I was curious what is known about how PARP1 finds it's nicked DNA target? Is it possible that the reduced binding to the nicked substrate is due to a reduction in the efficiency of finding the substrate (i.e. sliding or inter- or intra-strand transfer?).

This is a really elegant biochemical study--congratulations on the work! I was wondering if you could or have confirmed this compaction using an alternate methodology, like sedimentation velocity analytical ultracentrifugation?

Congratulations on the elegant work! I was wondering if you considered treating the cells with glucocorticoid to examine if there is a reversal of alternative splicing and/or expression vs. treatment with IL-beta?

Have you tested the neuronal responses of iPruriceptors to other pruritogens, like serotonin or TSLP? If not, does the expression pattern of differentiated iPruriceptors indicate that the receptors for other known pruritogens are present on iPruriceptors?

I'm curious if you have performed the multielectrode array assay on iPruriceptors in parallel with hDRGs for direct comparison?

Congratulations on developing this wonderful resource for studying itch! I have a few questions. Were the two groups of images being compared (2 left panels vs. 2 right panels) captured using similar microscopy techniques? At first glance, the left panels appear to have been captured using possibly DIC microscopy and the right panels captured using brightfield?

This is a very intriguing idea linking TRPV1-mediated calpain activation to downregulation of TRPV1! While your engineered HEK and CHO cell systems work well, can you perform this assay in more biologically relevant cells, such as DRGs, or cells more closely related to neurons, like keratinocytes, and examine endogenous proteins?

What do you mean by "regular dose"? It might be helpful to think about this in terms relative to the Kd of capsaicin for TRPV1.

Do you have a negative control for this experiment, where TRPV1 is absent from the cells?

Can you be more direct here and use this assay to show calpain is cleaving TRPV1 and that this leads to receptor internalization? For instance, addition of capsaicin should lead to activation of calpain that should cleave TRPV1, according to your theory. If so, after activation, can you detect the cleaved version of TRPV1 being internalized?

Given that mutation of residues that were predicted to be critical for cleavage had no effect, it would be important to confirm that this site is cleaved in vivo. This could rule out that the cleavage you observe in vitro is not an artifact due to incubation with high levels of enzyme or overepxression of substrates.

Wording-"were" may be omitted?

Can you use information on related, parasitic nematodes which can suppress host immune responses to help elucidate conserved biosynthetic pathways that may give rise to molecules that help evade host immune responses?

This is very elegant work--congratulations! I was wondering if you have any data on whether inhibition of TRPV+ neurons is reversible? If you culture the neurons longer and/or provide additional wash steps, can these neurons again respond normally (similar to control) to capsaicin?

Are there canonical RAR/RXR or RAR/RAR binding site in the promoter of engrailed?

Performing an equilibrium binding assay between your wild type and mutant receptors and radiolabeled atRA could discriminate between difference in atRA binding affinity rather than potential differences in the receptors’ ability to bind cofactors, or perhaps bind its heterodimer partner.

If RAR does not bind atRA, how do you explain the similarity here between treatments with atRA and Ro-41-5253?

This is a really intriguing study on the conservation of RAR in guiding developmental patterning across species. Congratulations! I'm curious if cofactors that bind NHRs to activate (coactivators) or repress (corepressors) transcription are conserved in C. gigas? I'm wondering what type of transcriptional responses you anticipate for addition of agonist and antagonist vs. no ligand? Could you see active repression in the absence of ligand, which may complicate your interpretation, especially if, as you suggest later, atRA may not bind RAR in this species.

As you note, there are potential drivers of AD and psoriasis. I know this may be asking a lot, but it would be useful to see how these pathways amplify immune signaling by allowing the user to “initiate” an upstream event and, in a temporal fashion, watch as it propagates through the nodes of the system leading to consequences that reinforce the disease state.

