites were predicted for both spiHvCNs, whereas N-glycosylation sites were predicted only for spiHvCN1.1  in the oral fraction and the total colony  of S. pistillata. The results showed no differential expression of spiSLC9A1, spiSLC9B1, spiSLC9B2 or spiV0V-ATPase subunit-a  in the oral fraction than in the total colony, and spiSLC9A8  in the total colony than in the oral fraction. Finally, spiHvCN1.1  in the total colony than in the oral fraction, whereas spiHvCN1.2  in the oral fraction than in the total colony.Quantitative real-time PCR was used to detect H+ transporter mRNA expression, real-time qPCR analysis of the spiSLC9A-B, spiV0V-ATPase subunit-a and spiHvCN genes was carried out in S. pistillata micro-colonies exposed to control and acidified seawater  for two different durations: 1 week and 1 year. The results showed that there was no difference in the expression of these genes between micro-colonies exposed to pH 8.1 and pH 7.2 for 1 week  at pH 7.2 than at the control pH of 8.1  sequences. GZ sequences mediate close helix-helix folding within transmembrane structures and facilitate the formation of membrane pores. This feature might facilitate access to the mitochondrial inner membrane, where these proteins are highly abundant [Strongylocentrotus purpuratus, Ciona intestinalis, Lepisosteus oculatus and Drosophila melanogaster [Homo sapiens, suggesting that voltage activation differs between these species and humans. The CNBD also regulates the activity of spiSLC9C, probably through the binding of cyclic adenosine monophosphate (cAMP) produced by the soluble adenylyl cyclase enzyme (sAC), as reported in the sea urchin [The spiSLC9s characterized in an SLC9s , 48, suc residue ; P167 an residue  and infl residue ; and R44” domain . For theabundant . Finallynogaster . Some ofa urchin .+/H+ exchangers. Their shared properties with previously characterized SLC9s indicate that they retain similar functions and provide insights into their activation mechanism and regulation in S. pistillata.Overall, the conserved features of spiSLC9s align with their roles as Na0 V-ATPase subunit-a homologs in Anthozoa  [+ to translocate across the membrane [0 V-ATPase subunit-a through the use of random and site-direct mutagenesis. Among these residues, R735 is known to play an essential role in proton transport, as mutations of this residue result in complete inactivation of the ATP-dependent H+ transport of the V-ATPase [S. pistillata suggests that R735 fulfils its role in H+ transport  . TMs aremembrane . PreviouV-ATPase . The conort Fig. b. Other 0 V-ATPase subunit-a and its human and yeast homologs align with its role as the subunit-a of the V0 V-ATPase. Furthermore, the identification of homologs of all the V-ATPase subunit-encoding genes in the genome and transcriptome of S. pistillata  was identified in all species examined in this study, including the four anthozoan orders, namely, Actinaria, Alcyonacea, Corallimorpharia and Scleractinia  in some Cnidaria. Genomic and transcriptomic searches of HvCN1.2 in public databases of Octocorallia and Hydrozoa (Hydra magnipapillata) did not produce any results (data not shown). The selective expression of HvCN1.2 only in Hexacorallia suggests that it is specific to this cnidarian subclass, and its presence in noncalcifying anthozoans  suggests that it is not linked to the appearance of aragonite biomineralization in Scleractinia [vCNs) characterized in S. pistillata are highly divergent. This finding is further supported by the length of the phylogenetic branch  and acidic residues (D and E) associated with voltage sensing, and the coiled-coil structure at the C-terminus [+ from the cell [vCNs might activate these channels and enable them to open faster and at less positive voltages than those required without activation, as previously reported in human leukocytes [The spiHterminus . The gatthe cell . Phosphoukocytes –62.vCN1.1 and spiHvCN1.2 share similar structures and organizations  [vCN1.1 (H193G) and are even less conserved in spiHvCN1.2 . Hence, we suggest that the replacement of Zn+ 2-coordinating residues with other residues potentially affects the Zn+ 2 sensitivity of spiHvCNs, as reported in vertebrates [vCN1.1 contains more Zn+ 2-coordinating residues, we propose that it is more sensitive to Zn+ 2 than spiHvCN1.2.SpiHons Fig. . Howeverig. vCN1 , 63, areectivity , is missn hHvCN1 , suggestnd H193) , 65. By tebrates , 67. SinvCNs align with their roles as voltage gated H+-channels. Common properties between the spiHvCNs and hHvCN preserve their voltage sensitivity and proton specificity, with some differences concerning their Zn+ 2 sensitivity. In addition, distinctive properties between different spiHvCNs suggest differential regulation, possibly linked to their localization/function. Future analyses, however, are required to validate these assumptions and provide insight into their physiological role.Overall, the conserved features of the spiH+ transporters in the two coral tissues helps identify the potential physiological processes in which they might participate.Whole coral colonies are typically used in conventional techniques of gene expression analysis, limiting the possibility of further differentiating specific gene expression between oral and aboral tissues. These tissues contain different cell subtypes; some of them are more abundant  or exclusively found  in the oral tissue, whereas others are exclusively found in the aboral tissue   . As seve+ transporters in the oral fraction to that in the total colony.To perform this task, we used a previously developed micro-dissection protocol , 69 to s+ transporters are more highly expressed in the oral fraction than in the total colony ; others are more highly expressed in the total colony than in the oral fraction ; and others are expressed at the same levels in both fractions (hereafter referred to as “ubiquitous”)  Fig. . Using H+ transporters include spiSLC9A6, spiSLC9A7 and spiHvCN1.2. spiSLC9A6 and spiSLC9A7 are organellar homologs  that accumulate in the host cytoplasm [vCN1.2 might favour the exit of H+ at the basolateral membrane of these cells, as reported in human osteoblasts [2− electron transfer. Additionally, the decreased Zn+ 2 sensitivity of spiHvCN1.2 compared to that of spiHvCN1.1 (see Discussion 3.1.3) could be correlated with the higher Zn+ 2 concentration levels in the oral (symbiotic) cells [The oral-specific Hytoplasm –75. Thuseoblasts , preventc) cells , 77–80.+ transporters are spiHvCN1.1 and spiSLC9A8. These transporters could play a role in the pHi regulation of calcifying cells. During the calcification reaction, H+ is produced in the ECM and needs to be removed to promote an alkaline environment favourable for calcification [+ 2-ATPase at the apical side of the calcifying cells has been suggested to be involved in pumping out H+ from the ECM [vCN1.1 might contribute to extruding excess H+ from the cytoplasm of calcifying cells, similar to the role it plays in coccolithophores [The aboral-specific Hfication , 81. The the ECM . On the hophores . Finallyhophores , 84, sinhophores .+ transporters  are ubiquitous. Our results suggest that the coral V0 V-ATPase subunit-a homolog is ubiquitously expressed and differs from those identified in humans. Indeed, the human V0 V-ATPase subunit-a isoforms have different tissue distributions with intracellular or apical/basolateral membrane localization [0 V-ATPase subunit-a, which do not discern the different isoforms , which activates the transporter at low pHi values, similar to humans [+ transporters SLC9, V-ATPase and HvCN, which were characterized in the present study, and the HCO3− transporters SLC4 and SLC26, which were characterized by Zoccola et al. in 2015.The rest of the Hlization , 86–89. o humans . Figure S. pistillata of several families of H+ transporters, which are known as pHi regulators in animal cells. Most importantly, we report for the first time a novel member of the HvCN gene family, HvCN1.2, in the cnidarian subclass Hexacorallia. The identification of conserved residues between coral and human H+ transporter homologs suggests functional conservation related to intracellular pHi regulation.This study provides the first molecular characterization in the coral + currents, etc.) with a larger number of replicates need to be carried out in the future to demonstrate the participation of these H+ transporters in coral acid-base cellular homeostasis. Moreover, we assessed the tissue specificity of the H+ transporter gene families in the coral S. pistillata, and we observed that their expression was not restricted to only one specific tissue , as reported for some members of the HCO3− gene family [+ transporters in the coral colony and suggest that they take part in homeostatic  and physiological processes . Finally, we investigated the impact of OA on H+ transporter gene expression in S. pistillata, and we identified one candidate gene (spiSLC9A1) involved in the coral response to ocean acidification that showed differential expression after long-term exposure to acidified seawater (1 year). The other H+ transporters did not show any significant changes at the gene level under seawater acidification conditions. Nevertheless, the modulation of gene expression can also occur at the protein level, which should be investigated in future studies. The influence of other environmental factors, e.g., temperature, remains to be tested and will enrich the understanding of coral phenotypic plasticity. This knowledge is especially relevant to our understanding of the ability of benthic animals to buffer the impacts of environmental changes, thereby providing more time for genetic adaptation to occur. In addition, responses to environmental changes are often species-specific [+ transporter gene expression, which could also be investigated in other coral species. Such comparative studies might aid in the development of molecular markers linked to pHi tolerance traits in coral reef populations.However, additional experiments  under the following controlled conditions: semi-open circuit, temperature of 25 °C, salinity of 38, light exposure of 200 μmol photons m− 2 s− 1, and a 12 h:12 h light:dark cycle. Samples were prepared from one mother colony as nubbins suspended on monofilament threads. After 3 weeks of cicatrisation, the first set of samples (n = 3) was used for the oral fraction micro-dissection experiment, and another set (n = 20) was used for the exposure to seawater acidification experiment.Experiments were conducted on the symbiotic scleractinian coral 2 to reduce the pH from the control value of pH 8.1 to the target value of 7.2. Twenty coral nubbins were randomly distributed among four experimental tanks : two tanks (pH 8.1 and 7.2) were reserved for 1 week of exposure, and two were reserved for 1 year of exposure. The experiments were repeated three times to ensure reliability. Similar results were obtained for each experiment (not shown).The seawater acidification setup was performed as described previously , 94, 95.