Genome–wide association studies (GWAS) have revealed a plethora of putative susceptibility genes for Alzheimer’s disease (AD), with the sole exception of APOE gene unequivocally validated in independent study. Considering that the etiology of complex diseases like AD could depend on functional multiple genes interaction network, here we proposed an alternative GWAS analysis strategy based on (i) multivariate methods and on a (ii) telescope approach, in order to guarantee the identification of correlated variables, and reveal their connections at three biological connected levels. Specifically as multivariate methods, we employed two machine learning algorithms and a genetic association test and we considered SNPs, Genes and Pathways features in the analysis of two public GWAS dataset (ADNI-1 and ADNI-2). For each dataset and for each feature we addressed two binary classifications tasks: cases vs. controls and the low vs. high risk of developing AD considering the allelic status of APOEe4. This complex strategy allowed the identification of SNPs, genes and pathways lists statistically robust and meaningful from the biological viewpoint. Among the results, we confirm the involvement of TOMM40 gene in AD and we propose GRM7 as a novel gene significantly associated with AD. APP, PS1 and PS2 mainly involved; a late-onset form (LOAD) that affects the vast majority of the patients in the elderly over 65s, whose causes remain still unknown1. Although LOAD has been defined as a multifactorial disease and its inheritance pattern has not been clarify yet, it is coming out the idea that it could be likely caused by multiple low penetrance genetic variants2, with a genetic predisposition for the patients and their relatives estimated of nearly 60–80%2.Alzheimer’s disease (AD) is the predominant form of dementia (50–75%) in the elderly population. Two forms of AD are known: an early-onset (EOAD) that affects the 2–10% of the patients and is inherited in an autosomal dominant way, with three genes APOE3. It encodes three known isoforms proteins , with APOE4 known to increase risk in familial and sporadic EOAD. This risk is estimated to be threefold and 15-fold for heterozygous and homozygous carriers respectively, with a dose-dependent effect on onset age2.The first well known gene associated to LOAD was 1. Anyways, none of the new identified loci reached the magnitude of APOEe4, as predisposing risk factor for AD, with the majority of the hereditable component of AD remaining unexplained4. Several different but not mutually exclusive explanations of such failure could coexist: AD could be caused by the concerted action of independent genetic factors, each having a small effect size that require to adopt multivariate methods and to increase sample size5; or it could be caused by the concerted actions of multiple genes (again characterized by low effect size) that act inter-dependently in still undefined pathways, that would need a pathway-based approach, as done for other complex diseases6. Alternatively, AD could be caused by vary rare but highly penetrant mutations that might be identified through DNA sequencing7.Large-scale collaborative GWAS and the International Genomics of Alzheimer’s Project have significantly advanced the knowledge regarding the genetics of LOADIn order to explore the first two possible scenarios, in this study we proposed an alternative GWAS analysis strategy based on (i) multivariate methods and on (ii) a SNPs-Genes-Pathways ensamble, in order to guarantee the identification of correlated variables, and reveal the possible connections existing among the identified relevant variables at different, but biologically connected levels.FS, in the genes analysis we used an association genetic test named SKAT and in the pathways analysis we considered Group Lasso with overlap. All these methods share the multivariate aspect, because they consider more features simultaneously  differently from the univariate methods, such as the t-test, that evaluate the statistical association of each single feature at the time. The final purpose is to identify lists or signatures of possible causal SNPs, genes and pathways that considered together might provide a convincing picture of heritable factors in the LOAD pathogenesis.Figure APOEe4, while in the discussion we comment the obtained results considering the possible integration of the signatures across the SNPs, genes and pathways levels and also across ADNI-1 and ADNI-2 dataset that we analyzed separately and considering only the genotype SNPs.In the Results section we show the signatures of SNPs, genes and pathways identified considering both the binary classification tasks, cases@controls and CDKAL1 is known to be associated to AD based on the literature8.The SNPs analysis performed on unimputed ADNI-1 dataset Table  identifiAPOE polymorphic alleles are the main genetic determinants of AD risk, being the individuals carrying one or two e4 alleles at higher risk to develop AD3. Considering that APOEe4 polymorphism was harbored in 120 AD of 179 and 58 Control of 214, a further analysis based on the binary classification 1 or 2 APOEe4 vs 0 APOEe4 presence (APOEe4 task) was performed in order to characterize a polygenic profile that could uncover small effect size gene variants associated with the disease in a cumulative manner. 