This is great resource to help display and condense the complexity of the etiology and maintenance of AD and psoriatic disease states. I may be missing this in your interactive tool, but it would be helpful to include the publications used to develop each node of the model as a link when selecting any particular node. Given the sometimes conflicting information available for association vs. causal factors in the literature, the links would be helpful to place particular weights on any given node.

What is the phenotype of a DSX knockdown in males and in females? I'm just curious if the larva/animals used to examine gene expression changes in DSX knockdowns are sick.

This is a very interesting study helping to elucidate the role of DSX in N. vitripennis development! Since there are male- and female-specific isoforms of DSX and DSX can act as both a transcriptional activator and repressor in both females and males, is it possible that a gene knockdown (not isoform specific) could obscure the sex-specific roles of DSX? Can you generate DSX knock ins that can only produce female specific isoforms and then examine the outcome on gene expression? (Presumably, a male-specific isoform can not be propagated.)

Is there a complete layilin knockout or other tissue-specific conditional knockouts that provide insight into layilin’s role in other cells/tissues?

Congratulations on the elegant study! I'm curious if there are layilin mutations associated with with chronic inflammation or highly metastatic carcinomas?

Could the increased transcriptional activity be due to enhanced stability of RORbeta? The wording suggests that these are independent outcomes.

Do you have any evidence of RORbeta binding in vivo? It would be interesting to examine RORbeta binding to endogenous RORbeta DNA binding sites to see if acetylation changes RORbeta’s propensity to bind target genes promoters.

Do you know if p300 was bound to the ROR-DNA complex (evidence of supershift with a p300 antibody)? I'm curious if p300 binding can alter RORbeta DNA binding after RORbeta is modified?

This is a very interesting study linking the complex interplay of RORbeta posttranslational modifications to RORbeta function-congratulations! Can you provide a list of these interacting proteins? I'm curious if any known interacting proteins were immunoprecipitated, such as co-repressors and other co-activator proteins?

Can your analysis detect potential changes in alternative splicing that may result from Pin4 binding?

Is there any correlation between the number of motifs in a particular 3'UTR and the likelihood of being bound by Pin4?

How predictive are these motifs for Pin4 binding? Are these motifs enriched on mRNAs bound by Pin4, or are they present but not bound throughout the genome?

Have you explored whether this phosphorylation-dependent structural rearrangement may play a role in the function of other NHR AF1 or F domains?

This is a very insightful study! I'm curious if you can model how this newly formed helical structure binds to AF1 interacting domains present on such proteins as TBP?

Is 10uM near the physiologic concentration for docosahexaenoic acid?

Based on your model, can you postulate different lipid structures or lipid classes that could alter pore function?

This is a very interesting study! Using your model, can you predict other channel-specific amino acids that are important for binding to lipid mediators? If so, it would be interesting to mutate these residues and show they have a similar effect as R63 mutants.

While you're elegant chimera approach suggests lipids are unlikely to have a destabilizing effect, can you rule out an indirect, isoform-specific activating effect?

Do you mean "stably"?

Congratulations on the revealing study! I was curious if you have evidence that the lipids are not generally affecting the biophysical proprieties of the membrane and indirectly activating mAC function?

Could you use ticks that are free from zoonotic pathogens to confirm that the pathogens are not causing the inflammatory response?

Do you have any evidence that longer feeding times promote a more severe allergic-like response on day 10 or 14, or if a longer feeding time may postpone the onset of inflammation?

I'm curious why you choose Ixoden ricinus rather than others species of ticks for this study? From my understanding, a preponderance of reported ticks bites that result in a red meat allergy in humans are caused by bites from Amblyomma americanum. I'm just wondering if there is an advantage to using I. ricinus.

The in vivo data is very compelling! I'm curious if there is potential for FosB KO in other cells types--do you know if Mcpt5 is specific only to mast cells?