+ transporters were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/protein) and used as a bait to mine (BLAST) the transcriptome, genome and EST databases of the following phyla: Cnidaria , Mollusca (Crassostrea gigas), Echinodermata (Strongylocentrotus purpuratus), Chordata , Porifera (Sycon ciliatum) and Placozoa (Trichoplax adhaerens). The following web servers were used: NCBI (http://www.ncbi.nlm.nih.gov/), EnsemblMetazoa (https://metazoa.ensembl.org/), ReefGenomics (http://reefgenomics.org) and Cnidarian Database (http://data.centrescientifique.mc/).The amino acid sequences of human Hhttp://www.cbs.dtu.dk/services/) using the TMHMM algorithm. On the same platform, phosphorylation and N-glycosylation prediction analyses were performed using NetPhos and NetNGlyc.Putative transmembrane helices in proteins were predicted by the Center for Biological Sequence Analysis Prediction Server . Based upon the produced amino acid alignments, maximum likelihood estimates of the topology and the branch length were obtained using PhyML v3.1 [Alignment of HyML v3.1 . The modyML v3.1 . Phylogen = 3) were set to rest in a glass petri dish filled with seawater and tricaine mesylate. Once polyps were extended, oral fractions  were cut from the coral colony  using micro-dissection scissors under a binocular microscope. Both fractions  were then used for RNA extraction.Coral micro-dissection was performed as indicated previously , 69. BriTotal RNA extraction and cDNA synthesis were performed as described previously . Brieflyn = 3) was used due to technical limitations. For the exposure to seawater acidification experiment, a sample size estimation was performed, and n = 5 samples were used. For both experiments, the normal distribution of the data was evaluated using Shapiro-Wilk’s test. Samples with a standard deviation ≥5 were excluded from the analysis. This was the case for spiSLC9C, whose qPCR results showed great variability, probably due to its extremely low expression values. A t-test was used to identify differentially expressed genes between S. pistillata fractions  and pH treatments (pH 8.1 and pH 7.2). We considered 0.10 ≤ p-values ≤0.11to indicate near-marginal significance (•); p-values ≤0.1 as significant (**); and p-values ≤0.05 as highly significant (*).Statistical analysis was performed using R v3.5.2 software. For the oral fraction micro-dissection experiment, a fixed number of samples  of H+ transporter proteins in S. pistillata.Additional file 4. Sequence comparison of the H. sapiens SLC9A1 and S. pistillata SLC9A proteins.Additional file 5. Sequence comparison of the S. pistillata and H. sapiens SLC9B proteins.Additional file 6. Sequence comparison of the S. pistillata and H. sapiens SLC9C1 proteins. The boxes represent human voltage-sensing domains (S1-S4), and the asterisks indicate conserved positively and negatively charged residues relevant to voltage sensing. The rectangles indicate conserved positively charged residues in S4 that are present in S. pistillata but missing in hSLC9C1. The triangles indicate residues involved in the cyclic nucleotide-binding domain.Additional file 7. Number of phosphorylation sites and N-glycosylation sites predicted for S. pistillata H+ transporters.Additional file 8. A. V0 V-ATPase subunit-a isoforms in coral S. pistillata. B. Human and coral homologs of V-ATPase subunits.Additional file 9. Statistical analysis of the relative mRNA quantification (Rq) of SLC9s, V0V-ATPase subunit-a and HvCNs (N = 3) in S. pistillata total  and oral  fractions.Additional file 10. Statistical analysis of the relative mRNA quantification (Rq) of SLC9s, V0V-ATPase subunit-a and HvCNs (n = 5) at pH 8.1 and 7.2 after 1 week and 1 year of pCO2 exposure. The asterisks indicate significant differences (** p < 0.05).Additional file 11. List of S. pistillata H+ transporter genes with RT-PCR primers and product sizes.


Streptococcus pyogenes Cas9 (SpCas9)-based adenine base editors (ABEs) with minimized RNA off-target activities display constrained editing scopes with efficient editing activities at positions 4-8. Here, functional ABE variants with diversified editing scopes and reduced RNA off-target activities are identified using domain insertion profiling inside SpCas9 and with different combinations of TadA variants. Engineered ABE variants in this study display narrowed, expanded or shifted editing scopes with efficient editing activities across protospacer positions 2-16. And when combined with deaminase engineering, the RNA off-target activities of engineered ABE variants are further minimized. Thus, domain insertion profiling provides a framework to improve and expand ABE toolkits, and its combination with other strategies for ABE engineering deserves comprehensive explorations in the future.Base editing tools with diversified editing scopes and minimized RNA off-target activities are required for broad applications. Nevertheless, current  Current SpCas9 adenine base editors that minimise RNA off-target activities have constrained editing windows. Here the authors use domain insertion of TadA into Cas9 to narrow, expand or shift the editing window with RNA off-target minimization simultaneously Theoretically, ~47% disease-associated single-nucleotide polymorphisms (SNPs) are G·C to A·T mutations, which could be corrected by ABEs4, making ABEs promising tools for gene therapy.Base editing tools, composed of ABEs mediating adenine to guanine (A–G) conversion and CBEs mediating cytosine to thymine (C–T) conversion without inducing double-strand breaks (DSBs), are powerful tools for targeted nucleotide editing in genomic DNA10. Generally, existing ABEs are mainly generated through “head-to-tail” fusion strategy, which means adenosine deaminases are fused to either the amino terminus (N-terminus) or the carboxy terminus (C-terminus) of Cas nickase (nCas) or catalytically inactive Cas (dCas) proteins3. As the N-/C-termini of Cas proteins are relatively fixed, it often leads to constrained deaminase locations and restricted editing scopes . Recently, circular permutation has been deployed for SpCas9 termini repositioning and several active SpCas9 circular permutants (Cas9-CPs) with altered N-/C-termini have been established11. As altered N-/C-termini bring significant changes to deaminase locations, the derived Cas9-CP-ABEs display expanded editing scopes12. Additionally, variant adenosine deaminase mutants have been generated and derived ABE variants display superior activities and expanded editing scopes13. Nevertheless, the editing scopes of derived ABE variants remain constrained, and alternative strategies are needed to further diversify the editing scopes.Diversified editing scopes will enable ABEs to access more target nucleotides and thus expand their capabilities for the correction of disease-associated SNPs. As single-stranded DNA (ssDNA) substrates are required for effective editing, the accessibility of ssDNA loop to deaminases is critical for the diversification of editing scopes, in addition to the features of deaminases and protospacer adjacent motif (PAM) of CRISPR-Cas systems17. One major issue is that original ABEs could induce obvious off-target mutations at transcriptome level17. Though ABE variants with minimized RNA off-target activities have been developed through deaminase engineering, their editing scopes were severely restricted, with editing windows mainly at positions 4–8 (the PAM was counted as 21–23 unless otherwise indicated). Therefore, there is an urgent need to develop ABE variants with diversified editing scopes and minimized RNA off-target activities.Moreover, safety concerns have been raised about the off-target activities for ABEs, which may limit future applications18. The relative distances of docking sites (DSs) inside Cas9 to ssDNA loop are more variable as compared to N-/C-termini, and domain insertion inside Cas9 may limit the excessive flexibility of functional domains, so it is possible that domain insertion could achieve scope diversifications and off-target minimization simultaneously.It has been demonstrated that the structure of SpCas9 protein is flexible enough to permit functional domain insertion without interfering its DNA binding ability19. The identified functional ABE-nSpCas9-DS variants retain high editing activities and display diversified editing scopes, with adenine nucleotides at protospacer positions 2–16 now targetable. Indeed, several engineered ABE-nSpCas9-DS variants such as ABE-nSpCas9-DS770 display obvious shifted editing scopes with maximal activities at positions 9–16, which has not been achieved previously. Transcriptome analysis further demonstrate that ABE-nSpCas9-DS variants display significantly reduced RNA off-target activities as compared to N-terminal counterparts, and RNA off-target activities could even be eliminated by domain insertion in combined with engineered deaminases. Additionally, in consistent with previous studies20, we also noticed that several functional ABE-nSpCas9-DS variants display obvious cytosine deamination activities and the editing scopes display a DS-dependent manner. Taken together, our study provides a series of ABE toolkits with diversified editing scopes and improved security. We also confirm that domain insertion is a powerful strategy and its combination with other strategies for ABE engineering deserves comprehensive investigations in the future.Here we initially screen for functional ABE variants using domain insertion profiling across 24 potential DSs in SpCas9 nickase , which are distributed across different domains of Cas9, with the majority in discrete regions and in close to ssDNA loop19 , in which TadA served as a scaffold for substrate without ssDNA deamination activity while TadA* is an evolved adenosine deaminase with obvious ssDNA deamination activity. Considering that insertion inside nSpCas9 would lead to domain repositioning and TadA may interfere with deamination process, we initially chose TadA*-TadA* homodimer to screen for functional ABE variants using domain insertion profiling. In all, 24 potential DSs -nSpCas9-DS variants (535-/583-/770-/793-/801-/895-/905-/915-/1010-/1029-/1249-TadA*-TadA*) displayed ≥20% A–G conversion efficiency at specific adenine nucleotide(s)  Fig. , with 10(s) Fig. .We wondered whether TadA*-TadA* dimer insertion would interfere with SpCas9’s DNA cleavage activity. ABE (TadA*-TadA*)-SpCas9-DS variants were generated and their DNA cleavage activities were assessed against two sgRNAs. It was revealed that insertions at different DSs have distinct impact on SpCas9’s DNA cleavage activity  mutations (H57A/E59A) into either TadA* monomer. We generated 10 ABE (TadA* (LOF)-TadA*)-nSpCas9-DS and 10 ABE (TadA*-TadA* (LOF))-nSpCas9-DS variants for A–G conversion analysis against sgRNA-1. Overall, the former TadA* deaminase activity is almost dispensable as all ABE (TadA* (LOF)-TadA*)-nSpCas9-DS variants displayed comparable editing activities and slightly narrower editing scopes as compared to ABE (TadA*-TadA*)-nSpCas9-DS counterparts -nSpCas9-DS variants and assessed their activities against sgRNA-1. It was shown that most ABE (TadA*)-nSpCas9-DS variants retained the adenine deamination activities except for 583-/895-TadA*  in functional ABE-nSpCas9-DS variants by introducing rts Fig. . In contrts Fig. . As bothrts Fig. . As it hRNA off-target activities hindered the wide applications of ABEs. We wondered whether domain insertion could reduce the RNA off-target activities. Functional ABE (TadA*-TadA*)-nSpCas9-DS variants (535-/583-/770-/793-/801-/895-/905-/919-1029-/1249-TadA*-TadA*), which displayed ≥40% A–G conversion efficiency, were all assessed by preliminary transcriptome analysis and it was revealed that domain insertion significantly reduced their RNA off-target activities as compared to N-TadA*-TadA* -nSpCas9-DS variants (535-/770-/801-/1029-/1249-TadA*-TadA*), which displayed representative diversified editing scopes, and TadA* mutants including TadA* (K20A/R20A), TadA* (V106W), TadA* (V82G) and TadA* (F148A) were selected to generate ABE-nSpCas9-DS variants 535-/770-/801-/1029-/1249-TadA*(LOF)-TadA*(K20A/R20A)/-TadA*(V82G)/-TadA*(V106W)/-TadA*(F148A). As the former TadA* deaminase activity is unnecessary, TadA*(LOF) was used to reduce RNA off-target activities as demonstrated previouslyFirstly, systematic editing scope analysis was performed against four sgRNA sites for selected 535-/770-/801-/1029-/1249-TadA*-TadA* and derived 535-/770-/801-/1029-/1249-TadA*(LOF)-TadA*(K20A/R20A)/-TadA*(V82G)/-TadA*V106W)/-TadA*(F148A) variants. 535-TadA*-TadA* displayed similar editing signatures to N-TadA*-TadA* and 1029-TadA*-TadA* displayed slightly expanded editing scopes with high editing activities at A2 for at least one sgRNA  and TadA*(V82G) reduced RNA off-target activities similarly while TadA*(F148A) could eliminate RNA off-target activity, we chose N-/535-/770-/801-/1029-/1249-TadA*(LOF)-TadA*(V82G)/-TadA*(V106W) variants for RNA off-target analysis. In consistent with previous studies, the RNA off-target activities of N-TadA*(LOF)-TadA*(V82G)/-TadA*(V106W) were significantly reduced as compared to N- TadA*-TadA* but still induced about 22- and 7.5-fold higher numbers of edited adenines than background, respectively -TadA*(V82G)/-TadA*(V106W) variants were further reduced as compared to their N-terminal counterparts Fig.  and 12. 23. We found that for 535-/583-/1029-TadA*(LOF)-TadA* displaying maximal activities at A5, 583-TadA*(LOF)-TadA* displayed minimal editing activities at both on-target sites. 535-TadA*(LOF)-TadA* displayed comparable off-target effects at all six off-target sites as compared to N-terminal counterpart while higher off-target effects were observed at sgRNA-HPRT off2 site for 1029-TadA*(LOF)-TadA* -TadA*)-nSpCas9-DS variants using sgRNA-FAN (FANCF site) and sgRNA-HPRT (HPRT site) at two sites with well-defined DNA off-target sites by either GUIDE-seq or Digenome-seq platform A > CA > AA/GA (underline for target) -TadA*(V82G) variants for editing analysis against another 20 genomic sites to characterize their editing signatures in diverse contexts, which would be valuable for their future applications. It was shown that 770-TadA*(LOF)-TadA*(V82G) displayed quite efficient A–G conversion activity, with an average editing activity >40% at positions 9–16 Fig. . For 801et) Fig. . 801-Tad/GA Fig. , but the AA Fig. .Fig. 5Co6. Indeed, Analysis of human pathogenic single-nucleotide polymorphisms (SNPs) in Clinvar database4 revealed that the percentage of correctable pathogenic SNPs by ABE tools with editing scopes across 4–8, 4–14, and 2–16 was 30%, 51%, and 61%, respectively -nSpCas9-DS variants generated in this study could achieve efficient A–G conversions across protospacer positions 2–16. Such broadened editing scopes would make more genomic sites targetable as compared to previous ABE tools with editing scopes across positions 4-8 or 4–1420  in ssDNA loop, while deaminase engineering would change the cytosine deamination activities, with V82G enhancing while V106W inhibiting their cytosine deamination activities generally.Additionally, we noticed that N-TadA*-TadA* induced about 3% of C6 conversion at sgRNA-B site, in consistent with previous studies showing that ABEs could induce cytosine deamination at specific sites20 Fig. . Similar20 Fig. . Cytosin20 Fig. . It was 20 Fig.  and V10620 Fig. , while K20 Fig. , indicat20 Fig. . We also20 Fig. . ABE-nCa20 Fig. . We also20 Fig. . This mi20 while our ABE-nSpCas9-DS variants could also target CC (underline indicating the target site) in addition to TC with an expanded editing window (positions 5–13). These differences might be attributed to the following reasons. Firstly, TadA* are quite different from TadA in DNA binding and deamination abilities, which might lead to the changes of preferred target motif. In addition, TadA* dimer insertions at different DSs changed their relative distances to ssDNA loop and targetable cytosine(s), and thus resulted in editing window expansions. Systematic analysis would be needed to clarify the impact of DS locations in combined with deaminase engineering on ABE cytosine deamination activities, which might interfere with canonical adenine deamination activity, to provide guidance for the selection of ABE variants. ABE cytosine deamination activity also deserves further explorations to assess their applicability for cytosine conversions.It was noted that our results are not entirely consistent with previous results, which showed that ABE(N-TadA-TadA*)-mediated cytosine conversion preferred TCN motif and a narrow editing window (positions 5–7)11. Both strategies are powerful for Cas9-based protein engineering. Indeed, the relative position of fusion domains could be changed when they were linked to either SpCas9-DSs or Cas9-CPs as compared to wild-type Cas9. For Cas9-CPs-based fusion proteins, they were still constructed through conventional “head-to-tail” fusion strategy, and thus the fusion domain is quite flexible as only one side is linked to Cas9-CPs. On the other hand, for SpCas9-DSs-based fusion proteins, domains were inside SpCas9-DSs, which would limit their excessive flexibility. Domain flexibility differences between Cas9-CPs- and SpCas9-DSs-based fusion proteins would affect protein properties such as protein stability, activity and specificity. As both strategies have been successfully applied for ABE engineering, we noticed that ABE (TadA-TadA*)-nSpCas9-DS1029/1249 displayed similar editing scopes as compared to CP1028/1249-ABEmax (TadA-TadA*) based on limited data, which indicated that similarity also existed between Cas9-CPs- and SpCas9-DSs-based tools6. Here we found that domain flexibility might be critical for RNA off-target effects, further analysis of RNA off-target differences between Cas9-CPs- and SpCas9-DSs-based ABE variants would be valuable to elucidate their characteristic differences. Additionally, Cas9-CPs without DNA cleavage activities but retaining DNA binding abilities remained to be discovered and evaluated in the future, as our results suggested that under specific conditions, DNA cleavage activity might be dispensable for SpCas9-DSs-based tools. Additionally, modest positive correlations have been observed between N-terminal-based conventional ABE/CBE activities and DNA cleavage activity of Cas924 while ABE-nSpCas9-DS770 to ABE-nSpCas9-DS919 described here maintained high editing activities without any DNA cleavage activity. As regions 770-919 mainly cover the HNH domain in Cas9, it is possible that deaminase inside here might induce specific conformation changes to promote base editing process. In summary, this study demonstrate that domain insertion provides a framework for further improvement of base editing tools and would generate more diversified and improved toolkits when combined with other strategies such as deaminase engineering. As different DSs inside Cas9 might confer distinct structural hindrance to modulate the functions of inserted domains, such divergence could be utilized to develop diversified tools such as ABEs with unique editing scopes and specificities to achieve more precise editing effects.In this work, we generated a series of functional ABE variants with diversified editing scopes and minimized RNA off-target activities using domain insertion profiling, which confirmed that domain insertion is an effective and powerful strategy for the development of Cas9-based fusion proteins. When combined with other strategies such as deaminase engineering, the editing scopes and RNA off-target activities of ABE tools were further improved, which allow efficient A–G conversions at currently untargetable sites and address a major safety concern that hindered the broad applications of ABEs. We also notice that the editing signatures of ABE variants are dependent on both docking sites and inserted deaminases, indicating that ABE toolkits could be further diversified by different combinations of TadA* variants. As ABE variants could display distinct editing signatures with different TadA* combinations at the same DSs, for every functional domain to be assessed, it is necessary to screen all potential DSs comprehensively to avoid omitting functional ABE variants. In addition to SpCas9-DSs, recent studies have established Cas9-CPs for fusion protein construction2 incubator (Thermo Scientific Heraeus) using DMEM (Gibco/Life Technologies) containing 10% FBS (Gibco/Life Technologies) at 37 °C. For cell transfection, HEK293T cells were initially plated into 6-well or 24-well plates (Thermo Scientific) and cultured for 20 h. Then transfection was performed using Lipofectamine 3000 (Thermo Scientific) mixed with plasmid expressing specific base editor and plasmid expressing specific sgRNA (mole ratio 