39 SNPs, which map to 47 genes or intergenic regions, have been identified in the APOEe4 task  repairing mechanisms9. This gene is the closest to different SNPs found discriminant in the two tasks: in cases@controls task rs6053572 is located in the intergenic region between GPCPD1 and SHLD1 genes while in the APOEe4 task rs236137 and rs1287032 are located in the intergenic region between SHLD1 and CHGB  had in common GB Table .SNP-based analysis on unimputed ADNI-2 dataset Table  identifiLOC101928063, rs383133 in ZNF221, rs415499 in ZNF155 and rs365745 that causes a missense mutation in ZNF221 gene. Interestingly, LOC101928063 and its rs367209 SNP, was found statistically relevant also considering ADNI-2-cases@controls task. Although none of these genes are already known to be associated to AD, according to the AlzGene database 10 , these SNPs are located in a linkage region  known to be associated to AD.When we considered the APOEe4 task, only chromosome 19 was found statistically significant, with very high values of both balanced accuracy (0.94) and MCC (0.90) Fig.  and withGRM7, encoded for metabotropic glutamate receptor 7. ADNI-2 cases@controls task identified the intergenic SNP rs266410 between MRPS35P and GRM7-AS3 (the antisense version of GRM7), while ADNI-1 APOEe4 task the SNP rs9311976, located in an intron inside GRM7.Considering SNP signatures of ADNI-1 and ADNI-2, we identified as a common gene TOMM40, with rs2075650, was found significantly associated to AD, applying all the tests , applying a very conservative threshold for selecting a list of genes and SNPs highly relevant for AD (see Supplementary Information). In ADNI-1 dataset, TOMM40, the intergenic region between LOC100129500 and APOC1, and the intergenic region between TOMM40 and APOE , whose relevance in neurodegenerative processes were well recognized 13. With the ADNI-1 cases@controls task, no groups reaching statistical significance were found. At variance, different pathway groups, associated with AD risk (APOEe4 task), achieved a good test score .In addition, we also performed a functional characterization in KEGG database (see Supplementary Information), in order to further biologically characterize the gene lists derived from the SNPs signatures identified before. A successful analysis was obtained only for ADNI-1 APOEe4 task and ADNI-2 cases@controls task, whose SNP signatures reported a long SNPs’ list Table . In ADNIGRIN2A, GRM7, GABRG3 and CYSLTR2 genes.In ADNI-2 the most important pathways related to AD were “Chemokine signaling”, “Calcium Signaling”, “Axon Guidance” and “Neuroactive ligand-receptor interaction” Table . The latAPOE gene is the sole exception unequivocally validated in independent studies.Despite the promise of GWAS to reveal the genetic contribution to AD susceptibility, the majority of its heritable component remains unexplained. The major factor contributing to hamper the identification of genetic burden lies in the complexity of GWASs data management, together with the genetic heterogeneity of AD. In fact, although GWAS studies have revealed a plethora of putative susceptibility genes for AD, The final purpose of the alternative strategy that we present in this study is to contribute in uncovering a robust heritable AD signature in the analysis of GWAS data. The key points of this strategy are the following: a new representation of the genotyped SNPs data; the telescope approach, since the data were analyzed at SNPs, genes and pathways levels; the choice of multivariate machine learning methods ad hoc for the three levels; the analysis of two separate dataset, each addressing two relevant binary classification tasks: cases@controls and APOEe4.1l2FS was used to analyzed SNPs data because it is a sparse method, meaning that the solution to the classification problem is searched among a precise selection of the most relevant SNPs. This feature is essential to discard all those SNPs that are background noise or that are weakly associated to the addressed classification problems. The precise choice of the algorithm together with the SNP data transformation improved the selection of the most relevant SNPs from both the statistical and biological viewpoints. For the analysis of the gene level we select SKAT method because it provides the user the possibility to weight the SNPs differently based on their frequency occurrence in the subjects of the SNP dataset. Group Lasso with overlap was chosen because it is characterized by a feature that we seek in the analysis of the pathways: while looking for the most discriminant pathways within a group of them, which in this work constitute a single SNPs data matrix, we wanted the algorithm to consider the involvement of a gene in more then one pathway of the group. Finally the motivation behind the choice of addressing two classification tasks and the separate analysis of ADNI-1 and ADNI-2 dataset is to compare the results, between different biological questions and between two independent studies respectively. Besides the cases@controls classification task, where the disease is the discriminant, we considered also the APOEe4 task because studies have shown that individuals with two copies of the e4 allele are at even greater AD risk, and the odds ratios for developing AD based on APOE is 5 times greater in APOEe4 homozygotes compared to heterozygotes15. Therefore, a binary classification based on the presence of almost one APOEe4 allele could (i) uncover a cumulative polymorphic risk variants contributing to AD predisposition, and/or (ii) highlight superimposable genetic fingerprint, allowing a better understanding of APOE genotype contribution in the disease etiology. In addition, this classification might give useful insights for better addressing the therapeutic strategies, since multiple studies over the past two decades have demonstrated that APOE variants may affect the therapeutic response to anti-dementia drugs19. In this context, very recently, Berkowitz et al. 15 claimed that in the prospective of clinical precision strategy, the APOEe4 carrier status could have a very important impact on AD prevention interventions.The new data representation have improved the classification performances of the applied machine learning algorithms because (i) it changed the nature of the data , improving their interpretability for the considered machine learning methods and (ii) it made a more evident and biologically sound prioritization of some SNPs with respects to others. The analysis at SNPs, genes and pathway levels