This is a very intriguing paper on the role of FosB in regulating mast cell function! I have a few questions. In figure 1J, are the two lanes replicates of extracts from LAD2 cells, or, like in figure 1B, is one lane vehicle and one lane stimulated? If the latter, the bands in the separate lanes do not look appreciably different. Is it that you see expression but no change in expression with stimulation?

LPA signaling has been implicated in promoting mitochondrial homeostasis as well as promoting mitochondrial apoptosis in pathologic conditions. Since STL-1 can bind LPA, I’m wondering if you can comment on how extra and intra-cellular LPA signaling may impact STL-1 function?

It will be interesting to model how pH changes can change STL-1-lipid interactions and generate point mutations to disrupt binding and observe outcomes.

Thanks for posting your great study on linking STL-1 to phosphatidic acid signaling and mtUPR. Can you confirm that tm1544 or other point mutants behave as a true null alleles (e.g. place tm1544 over a larger chromosomal deletion and confirm that tm1544/- phenocopies -/- animals?). Since the hps-6p::GFP reporter behaves differently when crossed with the different stil-1 mutants, is it possible that the mutations are not equivalent and regulate the hsp-6 and hsp-60 reporters differently?

Are there any naturally occurring mutations associated with diseases that map to regions that your model predicts would disrupt interdomain interactions? Or conversely, can you generate mutations that your model predicts would disrupt interactions and measure the effects on transcription use simple reporter assays and full-length constructs?

This is a really interesting study on the complex interplay between NHR domains! I was wondering if you tested interactions between these domains using in vitro protein-protein interaction assays that can sidestep possible complications of the heterologous cell-based Gal4 system (i.e. “pull-down” assays, etc.)?

While epidermal stimulation can broadly mediate SSN activity, how do you envision epidermal stimulation prioritizing nocifensive behavior? Does ensheathment of C4da and C3da neurons by epidermal cells explain the preferential response?

This is an elegant and thorough study mechanistically describing the role of epidermal cells in regulating nociceptive behavior as well as SSN activity more broadly, in Drosophila. Congratulations on your beautiful work! I’m curious why direct optogenetic activation of epidermal cells leads to a similar fraction of larva bending relative to direct activation of nociceptors, versus less than half of larva rolling in response to direct activation of epidermal cells relative to direct activation of nociceptors?

This is a nice proof-of-principle application for TRESK agonists to treat acute and chronic pruritus. It will be interesting to see how long TRESK agonists effects last after initial or repeated treatments--is the effect longer term than 2 hours?

I'm curious if activation of TRESK, though single or repeated injection of cloxyquin, resulted in any phenotypes other than reducing pruritus (concomitant reduction in cold or mechanical sensitivity, or a change in any migraine-like pain-associated behavior.)?

Given the potential role of glucocorticoid receptors in antagonizing NF-kB signaling, is there any evidence of discrete or cryptic GR response elements in the promoters of NF-kB-driven transcriptional modules? I'm curious if different GR agonists could reverse promoter-specific p300-dependent acetylation and the associated neurodegenerative phenotypes.

Are there other attributes of astrocyte health that could be assessed to examine if p300 downregulation is not having detrimental effects?

This is a beautiful study showing that epigenic memory in astrocytes can prime cells towards CNS pathologies. From examination of unregulated genes and DNA binding sites in the promotor of p300 or ACLY, are there any candidate transcription factors that could be responsible for the upregulation by IL-1b and TNF?

If unc-104(e1265) is a partial loss-of-function allele, is there a deletion, either gene-specific or chromosomal, that one can use to generate a true unc-104(Q94L)/unc-104(-) animal to test for a hypomorphic allele?

I’m curious if you examined the expression levels of the mutant KIF1Bbeta protein? i.e. does the Q98L mutation alter protein expression levels?

This is an elegant model to test the function of kinesins conserved across species! I just have a few questions. Is unc-104(e1265) a partial loss-of-function allele – a point mutation that demonstrates reduced PI(4,5)P(2) binding?