4:1). For base editing analysis, cells were cultured for 72 h for flow cytometry and genome isolation using lysis buffer ). For transcriptome analysis, cells were cultured for 48 h for flow cytometry and RNA isolation using Trizol reagent (Life Technologies).HEK293T cells ) were cultured in a 5% CO21. Sanger sequencing analysis was performed in biological duplicates and detailed Sanger sequencing data was summarized in Supplementary Data GFP/mCherry double-positive cells were isolated using a Moflo XDP (Beckman Coulter) and flow cytometry data was analyzed using SUMMIT Version 5.2.0 software. Cells were sorted into 200 μL lysis buffer ) for genome isolation. PCR primers were designed and synthesized (GENEWIZ). Amplification products were prepared using Takara LA Taq for sanger sequencing (Shanghai Majorbio Bio-Pharm Technology) and editing efficiency was quantified using EditRhttps://ice.synthego.com/)25.Genome samples were prepared as described in base editing analysis. Amplification products were prepared using Takara LA Taq for sanger sequencing  and Indel efficiency was quantified using online tool ICE analysis  site with >40% editing efficiency were included for preference evaluation. A sites were classified according to NA or AN types. Statistical analysis was performed with two-tailed unpaired t-test and p < 0.05 was considered significant. Detailed Sanger sequencing data was summarized in Supplementary Data Sequence preference for 770- and 801-TadA*(LOF)-TadA*V82G) was analyzed as described previously with slight modificationsG was anaPKKKRKVAAAGSGPKKKRKVAAAGSS) containing two NLS signals (underline). For TadA* monomer insertions, a GSS linker was used between the docking site and the N-terminus of TadA* monomer. The docking site was linked to the C-terminus of both TadA* dimer and monomer via ART amino acids. Plasmids for sgRNA expression were generated as described previously10. In brief, U6-sgRNA-EF1apha-UGI-T2A-mCherry plasmid was linearized with BsaI, and paired oligonucleotides were synthesized, annealed, and inserted for sgRNA expression vector constructions. All sgRNA information was listed in Supplementary Table Human codon optimized deaminases TadA and TadA* were synthesized commercially (Genescript). For mutagenesis, KOD-Plus system was used to generate TadA*K20A/R21A), TadA*(V82G), TadA*(V106W), TadA*(F148A), TadA*(LOF), and docking sites inside SpCas9 (PX461 (Addgene #48140)) for fusing protein construction. For TadA* dimer insertions, docking site, and the N-terminus of TadA* dimer was connected via a linker  and total RNA was isolated using Trizol reagent for RNA-seq. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries with standard Illumina protocols. RNA-seq was performed using Illumina HiSeq X Ten platform in Shanghai Novelbio Ltd. For each library, ~50–60 million reads were generated. We applied the reads filtering towards the raw reads after sequencing to achieve the clean data following the criteria: a) 10% base quality <15 b) 13% base quality <20. RNA sequencing data was aligned to the human reference genome (GRCh38) using STAR (v2.5.2b). Variants were called using the GATK best practices pipeline using Picard and GATK 3.8. Single-nucleotide variants (SNVs) were filtered to include loci with reads >10 and labeled as A–G or T–C for to evaluate RNA off-target activities.t-test was chosen for comparisons and statistical significance was considered as p value < 0.05. GraphPad Prism was used for statistical analysis of this study.Statistical details are provided in the text, figure legends, and methods. Error bars were standard error of the mean (S.E.M.) from independent experiments/samples. Two-tailed unpaired Student’s Further information on research design is available in the Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting SummaryPeer Review FileDescription of Additional Supplementary Files


Our results showed that whether exported by the CRM1 or NXF1 pathway, HIV-1 RNAs mainly use diffusion for cytoplasmic travel. Furthermore, HIV-1 RNAs exported using the CRM1 or NXF1 pathway are well mixed in the cytoplasm and do not display export pathway-specific clustering near centrosomes. Thus, the export pathways used by HIV-1 RNAs do not alter the cytoplasmic transport mechanisms or distribution.The unspliced HIV-1 full-length RNA (HIV-1 RNA) is packaged into virions as the genome and is translated to generate viral structural proteins and enzymes. To serve these functions, HIV-1 RNA must be exported from the nucleus to the cytoplasm. It was recently suggested that export pathways used by HIV-1 RNA could affect its cytoplasmic transport mechanisms and distribution. In the current report, we examined the HIV-1 RNA transport mechanism by following the movement of individual RNAs and identifying the distribution of RNA using  cis-acting element, Rev response element (RRE). When Rev/RRE is replaced with the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), HIV-1 RNA is exported through the NXF1 pathway. To determine the effects of the export pathway on HIV-1 RNA, we tracked individual RNAs and found that the vast majority of cytoplasmic HIV-1 RNAs travel by diffusion regardless of the export pathway. However, CTE-containing HIV-1 RNA diffuses at a rate slower than that of RRE-containing HIV-1 RNA. Using in situ hybridization, we analyzed the subcellular localizations of HIV-1 RNAs in cells expressing a CTE-containing and an RRE-containing provirus. We found that these two types of HIV-1 RNAs have similar subcellular distributions. HIV-1 RNA exported through the NXF1 pathway was suggested to cluster near centrosomes. To investigate this possibility, we measured the distances between individual RNAs to the centrosomes and found that HIV-1 RNAs exported through different pathways do not exhibit significantly different distances to centrosomes. Therefore, HIV-1 RNAs exported through CRM1 and NXF1 pathways use the same RNA transport mechanism and exhibit similar cytoplasmic distributions.HIV-1 full-length RNA (referred to as HIV-1 RNA here) serves as the viral genome in virions and as a template for Gag/Gag-Pol translation. We previously showed that HIV-1 RNA, which is exported via the CRM1 pathway, travels in the cytoplasm mainly through diffusion. A recent report suggested that the export pathway used by retroviral RNA could affect its cytoplasmic transport mechanism and localization. HIV-1 RNA export is directed by the viral protein Rev and the  Retroviral DNA is integrated into the cellular genome to become a provirus and a permanent part of the cell . Using tcis- and trans-acting elements to facilitate the export of partially spliced and unspliced RNA. HIV-1 encodes an accessory protein, Rev, that binds and multimerizes on a highly structured RNA element in the env gene, termed Rev response element (RRE)  –13. Rev-t (RRE) –, 15. In t (RRE) ––18. Thus (RRE) ––. Similaroduction . Interesoduction . Hence, oduction , 22. TheA export , 24; thuAfter exiting the nucleus, RNAs travel to various subcellular locations to serve their functions. The transport of cellular mRNA in the cytoplasm can be complex. Some mRNAs are transported in the cytoplasm predominantly by diffusion, whereas other mRNAs can be actively transported along the cytoskeleton by motor proteins to specific locations –30. By t–in situ hybridization, we analyzed cells dually infected with two proviruses, one containing CTE and the other containing RRE. Our results showed that CTE- and the RRE-containing HIV-1 RNAs have similar subcellular distributions. We measured the distances between individual RNAs to the centrosomes and found little if any difference between the CTE- and RRE-containing HIV-1 RNA. Thus, using live-cell imaging and in situ hybridization approaches, we observed that HIV-1 RNA exported through different pathways use similar RNA transport mechanisms and exhibit similar cytoplasmic distribution.To better understand how the nuclear export pathway affects HIV-1 RNA behavior, in this report, we compared the cytoplasmic trafficking of HIV-1 RNA containing the authentic RRE or the MPMV CTE in place of RRE. Using single particle tracking, we followed individual RNA molecules in the cytoplasm and found that the vast majority of the HIV-1 RNAs use diffusion as their transport mechanism. Interestingly, CTE-containing HIV-1 RNA diffuses at a lower rate than RRE-containing HIV-1 RNA. Using cis-acting elements necessary for viral replication and expresses Tat, Rev, and Nef. A T-to-A substitution of the gag translation start codon in 1-AAG and 1-AAG-CTE converts AUG to AAG, which abolishes Gag translation but does not interfere with RNA function, as the mutant RNA can be efficiently packaged into viral particles and undergoes one round of replication when essential proteins are provided in trans (pol gene in which 18 copies of stem-loop sequences (BSL) were inserted; BSL RNA is specifically recognized by the bacterial protein BglG  fusion protein , which be images , multiple images . To bett10.1128/mBio.01578-20.1MOVIE S1Movie S1, AVI file, 8.6 MB.1-AAG-CTE RNA movement in the cytoplasm of a cell. HeLa cells were transfected with plasmids 1-AAG-CTE and Bgl-YFP . As we previously described . The stagnant fraction is defined by the localization precision of our system, which was 40 nm, and yielded a diffusion coefficient of 0.01 μm2/s. Signals in the small stagnant fractions . A total of 528,579 one-step jump distances were obtained from the 19,530 1-AAG-CTE RNA tracks, and 635,465 one-step jump distances were obtained from the 35,766 1-AAG RNA tracks. The distributions of these one-step jump distances are shown in escribed , we obtaP = 0.0038 for 1-AAG-CTE and P = 0.0037 for 1-AAG RNA, chi-squared test). However, 1-AAG-CTE RNAs do not move in a directional manner more frequently than 1-AAG RNAs . Thus, the vast majority (>99%) of the HIV-1 RNAs use diffusion as a transport mechanism in the cytoplasm regardless of whether they were exported by the CRM1 or the NXF1 pathway.To analyze the directionality of RNA molecules, we performed a previously described persistence index analysis , which e2 > 0.9), indicating that diffusion is the major transport mechanism. The diffusion coefficient calculated from the ensemble MSD values is 0.059 μm2/s  but similar to 1-AAG-CTE RNA . Thus, similar to the movement of 1-AAG-CTE RNA in the absence of Gag, the vast majority of the tracks moved in a diffusive manner, and a very minor portion, less than 0.1%, moved in a directional manner. Taken together, the MSD, the one-step jump distance, and the persistence index analyses revealed that Gag expression