allowed the comparison of the results at these three levels and the successive identification of common related features, increasing their robustness in the association with AD. We chose the most appropriate machine learning method based on the characteristics of the analyzed data: lConsidering the single signatures and datasets, in this study we identified lists of SNPs and genes, some of which are already reported in literature  the different Illumina GWAS platforms, (ii), the lack of imputing procedure before the independent analysis of the two dataset (iii) and the difference in the genotype of APOE gene between ADNI-1 and ADNI-2 datasets. ADNI-1 and ADNI-2 datasets measured 620,901 and 730,523 SNPs respectively, of which only 300,000 were in common. An imputation procedure of ADNI-1 and ADNI-2 could have increased the SNPs overlap between the dataset and therefore could have increased the number of relevant SNPs genes and pathways and in turn allowed the validation of all the SNPs signatures identified in ADNI-1 and ADNI-2.APOE gene was genotyped separately from the other genes present in the platform, differently from ADNI-2, had surely influenced the obtained results in the SNPs and genes analyses.In addition, the fact that in ADNI-1 dataset TOMM40. On the other hand, the SNP and the pathway analysis of both ADNI-1 and ADNI-2 uncovered GRM7 gene as significantly associated with AD or AD susceptibility genetic profile.The only heritable susceptible gene confirmed across SNPs and genes in ADNI-1 and across pathways in both datasets was TOMM40 is located in 19q13.32 locus, a known linkage region for AD10. Its encoded protein plays a key role in the mitochondria functionality being essential for import of protein precursors into mitochondria. The SNPs-based analysis identified two SNPs rs2075650 and rs8106922 harbored on TOMM40 gene, for ADNI-1 APOEe4 task. At the same time the SNP rs2075650 located in one intron of TOMM40, has been identified by the genes analysis of both controls@cases and APOEe4 task of ADNI-1 dataset. The literature confirms that TOMM40 gene is deeply involved in AD pathology22, and in particular that rs2075650 SNP is already known to be a contributing factor for AD .TOMM40 was also confirmed by pathway analysis in both dataset for the APOEe4 task. In particular, the protein encoded by TOMM40 is involved in the “mitochondrial protein import” pathway. The fact that SNPs, genes and pathways analyses highlighted the strong association between TOMM40 variants and APOEe4 genotype is due to TOMM40 location in the tight gene cluster TOMM40-APOE-APOC1-APOC4-APOC2 that is a strong linkage disequilibrium (LD) block 25. Furthermore, it has been reported that the APOE-TOMM40 genomic region has been associated with cognitive aging26 and with pathological cognitive decline27. APOE-TOMM40 genotypes have been also shown to modify disease risk and age at onset of symptoms29.TOMM40 did not emerge, probably due to the lack of datasets imputation. On the other hand, in ADNI-2 APOEe4 task, TOMM40 appeared in the pathway “mitochondrial protein import”.Interestingly, although ADNI-2 showed a long list of susceptible genes in SNPs analysis, GRM7 represents a novel possible candidate gene that needs to be experimentally validated, for the association with AD. It is located in 3p26.1 locus, and encodes for the metabotropic glutamate receptors 7, involved in the presynaptic neurotransmitter regulation30. GRM7 was identified in the SNPs and pathways analyses of both ADNI-1 and ADNI-2 dataset. In particular, the SNP rs9311976 on GRM7 gene was found in ADNI-1 APOEe4 task, while rs266410 in the inter region MRPS35P1/GRM7-AS3 (the antisense of GRM7) of ADNI-2 controls@cases task. GRM7 was also identified in “GPCR ligand binding” pathway, belonging to the REACTOME group 9a in the APOEe4 tasks of both datasets , the Health and Retirement Study (HRS), and the Indiana Memory and Aging Study (IMAS)39. In addition, GRM7 has been confirmed associated with AD in a meta-analysis of GWAS studies where glutamate signaling genes were found overrepresented in KEGG pathway enrichment analysis40.TOMM40 and GRM7 polymorphic variants as strongly associated with AD. Their relevance was confirmed by the identification of the mitochondrial import and glutamatergic signaling pathways, identified by pathway analysis, in which TOMM40 and GRM7 are respectively involved. Furthermore, the fact that these genes were found strongly associated with APOEe4 status at the SNPs, genes and pathway levels, corroborated its significance in the context of a cumulative polygenetic susceptibility to AD.In conclusion, the alternative GWAS analysis strategy applied in the analysis of two unimputed ADNI datasets, identified Although a possible limitation of this work could be found in the preprocessing phase and specifically in the absence of the imputation step, that could had improved the overlap of ANDI-1 and ADNI-2 dataset and the validation of the identified SNPs signatures, we strongly believe that this alternative approach of GWAS analysis presented in this study could provide a valuable way to uncover the genetic hereditability of multifactorial diseases like AD. In the future we plan to (i) test our approach applying the imputation step before the reanalysis of ADNI-1 and ADNI-2; (ii) to validate the results obtain in ADNI-1 in ADNI-2 and viceversa, following the strategy adopted in this work; (iii) to validate the identified SNPs, Genes and Pathways signatures in other independent GWAS dataset; (iv) to integrate covariates data, such as clinical characteristics of the patients to the analysis of the most discriminant SNPs, Genes and Pathways identified before.https://adni.loni.usc.edu/). The primary goal of ADNI has been to test whether serial magnetic resonance imaging (MRI), positron emission tomography (PET), other biological markers, and clinical and neuropsychological assessment can be combined to measure