A Materials and Methods section may be missing.

I may be missing this, but I don't see a description of how this assay is performed in the figure legend or a materials and methods section in the primary or supplemental text. Could it be that this change in mobility is due to another modification and, if so, could you confirm with mass spectrometry?

This is a really interesting study linking NLRP3 palmitoylation to inflammation. Here, could you use a more sensitive readout for cell cytotoxicity to confirm cell death is not obscuring your results?

Huang, F., et al. 2013 showed that in Slack knockout rats, the knockouts were more sensitive to heat stimuli than control rats at 50oC. Your methods mention a infrared intensity setting of 25. What temperature corresponds to a setting of 25?

I'm curious if the antipruritic activity of compound 6 was long lasting. Did you examine if hours or days after administration of compound 6, the mice continued to show a reduction in scratching vs. control in your chronic itch models?

This is really nice dose-dependent reduction in scratching! I'm curious how you choose the timing for compound 6 administration? And did you try subcutaneous injections of compound 6 as well as systemic administration? I'm wondering if s.c. injections may help reduce potential undesirable off-target effects of compound 6 when compared to systemic administration.

Are there any mutants that phenocopy ok288 that may point to possible candidates in neuronal cells that cue localization of KCC-3?

This is a very thorough and detailed study digging into the mechanism of KCC-3’s apical localization in microdomains on AMsh glia. Beautiful work! I'm curious if you’ve looked at versions of KCC proteins that completely lack the N or C terminal variable regions? Assuming they still fold properly and are inserted in the membrane (maybe a stretch), the localization of these KCC mutants might help support these domain’s roles in targeting KCC apically or basolaterally.

Does this fall into the "other" category? And what alternative types of localization fall into "other"?

Since the balance and transition from repair to homeostasis likely exists to allow sufficient time for healing from injury, I'm curious if the accelerated recovery with overexpression of nAChRbeta3 results in abnormal repair or healing of the gut epithelium or any other abnormalities?

Congratulations!--This is an elegant tour de force investigation into how injured gut epithelium transitions from injury and a state of repair back toward homeostasis. You’ve clearly shown that ECs downregulate Ace and upregulate nAChRbeta3 to become more receptive to ACh. I’m curious if you have any ideas about what signals ECs to downregulate Ace and upregulate nAChRbeta3?

This is a beautiful study describing the regulation of the FLC locus. Do you believe APRF1, TOPP4, and LD are specific regulators of only FLC? Or do they play a more general role in regulating repression/activation at many loci? If so, do you have any genome-wide information on how APRF1, TOPP4, and LD mutants effect other genes, particularly those involved in sensing environment cues, like you've examined with flowering? And, if their role is not restricted to regulating FLC, what other phenotypes do you observe with these mutants--do the phenotypes give you insights into other processes they may regulate?

It will be exciting to see if these putative subunits possess phosphatase activity, in vitro, and further dissect how this phosphatase activity might be regulated.

The evidence that APRF1 is regulating flc expression is strong; however, given APRF1's likely role in regulating a wide range of genes, do you have evidence that APRF1 is directly associating with the flc locus to regulate expression and that the regulation is not indirect?

This is a great tool to help explore and exploit cell-cell specific contacts! I'm curious how providing lower levels of the the receptor fusions by stable integration, rather than transient transfection, would effect the reporter readout? Future studies using transgenic organisms that introduce constructs through single copy integration, would benefit greatly from your tool.

In this case, is it possible that TL-betaCat is limiting?

Can you quantify what percentage of SNAI1 was released from the plasma membrane upon photo stimulation? I'm curious if transcription/translation of TL-SNAI1 is rapid enough to ensure a pool of plasma bound SNAI1 that can lead a source of continual transcriptional activation upon constant photostimulation?

Your TANGO-light system is a really intriguing adaptation of TANGO and optogenetics! I'm curious in this case, how long did you have to stimulate cells with a 0.05s/5s pulse?