does not have significant effects on the mobility or directionality of the cytoplasmic HIV-1 RNA exported via the NXF1 pathway. These results are reminiscent of our previous report wherein Gag did not affect the diffusion rate of cytoplasmic HIV-1 RNA containing authentic RRE that was exported by the CRM1 pathway.The HIV-1 structural protein Gag is an RNA-binding protein, and its presence can potentially affect cytoplasmic RNA transport. To examine the potential effects of Gag on the cytoplasmic trafficking of CTE-containing HIV-1 RNA, we used two HIV-1 constructs that are structurally similar to 1-AAG-CTE but contain AUG at the Gag translational start codon; 1-Gag-CTE expresses an untagged Gag and 1-Gag-mCherry-CTE expresses an mCherry tagged Gag . We tran59 μm2/s , similar59 μm2/s  without 59 μm2/s , which i10.1128/mBio.01578-20.3MOVIE S3Movie S3, AVI file, 1.8 MB.HIV-1 RNA movement in the cytoplasm of a cell expressing functional Gag. HeLa cells were transfected with plasmids 1-GagmCherry-CTE/1-Gag-CTE  element; the rtTA gene was inserted into the nef gene, and the tet operator (tetO) binding sites were inserted into HIV-1 LTR. Thus, the expression of HIV-rtTA is not regulated by viral protein Tat but can be induced by the addition of doxycycline. Importantly, HIV-rtTA generates a full-length RNA with an intact 5′ untranslated region, identical to wild-type HIV-1 except for several point mutations in TAR. HIV-rtTA was modified to generate Gag-mCherry-RRE, which expresses a Gag fused to mCherry fluorescent protein, Tat, and Rev and contains an authentic HIV-1 RRE. The construct Gag-YFP-CTE has a similar structure . Infected cells were then enriched by repeated cell sorting until most of the cells (>90%) were dually positive with mCherry and YFP expression. Because cells were infected at low MOI, most of the infected cells contain only one of each provirus.in situ hybridization assay, RNAscope, to detect mCherry and yfp sequences, respectively. To determine whether this assay could distinguish between RNAs carrying mCherry or yfp sequences, we first tested cells infected with either Gag-mCherry-RRE or Gag-YFP-CTE. After the RNAscope procedure, we treated cells with DNA stain, DAPI , and captured images of cells using a confocal microscope in three channels to detect signals of probes targeting mCherry or yfp sequences and DAPI signals. A set of representative images of the equatorial planes is shown in mCherry probes but not yfp probes. Similarly, RNA signals were detected using yfp probes but not mCherry probes in cells infected with Gag-YFP-CTE. Thus, this assay can detect HIV-1 RNA derived from our constructs; furthermore, it can distinguish RNAs derived from Gag-mCherry-RRE and Gag-YFP-CTE.To detect HIV-1 RNA derived from Gag-mCherry-RRE and Gag-YFP-CTE, we used an To examine the kinetics of HIV-1 RNA expression, we treated the cells that were dually infected with Gag-mCherry-RRE and Gag-YFP-CTE with doxycycline, fixed cells at different time points, performed RNAscope, and captured images. Representative images are shown in To quantify the expression kinetics, a series of z-stack images were collected for each field of view, and the images of the equatorial planes were selected for analyses. The number of RNA signals from the equatorial plane of each cell was determined, and results from 24, 45, 83, 56, and 34 cells for the no-dox (0 h), 4-h, 7-h, 12-h, and 24-h time points, respectively, are summarized in P = 0.23; RRE to CTE observed versus random, P = 0.78; one-way analysis of variance [ANOVA]). Additional data from analyses of two other cells are shown in It has been suggested that the use of a particular RNA export pathway could affect the distribution of RNA in the cytoplasm . We reas10.1128/mBio.01578-20.4FIG S1FIG S1, TIF file, 1.4 MB.The spatial relationship between Gag-mCherry-RRE and Gag-YFP-CTE RNAs in the cytoplasm of two cells in addition to Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P = 0.98; centrosome 2, Gag-mCherry-RRE RNA versus Gag-YFP-CTE RNA, P = 0.99; one-way ANOVA). Results from analyses of 27 additional cells are shown in It was suggested that the MPMV CTE element links HIV-1 RNA to the microtubules in the cytoplasm, driving them to cluster at the centrosome . AnalyseTaken together, these results indicate that the vast majority of the HIV-1 RNA exported by CRM1- or NXF1-mediated pathways use diffusion to travel in the cytoplasm. However, the export pathways used by HIV-1 RNAs affect the mobility of the RNAs in the cytoplasm; HIV-1 RNA using the NXF1 export pathway diffuses at a lower rate than its counterpart exported by the CRM1 pathway. HIV-1 RNAs are exported efficiently by both CRM1 and NXF1 pathways, although RNAs exported through the CRM1 pathway accumulate in the cytoplasm more slowly than those exported through the NXF1 pathway, most likely because sufficient Rev expression is needed for the RNA transport. HIV-1 RNAs exported via the CRM1 or the NXF1 pathway have similar cytoplasmic distributions and do not cluster near the centrosomes.HIV-1 RNA must be exported into the cytoplasm to carry out its functions. Proper nuclear export not only allows HIV-1 RNA to localize in the cytoplasm, but also affects RNA functions and downstream events in viral replication. It has been shown that although HIV-1 RNA lacking RRE or Rev can be detected in the cytoplasm, it was not efficiently packaged or translated –43. Simi–To compare the movements of the RRE- and CTE-containing HIV-1 RNAs, we performed single-molecule tracking to follow a large number of RNA tracks. We then examined whether individual RNAs traveled in the same direction in consecutive steps using persistence index analyses. We found that >99% of the CTE- and RRE-containing HIV-1 RNA travel in a random-walk manner; furthermore, the proportion of CTE-containing RNA engaging in directional movement (0.076%) is not significantly different than that of RRE-containing RNA (0.039%). These results are different than the previous report indicating that CTE-containing HIV-1 RNA was transported in a directional manner . At this2/s) is faster than the rate of 1-AAG-CTE RNA (0.065 μm2/s). These results were generated from measuring the distances traveled by these two types of RNAs in four steps and used the results to calculate ensemble MSDs . The relationship between mass and the rate of diffusion is cubed, suggesting that the mass of the CTE-containing RNA is significantly larger than that of the RRE-containing HIV-1 RNA. However, 1-AAG-CTE RNA is only 78 nucleotides (nt), or 1.5%, longer than the 1-AAG RNA; thus, the difference in diffusion rates cannot be attributed to the difference in length between the two RNAs. Most of the cellular mRNAs are associated with proteins to form ribonucleoprotein (RNP) complexes. It is possible that the CTE- and RRE-containing HIV-1 RNAs have distinct protein compositions; many aspects of the proteins associated with cytoplasmic RNAs are undefined, including when the NXF1/NXT1 complexes dissociate from the exported HIV-1 RNA. Alternatively, it is also possible that HIV-1 RNAs exported through NXF1 pathways have distinct RNA folding and structures from RNA exported via CRM1 pathway, resulting in the difference in diffusion rates. Future experiments will be needed to identify the reasons for the differences in diffusion rates.Although we observed that both CTE- and RRE-containing RNAs use diffusion to travel within the cytoplasm, we observed that these two types of RNAs have different diffusion rates. The diffusion rate of 1-AAG RNA . Thus, HIV-1 RNAs do not cluster near centrosomes in an export pathway-dependent manner. These results are distinct from the previous observations  from 1-GagmCherry-BSL  supplemented with 10% fetal bovine serum (HyClone), penicillin , and streptomycin , and cells were maintained in humidified 37°C incubators with 5% COTransient transfections were performed using FuGENE HD transfection reagent (Promega) according to the manufacturer’s recommendations. HeLa cell lines that contain Gag-mCherry-RRE and Gag-YFP-CTE proviruses were generated by sequential infection and cell sorting. Briefly, an HIV-1 construct was cotransfected into 293T cells with pC-help and pHCMV-G plasmids, which express HIV-1 proteins  and vesimCherry gene  or the yfp gene . To detect centrosomes, following the RNAscope procedure, cells were treated with phosphate-buffered saline with 3% bovine serum albumin for 45 min at room temperature, followed by incubation with a mouse antibody against human γ-tubulin (Sigma) for 2 h at room temperature. After washing with phosphate-buffered saline containing 0.2% Tween 20, cells were incubated with Cy5-tagged anti-mouse antibody for 30 min at room temperature and washed, and this was followed by DAPI stain for 5 min. The cells were then mounted with mounting solution and sealed for imaging. Images of 3 or 4 channels to detect DAPI, Alexa 488 (for yfp sequence detection), Atto 550 (for mCherry sequence detection), and Cy5 (centrosome) were acquired with a Zeiss Lsm780 confocal microscope. A customized Matlab program was used to select the equatorial plane from acquired z-stack images based on the area of nuclear DAPI stain. Gag-mCherry-RRE and Gag-YFP-CTE RNA signals were identified with the Localize program (An RNAscope assay was performed according to the manufacturer’s recommendation (ACDbio) with probes specific to the  program . A custoFor live-cell imaging, spinning disk confocal microscopy was performed using an inverted Nikon Ti microscope with the Yokogawa CSU-X1 confocal scanner unit and a 100 × 1.45-numerical aperture (100 × 1.45-NA) total internal-reflection fluorescence (TIRF) oil objective. Simultaneous imaging of YFP and mCherry was performed by using two precisely aligned cameras (Andor iXon Ultra) on a Cairn image splitter (Optosplit II). Camera alignments were performed using labeled HIV-1 particles as previously described . The YFPhttp://physics.georgetown.edu/matlab/) based on the available tracking algorithms (r2(t)], are linearly related to time (t) and the diffusion coefficient (D) by the formula r2(t) = 4Dt.Single-molecule tracking was performed with Matlab (Mathworks) code (gorithms  with maxgorithms . MSDs wegorithms . In freer) and width (dr) at time (t) from that position is given as r, r + dr) traveled by a single RNA during a given time. When a population contains multiple diffusive fractions, the jump-distance distribution cannot be fitted satisfactorily by the above function with a single diffusion coefficient. Such different mobility fractions can be detected and quantified by curve fitting, taking several diffusion terms into account (In jump-distance analysis, the probability that a particle starting at a specific position will be encountered within a shell of radius ( account .d represents the distance, p1 represents the initial point, and pL represents the last point. A computer program based on Matlab was generated to simulate free diffusion in 2D. Using this program, and based on Gaussian random-walk movement, we generated 100 sets of simulation with the same number and length distribution of trajectories and proportion of the three diffusion coefficients as the 1-AAG, 1-AAG-CTE, and 1-GagmCherry-CTE data sets, respectively. To identify potential directional mobility, we scanned the experimental trajectories and 100 sets of simulated trajectories for segments consisting of 10 to 48 consecutive steps with a persistence index ≥ 0.7.The persistence index of a track between two points was calculated by dividing the net distance of the track between two points by the total distance, using the formula