the progression of mild cognitive impairment (MCI) and early AD. In this study GWAS data and APOE genotype obtained in the ADNI-1 and ADNI-2 datasets 41 were used  and subjects at higher risk vs. subjects not at risk of developing AD, according with APOE status  (APOEe4 task) (Table Data used in the preparation of this article were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database  Table . Since tAn alternative approach was devised to analyze the datasets. Considering the two classification tasks addressed and the SNPs, Genes and Pathways levels, each ADNI dataset was analyzed six times Fig. ., l1l2FS and Group Lasso with overlap), designed to identify the SNPs or pathways which are most discriminative for the classification tasks while restricting the selection of SNPs and pathways, and we considered a different representation of the SNP data (see Supplementary Information); (2) for the SNP analysis we analyzed each chromosome separately while for the gene and pathway analyses we grouped the SNPs considering genes/intergenic regions and pathways relevant for AD respectively.In order to increase the signal over noise ratio, reducing the number of SNPs to analyze, we adopted the following strategy: (1) for the SNP and pathway analyses we employed two sparse methods  while making feature selection (see Supplementary Information). l1l2FS was used within PALLADIO43 , a machine learning framework that can be customized to consider various combinations of feature selections and classification methods : Burden, SKAT and SKATO (see Supplementary Information). SKAT45 is a supervised regression method that test the association between genetic variants in a region and a dichotomous or a continuous trait while adjusting for covariates. The dichotomous traits considered here were cases@controls and APOEe4 tasks. In the first application of this alternative GWAS analysis strategy we chose to exclude covariates such as age at onset, race, sex. Furthermore we chose to consider genes or intergenic regions leveraging on the mapping files SNPs-to-genes provided by the GWAS platform manufacturer .For the gene analysis, we considered three association tests available in the SKAT package (49 (see Supplementary Information).The threshold of genome-wide significance we established, was conservative and in accordance with other studies50(https://reactome.org/).We selected 9 groups of pathways more relevant for neurodegenerative processes Tables  and S5 iIn this study the pathways we selected and analyzed are the same for both dataset but some groups can differs in the number of pathways between the datasets because there is a low overlap of analyzed SNPs between the used platforms of ADNI-1 and ADNI-2 dataset.42. This latter is a machine learning method, able to consider the presence of overlapping groups of SNPs mapped to genes, involved in more than one pathway inside a group. The goal of “Group Lasso with overlap” is to induce a “sparse” selection at the group level, using all the pathways specified in the group. In this way, starting from a possibly long list of pathways inside a group, the algorithm selected a few (but informative) pathways that could be relevant for the problem at hand.Each group contained two or more pathways and each group represented a SNP matrix that, together with a label that characterizes each subject, was given as input to “Group Lasso with overlap”Supplementary Information.


Background/AimsCoronavirus Disease 2019 (COVID-19) has presented an unprecedented challenge for non-COVID related clinical trials of investigational medicinal medicines (CTIMPs). These challenges are considerable for trials run in high -risk groups, such as older adults. Clinical trials must ensure the safety of their participants, whilst also considering the potential, and often long-term, benefits of the trial intervention to public health. Here we sought to provide a brief perspective on the response and conduct of CTIMPs relevant to older adults and neurology in the UK to the COVID-19 pandemic.MethodsWe performed a cross-sectional study, surveying CTIMP teams running trials broadly relevant to older adults and neurology in the UK, as well as sponsors and Clinical Trials Units (CTU), to understand the response and preparedness to the pandemic.ResultsDue to the pandemic, active recruitment has been suspended in more than half of the trials. The primary driver for the temporary halt of recruitment activity was considerations of patient safety. Interestingly, the majority of trials, sponsors and CTUs did not consider pandemic or epidemic outbreaks in their risk assessments before January 2020.ConclusionThese findings support the need to re-evaluate the risk-management approach whereby clinical trials establish contingency plans for predicted but rare events to minimise the disruption to recruitment and clinical trial delivery. Clinical trials have faced an unprecedented challenge in the light of COVID-19. On March 11, 2020, the World Health Organisation (WHO) declared a pandemic which unequivocally complicated the conduct of clinical trials. Importantly, urgent strategies were required to ensure the safety of enrolled participants, weighing up the potential risks to participants of acquiring COVID-19 versus the often long-term, benefits of the trial intervention to public health [In the United Kingdom, the Health Research Authorities (HRA) published a response to the pandemic advising clinical trials to evaluate the risk-benefit of their trial in the broader context of the impact of COVID-19 on National Health Service (NHS) staffing, restriction of movement and government advice . The NatGuidance issued by the major UK non-commercial funding bodies stated that funding for trials in progress would not be affected by the temporary halt of the trial due to COVID-19, in the short term, and/or that the continuation of funding and no-cost extensions will be assessed on a case-by-case