In general, NHR recruitment of coactivators or corepressors is regulated by the position of the terminal helix 12 of the ligand binding domain. Helix 12 is necessary for the proper confirmation of NHR to recruit coactivators, while it is dispensable for recruitment of corepressors. In fact, amputation of NHRs which remove helix 12 convert the receptors into constitutive repressors. While a corepressor for nhr-49 has not been identified, it may be revealing to generate an nhr-49 allele that lacks helix 12. It would be unlikely to bind MDT-15 and may help dissect mdt-15 roles in regulating hsf-1 function, as well the cement the role of mdt-15 in nhr-49 mediated regulation of proteotoxic stress.

I appreciate the clarity in Figure 5G, but it may be helpful to include all of the genes/proteins involved in this pathway that were mentioned above, to aid in interpreting your conclusions.

It appears that there is a significant difference between the percent survival of wild type worms + control RNAi (Fig 3F) and wild type worms (Fig 3G) as well as cdb-1 + control RNAi (Fig 3F) and cdb-1 (Fig 3G). Is this difference due to the different bacteria used for dsRNA expression and normal growth, for instance, HT115 vs. OP50, or some other variation?

This is an elegant study implicating nhr-49 as an important link tying lipid metabolism to proteotoxic stress. I only had a few questions. Since mediator is a shared coactivator used by numerous transcription factors, could the difference in suppression be due to med-15 targets that are not regulated by nhr-49? And do you know if mdt-15 RNAi recapitulates a null allele of mdt-15?

A key way PRMTs function is through recruitment to promoters by transcription factors. With your RNAseq data, are there any expression patterns that may help elucidate the primary target promoters regulated by MS023, and therefore the key transcription factor or factors that are targeting the PRMTs? One of better characterized class of transcription factors that can recruit PRMT cofactors are nuclear hormone receptors (NHRs) and NHRs have been shown to be important regulators of stem cell differentiation. If you can identify transcription factor targeting PRMTs, and it is an NHR, use of NHR agonists or antagonists may reinforce the application of MS023 and further increase muscle stem cells’ potential to expand, in vitro.

This is a thorough study demonstrating the utility of the type I PRMT inhibitor, MS023, in allowing muscle stem cells to expand, in vitro. To convincingly show that PRMT1 is the key target mediator of MS023, is there a means to directly target PRMT1 for degradation or downregulation, such as tagging it with an auxin-inducible degron (transgenic mice)?

Antibodies made against asymmetric di-methyl-arginine may cross react and diminish actual changes in PIWI protein expression levels. Are there antibodies made against PIWI proteins in related species that could provide a more specific measure of PIWI protein expression levels in ISE6 cells, revealing what may be a more dramatic change in expression?

This is an intriguing finding. The number of genes whose expression changes seems small relative to the important and diverse biology processes that are predicted to be regulated by RpRPs and AGOs. When related genes are knocked down in other species, how many genes show significant changes in expression? In addition, is the phenotype of the RpRPs and AGOs knock downs in Ixodes scapularis known?

If available, comparison of sRNA conservation across multiple tick species would bolster the idea that these sRNAs are deeply conserved. It may also reveal interesting species-specific biological differences.

Has targeting of CRISPR-Csm to 5’ or 3’ UTRs been performed? Could this provide different levels of knock-down and further add your ability to tune RNA levels (in addition to altering spacer length)?

Being able to directly target complexes to the native RNA sequences is a significant benefit over the potentially disruptive insertion of RNA aptamer sequences into RNAs to visualize RNA dynamics; however, can binding of multiple CRISPR-Csm complexes to native RNAs disrupt function? Do you have any evidence that CRISPR-Csm complex binding does not disrupt RNA metabolism of ncRNAs or protein coding RNAs or expression of corresponding proteins? Additionally, do you have any evidence that you have the sensitivity to detect single RNA molecules (comparison to smFISH)?