The 3′-terminal stem-loop (3′SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3′SL was proposed to act as a replication silencer. The lower part of the 3′SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3′SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3′SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3′SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3′SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3′SL acts as an RNA thermometer that modulates flavivirus replication during host switching. Flavivirus within the virus family Flaviviridae. The WNV, which cycles between mosquitos and birds, is currently the arbovirus with the widest geographic distribution. Humans and horses are incidental hosts and they do not play a role in the transmission cycle  CTP, 10 nM of template RNA, and 15 nM of the recombinant, purified NS5. Supplementation was performed such that 200 nM AUF1 p45 was preincubated under the assay conditions with the template RNA, and NS5 was added subsequently. The reaction was carried out for 60 min at 37 °C and stopped by phenol/chloroform extraction and ethanol precipitation of the RNA. The RNA products were analyzed by electrophoresis on a 5% denaturing polyacrylamide gel (7 M urea), visualized by phosphor imaging and quantified by ImageQuant Software .The assay was performed in a total volume of 40 μL in buffer containing 50 mM Hepes/NaOH, 10 mM KCl, 5 mM MgClA. albopictus p30 and p32 in Western blots, a rabbit polyclonal antiserum was applied that was raised against the recombinant, purified full-length p32 protein . Secondary antibodies were purchased from LI-COR Biosciences . Western blots were performed using standard protocols and by following the instructions of the manufacturers.To detect 2). Fluorescence changes were monitored using a Fluoromax-4 spectrofluorometer  at 22 °C. The signal amplitudes of the 5′-FAM-EX-labeled RNAs  in assay buffer  and 5 pmol of protein arginine methyltransferase 1 . The reaction was performed at 30 °C for 2 h and 13 pmol AUF1 p45 (purified from E. coli) served as a positive control. The degree of methylation was analyzed by SDS-PAGE and phosphor imaging.Either 1 pmol of FLAG-p30 or FLAG-p32 (purified from C6/36 cells), or 1 pmol p30 or p32 (purified from rding to ) were intrunc-RNA . Then, 100 nM of non-labeled 5′UAR RNA was added and readings were taken for another 400 s. Changes in the fluorescence signals were monitored in a Fluoromax-4 spectrofluorometer  at 22 °C with the following parameters: excitation at 643 nm and emission at 667 nm. Fluorescence intensities relative to the starting fluorescence were plotted against the time and fitted by KaleidaGraph 4.5  to first-order reaction when protein was omitted (Equation (2)) and second-order reaction when protein was included (Equation (3)) yielding the corresponding rate constants kobs.offset is the fluorescence intensity at the start point of the reaction, Fmax is the maximum signal amplitude, kobs is the observed rate constant, and t is time.The assay was performed as previously described in . Briefly2). Then, 10 nM of 5′-Cy5-labeled 5′CS-RNA was added, and readings were taken for another 400 s. Changes in the fluorescence signals were monitored in a Fluoromax-4 spectrofluorometer  at 22 °C. The Cy3 fluorophore was excited at 535 nm wavelength and readings were taken at the Cy5 emission wavelength 680 nm. Fluorescence intensities relative to the starting fluorescence were plotted against the time and fitted by KaleidaGraph 4.5  to a second-order reaction (Equation (3), see above) yielding the corresponding rate constants kobs.The assay was performed as previously described in . BrieflySedimentation equilibrium measurements were performed as previously described in . AnalysiMRW.Measurements of dichroic properties of mosquito proteins p30 and p32 were performed using a JASCO J-810 spectropolarimeter. Far-UV circular dichroism spectra were recorded at a protein concentration of 2 µM in 20 mM Tris/HCl, pH 7.6, 150 mM KCl, and 1 mM TCEP at 22 °C using cuvettes with optical path lengths of 1 mm. Acquired protein spectra were corrected for buffer contribution and smoothed using the Spectra Manager I software (JASCO). The data were converted to mean residue ellipticity Θtrunc), which harbors the 3′UAR cyclization sequence, as well as the metastable element with the two flanking bulges  at 76.2 °C calculated from the first derivative of the transition curve , the upper bulge (3′SL Mut2), or both bulges (3′SL Mut3)  A. To maiperature B. Next, perature C. These Next, we tested how the mutations affected AUF1 p45-supported WNV negative-strand RNA synthesis. For this purpose, we performed a previously established in vitro replicase assay using the purified WNV RNA-dependent RNA polymerase (RdRp) NS5 and, as a template, a subgenomic RNA (sgRNA). The assay essentially generates two RNA products, namely a negative-strand RNA product, which results from de novo initiation of RNA polymerization on the template, and a hairpin product, which is synthesized by priming at the 3′-OH of the template and turn-around copy-back polymerization by the NS5 RdRp . De novoTogether, the data imply that (1) an increased stability of the WNV 3′SL (at least in the case of Mut1 and Mut3) prevents precise initiation of RNA synthesis by NS5 at the 3′-end of the viral RNA, and that (2) the host factor AUF1 p45 stimulates full-length de novo RNA synthesis only if a certain structural flexibility of the 3′SL is provided.To assess the role of the stability of the 3′SL on WNV replication in different host cells, we introduced each mutation individually into a WNV luciferase reporter replicon  that enaWe observed a different picture in the mosquito cell line C6/36. While the translation levels of all mutant replicons were again similar to those of the wild-type replicon, all mutant replicons turned out to be replication deficient B. AdditiNext, we asked whether the replication-defective phenotypes of the 3′SL mutant replicons in human and mosquito cells were simply caused by different cultivation temperatures. Therefore, we swapped the cultivation temperatures and performed the Huh7 experiment at 28 °C and the C6/36 experiment at 37 °C. Interestingly, the lag phase preceding replication of the WNV replicon was considerably longer when the Huh7 cells were cultivated at 28 °C see . With thIn comparison to the wild-type replicon, the 3′SL Mut1 and 3′SL Mut2 replicons showed again a delayed RNA replication phenotype. However, in contrast to the situation at 37 °C, the delay of replication at 28 °C was more pronounced and the mutant replicons barely replicated to wild-type levels. The replicon containing the 3′SL Mut3 mutation was replication-deficient at 28 °C, as it was at 37 °C A.The replication of the wild-type replicon RNA in the C6/36 cells that were cultured at 37 °C after transfection increased significantly already after 24 h and, accordingly, showed no lag phase B. TherefAedes albopictus (the mosquito species from which the cell line C6/36 was derived) containing two RNA recognition motifs (RRMs), an arginine-glycine-rich region (RGG), and a tyrosine-glycine-rich region (YGG), and therefore appear to be very similar to AUF1  with an antiserum raised against the full-length p32 protein revealed two proteins migrating at the same position as the proteins squid p30 and p32 purified from ene loci C. It hasto cells D.KD = 13 nM) and squid p32 (KD = 130 nM) was found to be eight-fold and 80-fold less efficient than with AUF1 p45, respectively. Binding of the random RNA by squid p30 (KD = 250 nM) and squid p32 (KD = 630 nM) was 1.6-fold and four-fold less efficient than with AUF1 p45, respectively  corresponding to the lower part of the WNV 3′SL, including the 3′UAR sequence. The second RNA is single-stranded and corresponds to a sequence of the WNV 5′UTR mainly comprising the 5′UAR cyclization sequence. Hybridization of both RNAs via the UAR elements requires a rearrangement of the SL, which dislocates the Cy5 fluorophore from the BHQ and can be measured time-dependently (exemplary kinetic traces shown in trunc–5′UAR interaction as compared with AUF1 p45, with squid p32 being more active than squid p30 (In the following set of experiments, we applied two assays to evaluate the RNA remodeling capabilities of the minal SL A. To enaminal SL A. Using quid p30 B. IntereIn a second approach, we investigated whether the mosquito squid proteins could promote the annealing of single-stranded complementary RNAs. For this purpose, we applied a previously established FRET-based RNA annealing assay using the conserved flaviviral 5′ and 3′CS cyclization sequences, which are labeled with a Cy5 and a Cy3 fluorophore, respectively (exemplary kinetic traces shown in These data show that the mosquito squid proteins share RNA remodeling activities similar to AUF1 p45, however, both the RNA chaperone and especially the RNA annealing activities of p30 and p32 are less efficient as compared with AUF1 p45.Since the temperature differentially affected the replication efficiency of WNV reporter replicon RNAs in human and mosquito cells, we wondered if the RNA remodeling activities of the human AUF1 p45 and the mosquito p30 and p32 were also temperature dependent. Therefore, we repeated the RNA remodeling assay, which is normally conducted at 22 °C, at 28 °C. Interestingly, we observed that the RNA chaperone activity of AUF1 p45 was strongly enhanced at 28 °C, whereas the RNA chaperone activities of p30 and p32 were not, or were only mildly, improved at higher temperature, respectively