basis -7. We reThis study aimed to determine the response to the COVID-19 pandemic at; a) clinical trial management level and, b) an institutional level. We sought to determine the degree to which the pandemic impacted trial activities. As well, we set out to understand the significant drivers behind decisions affecting the running of CTIMP trials and the pandemic preparedness in terms of pre-existing risk assessments.This is a retrospective, cross-sectional survey of clinical trials taking place in the UK. Ethics approval was received from the University of Bristol on the 5th May 2020 (reference no- 104302).The European Union Drug Regulating Authorities Clinical Trials (EudraCT) Clinical Trial Register database was searched on May 8, 2020 using advanced search terms defined as studies in the UK (Country), Elderly (Age range), Ongoing, Prematurely ended, Restarted or Temporarily halted  and Phase I, II and II . Gender selection was not specified. The study included trials that opened between March 1, 2015, and March 16, 2020. The search yielded a total of 2034 trials.The results were screened based on the patient population for conditions primarily affecting or relevant to older adults or neurology. The following conditions and studies were excluded: alcohol and substance abuse-induced disorders of the liver and lungs, anaesthesia, asthma, cystic fibrosis, dermatology, endocrinology including type 2 diabetes, gastrointestinal and metabolic disorders, haematology, infectious diseases, ITU admission, oncology, post-surgical interventions, psychiatric disorders that were not secondary to a known neurological condition, rare genetic disorders, rheumatology, trauma and transplant studies.A total of 265 CTIMPs met the inclusion criteria and were emailed an invite to take part in the study. Emails were obtained from publicly available sources, including trial registries and websites. The person responding on behalf of the CTIMP completed electronic consent before participating. CTIMPs were surveyed on their response to COVID-19 concerning recruitment, IMP management and follow-up activity. We explicitly enquired as to whether the CTIMP risk assessment referenced pandemic management before the pandemic (i.e. before January 2020).The sponsor organisations of the 265 CTIMPs were identified via EudraCT and invited to take part using contact details via publicly available sources such as trial registries. This survey included a total of 149 sponsor invitations (given that some sponsors provide support to more than one study). Also, 46 clinical trial units (CTUs) who are fully registered with the UK Clinical Trials Research Network (UKCTRN) were identified via the UKCTRN website. Of these, seven CTUs specialised in areas beyond the scope of the trial criterion (e.g. paediatrics or cancer research) and were excluded. A total of 39 CTUs were invited to take part in the survey.Sponsors and CTUs were surveyed on the advice provided to trials concerning recruitment during the COVID-19 pandemic as well as preparedness in terms of crisis management plans or risk assessment considering the effect of a pandemic on trial activity. We also collected data on the number of CTIMPs supported by the sponsors and CTUs, excluding COVID-19.The results are presented as percentages and descriptive statistics.The highest response rate was received from CTUs (46% response rate), compared to the response rates from trials (12% response rate) and sponsors (7% response rate) which were markedly lower. This is illustrated in Figure Trial characteristicsOf the 265 invited CTIMPs, 34 trials took part in the survey. The majority of the responses pertained to trials in the fields of Neurology or Psychiatry (44%) followed by Cardiology (41%) with the remaining trials falling under respiratory, ophthalmology or trauma in older adults. 5 trials were single-centre trials, and 29 trials were multicentre. 14 trials were international with sites in the UK, whilst 20 were UK based.On the 16th of March 2020, when the UK Government announced the ‘Stay Home’ directive, of the 34 trials, 3 were in set-up, 16 trials were actively recruiting, 2 trials were suspended, 12 had completed recruitment, 1 trial was stopped permanently.Overall, 24/34 (71%) trial teams had considered alternative ways to deliver the trial in order to reduce the risk to participants and/or healthcare staff. Of these 15/34 trials (44%) were able to implement changes to deliver the trial during COVID-19. Seven trials (21%) reported that they did consider this but did not implement the changes because the institution, funder or other organisation such as the NHS Trust had requested the suspension of the trial. Alternative methods of delivery were considered by 2 trial teams (6%) who were unable to implement these changes and in one case this was not considered to be applicable to the trial. Nine trial teams (26%) suggested that they had not considered alternative ways of delivering the trials.Impact of COVID-19 on recruitmentSixteen out of 34 trials (47%) temporarily halted recruitment during the pandemic, 4/34 trials 12%) continued recruitment with modification to procedures, and the remaining 14 trials (41%) were not actively recruiting e.g. studies in the follow -up phase. Data on recruitment decisions as well as the primary factor for the decision is shown in Figure % continuOf the four trials which continued recruitment during COVID-19, the primary driver in 3/4 (75%) of trials was potential participant benefit of treatment and participant safety 1/4 (25%). Concerning the factors influencing the decision to continue recruitment with modified procedures, a single trial identified participant benefit of the treatment, and one identified the risk associated with treatment withdrawal as influencing the decision. In 3/4 (75%) trials, funding was a factor. Impact on the Investigational Medicinal Product interventionThe impact of COVID-19 on IMP