TARDIS is a significant step toward generating library scale insertions in animals. I was curious about a few points. Is this observed lethality due to off-target, collateral damage of Cas9 induction? (i.e. In worms that lack arrays and that are not under selective pressure, is there any significant lethality after heat shock?)

As a means to improve efficiency of homology directed repair using large or difficult to insert templates, have you tried or considered using two Cas9 targeting sequences to promote the insertion of a repair template or library at a single landing site (the repair template flanked by two Cas9 targeting sequences)?

Do these patterns reflect the phenotypes of inserted fluorophore sequences at the endogenous genomic loci, if known? I’m curious how much variation may be due to the expression at this landing site.

This elegant study provides a rich data set and a pathway to explore sex-specific metabolomic data in C. elegans and its involvement in a myriad of biological processes.

Given the large expansion of the nuclear hormone receptor (NHR) family in C. elegans, it will be very informative to explore lipid fractions for potential NHR ligands that likely regulate homeostasis, metabolism and reproduction.

The decision to choose either male or hermaphroditic fate occurs very early in development, at around the 20 to 100 cell stage. It would be intriguing to examine if sex specific differences in metabolites is evident in the early embryonic stages of development, too. It may provide insights into how cell fate decisions are made and maintained.

There is no Figure 3H present in the publication. Possibly a typo?

Since substance P precursor levels are substantially elevated in skin after surgical incision (Fig 6A), and substance P has been shown to cause degranulation, releasing a number of factors including histamine, can you postulate why you don't see elevated levels of histamine at the site of incision (Fig 3B)?

You elegantly show that neuronally derived BH4 contributes to the increase in BH4 levels after injury but is not involved in pain hypersensitivity. If BH4 can directly activate nociceptors, it seems like you would have noticed a decrease in pain hypersensitivity when you reduced BH4 levels. Do you think there is something chemically unique about mast cell-derived BH4 vs. neurally-derived BH4 that would allow direct activation of nociceptors?

This is a very intriguing finding-extracellular release of the enzymatic machinery necessary for BH4 and serotonin synthesis could allow signal amplification after degranulation. Is there any evidence of extracellular BH4 synthesis in other contexts from other studies? Or could these enzymes be remnants of the synthesis of serotonin within the granules?

This is s great proof-of-concept example that Cas9-mediated nonhomologous recombination can be a platform in C. elegans to aid in genetically mapping variants.

Numerous studies in C. elegans using co-conversion have achieved a similar efficiency of imprecise repair at a Cas9-induced DSB as you achieved for isolating LOH offspring, which is remarkable. Do you have a sense of the cleavage and repair efficiency of Cas9-induced DSBs you've generated-not just LOH animals?

For comparison, from other studies, do you have a sense of the natural recombination efficiency of neighboring loci in this region of chromosome V?

It would satisfying to see this pattern of repair generalize across different regions of different chromosomes. There may be positional effects that influence the rate of Cas9-mediated recombination.

It would interesting to examine if one can recover non-homologous recombination events at a reasonable frequency when targeting highly repetitive regions in the genome that may be prone to favor microhomology-mediated repair from sequences in cis.

It would interesting to examine if one can recover non-homologous recombination events at a reasonable frequency when targeting highly repetitive regions in the genome that may be prone to favor microhomology-mediated repair from sequences in cis.

It would satisfying to see this pattern of repair generalize across different regions of different chromosomes. There may be positional effects that influence the rate of Cas9-mediated recombination.

For comparison, from other studies, do you have a sense of the natural recombination efficiency of neighboring loci in this region of chromosome V?

This is s great proof-of-concept example that Cas9-mediated nonhomologous recombination can be a platform in C. elegans to aid in genetically mapping variants.