A–C. The In order to maintain their natural transmission cycle, arboviruses are required to establish persistent non-lethal infections in the mosquito host that do not significantly interfere with the vector’s physiology, and therefore transmission efficiency. However, the relationship between vector-borne viruses and their arthropod vectors need not be benign, with the magnitude of virulence depending on the vector and virus taxonomic groups and the mode of transmission . EvolutiDuring the natural process of host switching, invertebrate and vertebrate species impose different selective forces on flaviviral populations with differential impacts on viral mutational diversity and relative fitness ,36,37,38In mosquito and human cells, we observed slower WNV replicon replication kinetics at 28 °C and faster replication kinetics at 37 °C, similar to a study that was done with infectious WNV . FlaviviThe temperature shift, upon entering the natural vertebrate host, releases the silencing effect of the 3′SL leading to increased cyclization and replication rates and high virus titers accompanied by cytopathic effects. A so-called RNA thermometer activity in order to switch from a stable to a flexible 3′SL could be involved in modulating the replication efficiency during host switching . RNA theWith increasing temperature, the structural flexibility, and with this accessibility of the 3′SL is believed to increase, which not only facilitates cyclization of the flaviviral genome, but is also likely to stimulate the activity of cellular host factors that modulate RNA conformations. In this study we demonstrate that a defined conformation, and, with this, a high structural flexibility of the 3′SL, enhanced the RNA chaperone activity of the flavivirus host factor AUF1 p45, which, in turn, favored RNA synthesis of WNV .We identified AUF1-homologous proteins in mosquito cells that are capable of remodeling viral RNA structures; however, it is not clear if squid p30 and p32 act as host factors for flavivirus replication in mosquitos similar to the situation with AUF1 p45 in human cells. Our attempts to identify a function of p30 and p32 during WNV replication in mosquito cells by knockdown or knockout experiments did not reveal a supportive role . There cIn addition to AUF1, there might be other host RNA-binding proteins that modulate the structure of the 3′SL and which are involved in the flaviviral RNA switch from linear to circular. Such a role was previously proposed for eEF1A which binds to the 3′SL ,46,47; hIn summary, we propose a molecular mechanism describing a constellation where temperature, a viral RNA structure element, and a host factor activity are functionally connected. It has been suggested that a fine balance between the linear and the circular conformation is crucial for flavivirus replication  and that


Psammonitocrellakumeyaayisp. nov. from the Nearctic Region  is proposed. The position of the genus within Harpacticoida and its relationship with the Parastenocarididae is discussed. The new species can be included within Psammonitocrella on account of a) the cylindrical furca, longer than the telson, b) the unmodified inner spine on the basis of the male first leg, c) loss of the outer spine on the second exopodal segment of the first leg, d) loss of the outer spine of the third exopodal segment of the second, third, and fourth legs, e) loss of the inner apical seta on the third exopodal segment of the second and third legs, f) transformation of the inner apical seta of the third exopodal segment of the fourth leg into a spine, and g) loss of the endopodite of the fourth leg. The new species differs remarkably from P.boultoni, and P.longifurcata in the loss of the outer spine of the second exopodal segment of the fourth leg, in the presence of a one-segmented fifth leg exopodite, and in the presence of an outer seta on the basis of the first and second legs. Both Psammonitocrella and the known species of Parastenocarididae have a one-segmented endopod on the fourth leg, and the endopods of the second and third legs are reduced to one or two segments. Psammonitocrella is currently allocated into the Ameiridae, and evidence suggesting a sister-group relationship with Parastenocarididae—both share the loss of the inner seta on the first endopodal segment of the first leg—indicates that the Parastenocarididae should be included into the Ameiridae. In an evolutionary context, Parastenocarididae could have evolved from a lineage of freshwater ameirids that became interstitial in continental waters and colonized aquifers and groundwaters.The freshwater harpacticoid  Ameiridae Boeck, 1865 is composed of about 300 species accommodated in 47 marine and freshwater genera  and Stygonitocrellaorghidani  incertae sedis . The sampling locality is described in Noodt’s field notebook and in the sample identification as “wenig fließendes stehendes Wasser”, an almost lotic or a standing water environment or with very low current. After an extensive toponymic search we concluded that Prof. Noodt was referring to the San Clemente Canyon in San Diego County. The San Clemente Canyon is nowadays included in the Marian Bear Memorial Park in the city of San Diego, a linear open space park along a canyon rich in temporary water bodies.The habitus was drawn from the whole specimen temporarily mounted onto one slide with glycerin as mounting medium; adhesive plastic discs were used to support the cover slip and prevent destruction of the specimen . Total lThe terms ‘furca’ and ‘telson’ are used according to Psammonitocrella. It is amended from The diagnosis represents the reconstructed ground pattern of A1= antennule, A2 = antenna, aes = aesthetasc, ap= apomorphy, benp(s)= basendopod(s), cph = cephalothorax, DAS= distal apical seta; DOS= distal outer seta; enp= endopod, exp(s)= exopod(s), enp1–3 = endopodal segments 1–3, exp1–3 = exopodal segments 1–3, Fu= furca, GF = genital field, IAS= inner apical seta/spine; ms= modified spine, md = mandible, mx1 = maxillule, mx2 = maxilla, mxp = maxilliped, P1–P6= legs 1 to 6, pl= plesiomorphy, Ur1 to 5= first to fifth urosomites.Abbreviations used in the text and figures: Ameiridae Boeck, 1865Family Taxon classificationAnimaliaHarpacticoidaAmeiridaeGenusHuys, 2009C3C6B473-52A0-5F1D-87A0-6F82AFA7F982Ameiridae. Body small, slender, and cylindrical, without distinct demarcation between prosome and urosome. Integument weakly chitinized, with or without lateral cuticular windows on P2–P3-bearing somites (presence of these cuticular windows is uncertain for P.longifurcata and P.boultoni); hyaline posterior fringe of all somites smooth. First pedigerous somite incorporated into cephalosome. Prosome ornamented only with sensilla; Ur ornamented with rows of small spinules. Genital (Ur2) and Ur3 separated in female; GF with single large copulatory pore, wide copulatory duct, and two small semicircular seminal receptacles; single small genital aperture covered by fused reduced P6, without armature or ornamentation. Telson unornamented or ornamented with small spinules and tube pores. Anal operculum unornamented or ornamented with small spinules, wide and convex, not reaching or reaching posterior end of anal somite. Fu slender, tapering distally or cylindrical, slightly divergent, longer than anal somite, with long tube pores in P.kumeyaayi sp. nov.; seta VII inserted subdistally, close to inner margin, less than half the ramus length; outer setae I and II inserted on the proximal half of Fu; seta III on the same plane as seta VII; seta VI minute; seta V without breaking plane; seta IV longer, as long as or shorter than ramus. A1 long and slender, eight-segmented in female, 10-segmented, haplocer, and geniculate in male; without seta on short first segment in female, with a seta on the first segment in male. A2 composed of coxa, basis, two-segmented enp and one-segmented exp; exp armed with three setae. Md with narrow cutting edge and two-segmented uniramous palp; basis unarmed; enp with three to five apical setae. Mx1 with praecoxal arthrite armed with three distal claws, one or two minute oral setae, and one or two accessory aboral setae; coxa with two or three apical setae; basis with two to four apical setae; enp present or absent. Mx2 with syncoxal endite armed with a single element, or endite absent; basal endite armed with three elements; enp1 drawn out into a claw, with an accessory seta; remaining endopodal segment represented by one or two setae. Basis of P1 with unmodified inner spine in male, and with or without outer seta; without any other sexual dimorphism in swimming legs; basis of P2 with or without outer seta. Enp of P1 three-segmented; enp of P2 and P3 one- or two-segmented, of P4 reduced to small knob or completely absent; P1enp1 unarmed, long, reaching distal margin of exp2 or nearly as long as exp1; P1enp2 unarmed, longer than enp3; P1enp3 with outer seta, with or without geniculation, inner seta probably geniculated in all species; if enp of P2 and P3 two-segmented, then first segment unarmed, second segment with one apical seta. All swimming legs with three-segmented exps; exopodal segments of P2–P4 subequal in length; P1–P4exp1 without inner seta; exp2 of P1 without outer spine and with inner seta; exp2 of P2 and P3 with inner seta and outer spine; exp2 of P4 with or without outer spine and with inner seta; exp3 of P1 with two outer spines, two geniculate distal setae, and without inner armature; P2–P4exp3 without outer spine; P2–P3exp3 with or without inner apical seta; inner apical seta of P4exp3 may be transformed into a spine. P5 similar in both sexes; fused to somite or free; with or without recognizable endopodal lobe, and with recognizable exopodal lobe, or exp one-segmented; endopodal lobe (if present) armed with one or two elements; exopodal lobe or exp with four, three, two or only one seta.Psammonitocrellaboultoni Rouch, 1992.Psammonitocrellalongifurcata Rouch, 1992; P.kumeyaayi sp. nov.Taxon classificationAnimaliaHarpacticoidaAmeiridae2EA1B414-643A-586D-A2CA-3B8A57537D8Ehttp://zoobank.org/16622CED-8D1F-4F39-9132-E7C66AD3A972Holotype: One male dissected and mounted onto 7 slides .Type locality: San Clemente Canyon, San Diego, California, USA .cph, with two sensilla on tip /8(4ms +1)/9(4)/10(4+acrothek). Acrothek consisting of two setae fused to aesthetasc.cer Fig. , 10-segmA2  of variable shape, size and ornamentation in left  represented by unarmed cuticular flap.kumeyaayi” refers to the Kumeyaay native American people, who inhabited the area of San Diego County for 10,000 years. Evidence of their presence still remains in San