regimes is shown in Figure Free-text feedback highlighted that the decision regarding the IMP regime continuation or alteration was driven by clinical and/or practical considerations for the participants.Follow up assessmentsData on follow up assessments are shown in Figure Trial Risk AssessmentNone of the trials surveyed identified 'epidemic', 'pandemic' or other 'public health emergency' as listed on the trial risk assessment. Twenty-seven trials (79%) stated that their risk assessment did not list these terms, whilst 7/34 (21%) trials were unsure. This data is shown in Figure CTU responseEighteen CTUs responded to the survey and were supporting 17 (range 0 and 14 per unit) actively recruiting CTIMPs on March 16 2020. Four CTUs had CTIMPs that remain actively recruiting (excluding COVID-19 studies), with 3 CTIMPs being relevant to older adults.CTU level guidance in response to the COVID-19 crisis advocated halting new recruitment and continue to follow-ups in 7/18 units (39%). Eight CTUs (44%) indicated that the trial team  should evaluate the risk-benefit of their trial with regards to continuing or halting recruitment. Two units (11%) encouraged continued recruitment where possible. These data are illustrated in Figure Prior to January 2020, 4/18 (22%) CTUs had an action plan or a crisis management plan in place describing the steps to be taken in the case of a pandemic (or epidemic), 4/18 (22%) CTUs reported non-specific contingency plans or guidance was in place for unexpected events but not specific to a pandemic (or epidemic), and 8/18 (44%) CTUs had no pandemic (or epidemic) management plan.One CTU reported pandemic or epidemic prior to January 2020 mentioned in their risk assessment at both trial and management level. Three CTUs (17%) reported mention of pandemic or epidemic in their risk assessment prior to January 2020 but only at the management level. The remaining 14/18 (78%) CTUs did not mention pandemic or epidemic in their risk assessment prior to January 2020. This data is illustrated in Figure Preparedness of SponsorsEleven sponsors replied to the survey. Of these, three were commercial sponsors, and 8 were non-commercial sponsors. On March 16 2020, the sponsors supported a total of 23 actively recruiting CTIMPs (range 0-4 per unit). Of these 2 CTIMPs remained actively recruiting with no suspension of activities excluding any COVID-19 studies.Two sponsors (18%) encouraged recruitment to continue where possible, four sponsors 36%) advised to halt new recruitment but continue to follow up activity, and three sponsors (27%) advised investigators to suspend all non-COVID related trial activity. The remaining two reported that the response was either led by the NHS trust or did not give specific advice to CTIMPs as no CTIMP was open. These data are shown in Figure % advisedPrior to January 2020, 3/11 (27%) sponsors had an action plan or a crisis management plan in place describing the steps to be taken in the case of a pandemic (or epidemic). One sponsor reported non-specific contingency plans were in place, but not specific to a pandemic. Seven sponsors (64%) reported that they had no pandemic (or epidemic) management plan in place prior to January 2020.Three sponsors (27%) reported pandemic or epidemic prior to January 2020 mentioned in their risk assessment at but only at management level. None of the sponsors reported mention of pandemic or epidemic prior to January 2020 in the risk assessment at both trial and management level (0%). The remaining eight sponsors (73%) did not mention pandemic or epidemic in their risk assessment prior to January 2020. This data is shown in Figure During the pandemic, there was a rapid shift in focus and prioritisation to research that tackled COVID-19 -4. The rTo further understand and quantify this broader impact, we surveyed clinical trial teams and sought data from supporting CTUs and sponsors. We identified >2000 potentially eligible trials. The response rates from clinical trials and sponsors were low and we anticipate that this may have been a different function of the pandemic. The majority of those that responded paused recruitment during COVID-19, but follow-up activity and IMP regimes continued mostly unchanged. This suggests that whilst recruitment activity was heavily affected, trials were mostly able to proceed or accommodate changes which allowed them to continue the trial activity for those participants enrolled before the COVID-19 pandemic. Such changes included modification to the follow -up procedures, adjusting time scales for follow-ups and in some cases changing the logistics of the IMP to allow for home delivery to participants. Participant safety or potential benefit was the primary driver that determined the course of action taken during the pandemic.Very few institutions (CTUs and sponsors) had considered the pandemic impact on trials before January 2020. At a trial level, no risk assessment mentioned pandemic or epidemic, suggesting little preparatory activity to mitigate the risk was undertaken before the outbreak.The UK National Risk Register of Civil Emergencies 2017  documentAs such, the preparedness of trials to respond and adapt to an epidemic, pandemic or local outbreak of infectious diseases which increases the risk to the vulnerable populations appears, in retrospect, to be a worthwhile exercise. For example, most trials were able to adapt follow up procedures to allow some trial activity to continue whilst recruitment was suspended in more than half of the trials actively recruiting prior to the pandemic.Strengths and limitationsThe study provides insight which may be helpful to the future planning and preparedness of trials not only to a potential second wave of COVID-19 but also to other local and national infectious disease outbreaks. The survey focused on the key aspects of trial activity for CTIMPs; namely, recruitment, follow up and IMP management. This allowed us to assess