Numerous studies in C. elegans using co-conversion have achieved a similar efficiency of imprecise repair at a Cas9-induced DSB as you achieved for isolating LOH offspring, which is remarkable. Do you have a sense of the cleavage and repair efficiency of Cas9-induced DSBs you've generated-not just LOH animals?

This is a very intriguing finding-extracellular release of the enzymatic machinery necessary for BH4 and serotonin synthesis could allow signal amplification after degranulation. Is there any evidence of extracellular BH4 synthesis in other contexts from other studies? Or could these enzymes be remnants of the synthesis of serotonin within the granules?

There is no Figure 3H present in the publication. Possibly a typo?

You elegantly show that neuronally derived BH4 contributes to the increase in BH4 levels after injury but is not involved in pain hypersensitivity. If BH4 can directly activate nociceptors, it seems like you would have noticed a decrease in pain hypersensitivity when you reduced BH4 levels. Do you think there is something chemically unique about mast cell-derived BH4 vs. neurally-derived BH4 that would allow direct activation of nociceptors?

Since substance P precursor levels are substantially elevated in skin after surgical incision (Fig 6A), and substance P has been shown to cause degranulation, releasing a number of factors including histamine, can you postulate why you don't see elevated levels of histamine at the site of incision (Fig 3B)?

Given the large expansion of the nuclear hormone receptor (NHR) family in C. elegans, it will be very informative to explore lipid fractions for potential NHR ligands that likely regulate homeostasis, metabolism and reproduction.

The decision to choose either male or hermaphroditic fate occurs very early in development, at around the 20 to 100 cell stage. It would be intriguing to examine if sex specific differences in metabolites is evident in the early embryonic stages of development, too. It may provide insights into how cell fate decisions are made and maintained.

This elegant study provides a rich data set and a pathway to explore sex-specific metabolomic data in C. elegans and its involvement in a myriad of biological processes.

As a means to improve efficiency of homology directed repair using large or difficult to insert templates, have you tried or considered using two Cas9 targeting sequences to promote the insertion of a repair template or library at a single landing site (the repair template flanked by two Cas9 targeting sequences)?

Do these patterns reflect the phenotypes of inserted fluorophore sequences at the endogenous genomic loci, if known? I’m curious how much variation may be due to the expression at this landing site.

TARDIS is a significant step toward generating library scale insertions in animals. I was curious about a few points. Is this observed lethality due to off-target, collateral damage of Cas9 induction? (i.e. In worms that lack arrays and that are not under selective pressure, is there any significant lethality after heat shock?)

This is an intriguing finding. The number of genes whose expression changes seems small relative to the important and diverse biology processes that are predicted to be regulated by RpRPs and AGOs. When related genes are knocked down in other species, how many genes show significant changes in expression? In addition, is the phenotype of the RpRPs and AGOs knock downs in Ixodes scapularis known?

If available, comparison of sRNA conservation across multiple tick species would bolster the idea that these sRNAs are deeply conserved. It may also reveal interesting species-specific biological differences.

Antibodies made against asymmetric di-methyl-arginine may cross react and diminish actual changes in PIWI protein expression levels. Are there antibodies made against PIWI proteins in related species that could provide a more specific measure of PIWI protein expression levels in ISE6 cells, revealing what may be a more dramatic change in expression?

Has targeting of CRISPR-Csm to 5’ or 3’ UTRs been performed? Could this provide different levels of knock-down and further add your ability to tune RNA levels (in addition to altering spacer length)?

Being able to directly target complexes to the native RNA sequences is a significant benefit over the potentially disruptive insertion of RNA aptamer sequences into RNAs to visualize RNA dynamics; however, can binding of multiple CRISPR-Csm complexes to native RNAs disrupt function? Do you have any evidence that CRISPR-Csm complex binding does not disrupt RNA metabolism of ncRNAs or protein coding RNAs or expression of corresponding proteins? Additionally, do you have any evidence that you have the sensitivity to detect single RNA molecules (comparison to smFISH)?