Clemente Canyon.The specific epithet “Eduardonitocrellamexicana, S.orghidani, and their closely related species Psammonitocrellaboultoni and P.longifurcata. This character was, however, never mentioned by Psammonitocrella. However, it is possible that integumental windows are present on some pedigerous somites within the genus Psammonitocrella, as indicated by the presence of lateral integumental windows on the second and third pedigerous somites of Psammonitocrellakumeyaayi sp. nov. The probable loss of lateral integumental windows in Psammonitocrellaboultoni and P.longifurcata should be considered as derived. The presence of lateral integumental windows is a potential synapomorphy for a larger group of groundwater ameirids as proposed by P4exp2 is an autapomorphy for the new species.Female unknown. The presence of integumental windows on the third and fourth pedigerous somites was reported by Psammonitocrella would have fitted nicely into the original diagnosis of the genus Stygonitocrella as defined by Stygonitocrella before Psammonitocrella revealed that both genera differ in the armature of P1exp2, P2–P4exp3, and shape of the furca, with major reductions in the armature of P1–P4 and furca elongation in Psammonitocrella. However, one could still assume that Psammonitocrella is a junior synonym of Stygonitocrella, and that the constituent taxa of the former belong to a derived group within the latter. This assumption is not supported by Stygonitocrella, and concluded that the generic diagnosis of the genus should be emended and the genus rearranged to include only four species: the type species S.montana  from Argentina, S.dubia  and S.guadalfensis Rouch, 1985 from Spain, and S.sequoyahi Reid, Hunt & Stanley, 2003 from the USA. Other species previously included within Stygonitocrella were reallocated into Eduardonitocrella Karanovic & Hancock, 2009, Reidnitocrella Karanovic & Hancock, 2009, and Megastygonitocrella Karanovic & Hancock, 2009. If the hypothesis proposed by Psammonitocrella should be considered as monophyletic and closely related to the monotypic genera Inermipes Lee & Huys, 2002, Neonitocrella Lee & Huys, 2002, and Eduardonitocrella (Stygonitocrellaorghidani was relegated to incertae sedis by Eduardonitocrella in the same publication). It is our opinion that P4enp, and one- or two-segmented P2 and P3enp. However, both reductions are known to occur in the Parastenocarididae, and the reduction of the P2–P4enp to a two-segmented structure occurs in or within some interstitial genera of ameirids  and in some genera assigned to the Leptopontiidae Lang, 1948 .According to Parastenocarididae would code in the cladistic analysis of Psammonitocrella—Psammonitocrella, and evidencing their sister-group relationship.In fact, the 250+ species of the Parastenocarididae and Psammonitocrella. In an evolutionary context this is relevant because this evidenced that the Parastenocarididae—one of the largest continental groundwater family of the Harpacticoida—did not evolve from pre-adapted marine interstitial ancestors, but from freshwater interstitial (groundwater) ancestors. This has deep implications for the understanding of the colonization and evolution of groundwater fauna. Because both the Parastenocarididae and Ameiridae had a family rank at that time, placing Psammonitocrella as sister group of the Parastenocarididae unequivocally implies its exclusion from the Ameiridae. Psammonitocrella, but also from including this genus into the Parastenocarididae, waiting for more robust evidence.One important achievement of Psammonitocrella as belonging to the Ameiridae without properly discussing Psammonitocrella into the Ameiridae. However, Psammonitocrella was considered to belong to the Ameiridae without proper phylogenetic analyses.Psammonitocrella cannot be solved without discussing the position of its sister group, Parastenocarididae, nor without including in the discussion the Ameiridae as a whole. Note that also Ameiridae in their discussion. They just defined the Ameiridae as displaying the synapomorphic modification of the basal inner seta of P1 in the males. It is interesting to note that all Ameiridae discussed by Parastenocarididae and Psammonitocrella. Parastenocarididae and Psammonitocrella, rather than a secondary reduction.The simple removal of a genus from one family to another without proper discussion is naive, and it does not solve the real problem. The phylogenetic position of Parastenocarididae and Psammonitocrella allows for only two systematic scenarios.The sister-group relationship between the Parastenocarididae + Psammonitocrella are the sister group of the Ameiridae (or any other family). In this case, a new family should be proposed for Psammonitocrella, and the Parastenocarididae would be composed only by those species with the synapomorphic characters proposed by P3; one-segmented male and female P2-P4enp; absence of outer spine on the P2 and P4exp2 of both male and female, etc.).1) orParastenocarididae + Psammonitocrella is a monophyletic group within the Ameiridae. In this case the family rank of the Parastenocarididae would be compromised. “Parastenocarididae” would be a junior subjective synonym of “Ameiridae”, and the Parastenocarididae + Psammonitocrella would be a derived group within the Ameiridae.2) Parastenocarididae evolved from a lineage of freshwater ameirids that became interstitial in continental waters and colonized aquifers and groundwaters. The analysis offered by Psammonitocrella as phylogenetically related to a group of freshwater interstitial ameirids, but they unfortunately ignored the Parastenocarididae.In an evolutionary context, it would make sense that the We advocate for the consideration of alternative 2 as the most realistic evolutionary working scenario.Psammonitocrella is an Ameiridae, the evidence suggesting its sister-group relationship with the Parastenocarididae—both sharing the loss of the inner seta on the enp1 of the P1—indicates that the Parastenocarididae should be included into the Ameiridae. But the relationships to marine taxa as discussed by Psammonitocrella, Parastenocarididae and Leptopontiidae share the loss of the inner seta of the enp2 of P1 and most importantly, the loss of the outer spine of the exp2 of P1—which is a rare reduction within Harpacticoida , and the mx1 has a reduced distal armature of three spines on the praecoxal arthrite of both taxa. Except for E.mexicana, the distal rim of the anal operculum of the species studied by Parastenocarididae.The family Ameiridae and Parastenocarididae. We believe that only a phylogenetic analysis of the Ameiridae and Parastenocarididae, with a large taxonomic coverage based on both traditional morphological and multi-gene datasets is suitable to undisputedly determine which one of the two hypotheses mentioned above is better supported by the data. Unfortunately, within the Harpacticoida the taxon sampling for molecular work is still in its infancy.Taking this into account, future studies are necessary to unfold the relationship between the Psammonitocrella can be supported by some reductions of the armature of P1 to P4, and the length of the furca. Of remarkable importance is the cylindrical furca which is also longer than the telson (ap), the basis of P1 in male with unmodified inner spine (ap), loss of the outer spine on the P1exp2 (ap), loss of the outer spine of the exp3 of P2 (ap), P3 (ap) and P4 (ap), loss of the inner apical seta (IAS) on the exp3 of P3 (ap) (IAS of the P4exp3 into a spine (ap), reduction of the P4enp to a small knob or its complete loss (ap).The monophyly of  P3 (ap) , transfoPsammonitocrella is composed of P.boultoni and P.longifurcata from Sycamore creek , and P.kumeyaayi sp. nov. from the San Clemente Canyon, San Diego . The new species differs remarkably from P.boultoni, and P.longifurcata in the loss of the outer spine of the P4exp2 (ap), in the presence of a one-segmented P5exp (pl), and in the presence of an outer seta of the basis of P1 (pl) and P2 (pl). The one-segmented exp of the P5 of P.kumeyaayi sp. nov. closely resembles that of P.boultoni. In the former species it is armed with four elements —one very reduced spine on the left limb—, and three setae are present in the exopodal lobe of the P5 of P.boultoni. However, the benp of P.kumeyaayi sp. nov. is armed with two spines, but it is armed with only one spine in P.boultoni. The enp of the P2 and P3 is two-segmented in both P.boultoni and P.kumeyaayi sp. nov., whereas the P2 and P3enp is one-segmented in P.longifurcata. The shape and length of the furca is similar in P.boultoni and P.kumeyaayi sp. nov., and it is much longer in P.longifurcata.Nowadays the genus P2 and P3 enps of P.boultoni and P.kumeyaayi sp. nov. are definitely plesiomorphic within the genus. The one-segmented P5exp and the presence of two spines on the benp of P5 of P.kumeyaayi sp. nov are also considered plesiomorphic. The long furca of P.longifurcata seems to be an apomorphic condition within the genus. The presence of the outer seta on the basis of the P1 and P2 are plesiomorphic for P.kumeyaayi sp. nov. Considering this, it seems possible that P.longifurcata and P.boultoni forms a monophyletic group within Psammonitocrella, sharing the absence of the outer basal seta of P1 and P2, what could be also supported by the fact that P.longifurcata and P.boultoni occur in sympatry. The absence of the outer seta on the basis of P1 and P2 is uncommon within Harpacticoida, whilst the reduction or loss of the enp may have occurred convergently many times. Appendage reductions, character losses, and miniaturization  are common denominators of the ‘darkness syndrome’ of many stygobionts . The new species can be clearly distinguished from its congeners by the absence of the outer spine on P4exp2 and the presence of a one-segmented P5exp.1) A new species of the freshwater ameirid genus P.kumeyaayi sp. nov. is probably the sister group of a monophylum formed by P.boultoni and P.longifurcata. However, without the study of the species described by 2) Psammonitocrella is an Ameiridae, the evidence suggesting its sister-group relationship with the Parastenocarididae indicates that the Parastenocarididae should be included into the Ameiridae.3) If Parastenocarididae and Leptopontiidae within the ameirid-like harpacticoids should be tested.4) Future phylogenetic studies should include both freshwater and marine ameirids, to test the hypothesis that some freshwater taxa are more related to marine ones, and that the invasion of the fresh water by this family followed multiple waves. In addition, the position of the