specifically the type of trial activity impacted by the novel health emergency. The study was conducted in a subgroup of people who are listed as 'high clinical risk' by UK government officials. Therefore, the CTIMPs surveyed would encompass those who are most likely to be impacted by the pandemic.The low response rate from trials and sponsors is such that we would seek to see the findings reproduced and considered in other populations and disease areas before definitive conclusions could inform future policy. Whilst we purposefully, but pragmatically, limited the search criteria to include studies relevant to older adults and neurology, some trials may have been omitted which could be of relevance given the criteria search on EudraCT, e.g. studies which were relevant to older adults but did not tag 'Elderly' on the database.The survey was conducted in May 2020, and therefore it was issued more than one month after the UK government's 'Stay Home' directive . Whilst Future impactsCOVID-19 has necessitated a realignment of focus on the immediate public health crisis facing the UK population. An indirect consequence of this is the potentially negative impact on the existing portfolio of clinical trials running in non-COVID conditions. Our work has demonstrated that rapid changes and flexibility of delivery partially mitigated the impact.A new framework for risk-adapted approaches to clinical trials may be needed until a vaccine, or efficacious treatment is available ,4. TrialThe majority of trials temporarily halted recruitment during the pandemic. Despite a lack or delayed recognition of the risk that a pandemic presented at all levels of trial oversight, the rapid implementation of novel delivery methods holds promise for the future. Our findings call for a re-evaluation of the risk-management approach to evaluate whether clinical trials may be able to find contingency plans in the future to allow less disruption to trial activity.


To minimise patient burden and to enhance response rates in research studies, patient-reported outcome measures (PROM) need to be as short as possible, without sacrificing reliability and validity. It is also important to have a generic measure that can be used for comparisons across different patient populations. Thus, the aim of this secondary analysis was to evaluate the psychometric properties of the Norwegian 5-item version of the Lee Fatigue Scale (LFS) in two distinct patient populations.Fatigue is a common symptom associated with a wide range of diseases and needs to be more thoroughly studiedn = 322) and patients with osteoarthritis on a waiting list for total knee arthroplasty (n = 203). Fatigue severity was rated by five items from the Norwegian version of the LFS, rating each item on a numeric rating scale from 1 to 10. Rasch analysis was used to evaluate the psychometric properties of the 5-item scale across the two patient samples.The sample was obtained from two different Norwegian studies and included patients 4–6 weeks after stroke  showed acceptable internal scale validity as they met the set criterion for goodness-of-fit after removal of two items with unacceptable goodness-of-fit to the Rasch model. The 3-item LFS explained 81.6% of the variance, demonstrated acceptable unidimensionality, could separate the fatigue responses into three distinct severity groups and had no differential functioning with regard to disease group. The 3-item version of the LFS had a higher separation index and better internal consistency reliability than the 5-item version.A 3-item version of the LFS demonstrated acceptable psychometric properties in two distinct samples of patients, suggesting it may be useful as a brief generic measure of fatigue severity.NCT02338869; registered 10/04/2014 (stroke study).Clinicaltrials.gov:  Fatigue is a common symptom associated with a wide range of chronic diseases  and has One of the frequently used PROMs for fatigue is the 13-item Lee Fatigue Scale (LFS). It has primarily been used among adults with cancer , 5 and hWe have previously evaluated the English version of the LFS in samples of patients with cancer  and peopIn order to evaluate whether the LFS is suitable for use as a generic measure of fatigue severity, there is a need for further exploration of the psychometric properties of the LFS. The aim of this study was to evaluate the psychometric properties of the Norwegian 5-item version of the LFS in two different patient populations, adults with stroke and adults with osteoarthritis.This study has a cross-sectional design and includes initial pre-intervention data from two longitudinal studies, a multicentre randomised controlled trial evaluating the effect of an intervention promoting psychosocial well-being following stroke  and a loA total of 322 adult stroke survivors recruited from 11 acute stroke or rehabilitation units in university hospitals and other local hospitals providing acute care in Norway were consented into the trial . The incInitial pre-intervention data were collected in structured in-person assessment interviews within 6 weeks after stroke onset. The data collector recorded the participant’s responses consisting of demographics and 5 items from the LFS , in addiA total of 203 patients with osteoarthritis who were admitted for total knee arthroplasty at a surgical clinic in Oslo, Norway were included in the study. The inclusion criteria were: adults ≥ 18 years of age, ability to read, write and understand Norwegian, and scheduled for unilateral primary total knee arthroplasty. Patients undergoing unicompartmental or revision surgery were excluded. A comprehensive description of the study participants and data collection measures has been reported elsewhere .The initial data collection was performed prior to surgery, after admission to the hospital. Patients independently completed paper questionnaires assessing demographic characteristics, 5 items from the original LFS and several other measures included in the osteoarthritis study . Psychomnot at all tired” to “extremely tired”, item 4 “not at all fatigued” to “extremely fatigued”, item 5 “not at all worn out” to “extremely worn out”, item 16 “carry on a conversation is no effort at all” to “carrying on a conversation is a tremendous chore”, and item 17 “I have absolutely no desire to close my eyes” to “I have a tremendous desire to close my eyes”. All items were rated on a numeric rating scale from 0 to 10; higher scores indicate higher fatigue severity. Items 1, 4 and 5 were also included in a short version of the LFS evaluated through Rasch analysis among women living with HIV [Fatigue severity was measured in both samples using the same 5-item LFS. The following items with the anchor phrases from the original 13-item version  were usewith HIV . Althougwith HIV .The analysis of the LFS was guided by the use of a Rasch rating scale model . The RasOutfit Mean Square (MnSq) value of less than 2.0 for each of the step calibrations [First, an evaluation of the psychometric properties of the rating scale was conducted. The criterion used was that the average measures for each response category on each item should advance monotonically, as evidenced by an brations .Infit MnSq values between 0.7 and 1.3 logits [The second step aimed to evaluate the fit of the item responses . Any ite3 logits . In the 3 logits .Infit MnSq values of 1.4 logits or higher or associated with a z-value of 2 or higher, accepting that 5% of the sample may by chance fail to demonstrate acceptable goodness-of-fit without threatening evidence of person response validity [The fourth step evaluated aspects of person response validity. The criterion for evaluating person goodness-of-fit was to reject validity –26. We aDIF) analyses were performed in order to evaluate the stability of the LFS response patterns in relation to diagnosis (stroke or osteoarthritis) using the Mantel-Haenzel statistics for polytomous scales using log-odds estimators [p-values < 0.01 were considered indicative of DIF.In the fifth step, Differential Item Functioning  was evaluated by calculating the scale’s person separation index . The sepP-values < 0.05 were considered statistically significant.In addition to the steps described above for psychometric analysis of the LFS, characteristics of the study samples were summarized and compared using SPSS statistical software, version 25 . DiffereCharacteristics of the two patient samples are described in Table MnSq statistics (#16 carry on a conversation and #17 close eyes), and two items demonstrated lower than acceptable MnSq (#1 tired and #4 fatigued). Because items with higher fit statistics are a greater threat to unidimensionality compared to items with lower fit statistics, these two items were initially excluded and the remaining three items  were re-analysed. In the second iteration, the three remaining items demonstrated an acceptable range for goodness-of-fit to the model.The LFS rating scale demonstrated acceptable outcomes in relation to the established criteria. In addition, all ten rating scale steps were used with a frequency above 100 scores for all scale steps. When analyzing the infit mean square statistics for the five included items, only one item out of five demonstrated acceptable goodness-of-fit  was within the set criterion of < 5% and was close to the criterion for the 3-item scale (5.6%). The number of respondents with maximum scores indicated negligible ceiling effects, but the number of respondents with minimum scores on both the 5-item and 3-item scales indicated a moderate floor effect.The DIF analysis indicated that two of the misfitting items showed DIF in relation to disease group, but once those two items were removed, the 3-item scale revealed no systematic differences in relation to diagnosis across any of the three remaining LFS items. The separation index of the LFS scale also increased from 1.82 to 2.49 after deleting the two items demonstrating misfit, indicating the 3-item scale was able to distinguish three statistically distinct groups of fatigue. Measures of internal consistency indicated that the 3-item scale met all set criteria and performed better than the 5-item LFS scale. See Table Findings from our study showed that a 3-item version of the LFS had better psychometric properties than the 5-item version. The 3-item LFS showed unidimensionality, accounted for a large proportion of explained variance, and was able to differentiate three statistically distinct fatigue severity groups. In a previous psychometric evaluation in a sample of women with HIV , all 13 As shown in this analysis, scales with more items are not always better. They may lack unidimensionality, which poses challenges to the generation of meaningful total scores and may indicate that the use of subscales is warranted. Moreover, the inclusion of poorly performing items may actually reduce the ability of the scale to distinguish distinct groups based on level of severity, as occurred in this analysis, in which the 5-item scale could only distinguish two severity groups, while the 3-item scale was able to distinguish three severity groups. Even though these groups are based on statistical calculations, future studies could explore the clinical relevance and potential cut-offs for determining such group allocations. This could be a logical step for future research now that there is evidence that the 3-item scale is sensitive enough to detect statistically distinct groups.Another interesting aspect is that the items in the 3-item version all demonstrated acceptable goodness-of-fit within the set ranges, indicating that the response patterns all contribute to the underlying measure, without evidence of over- or underfit as in the 5-item version, supporting validity evidence of internal structure. The three remaining items  are conceptually more similar than the excluded items that involve interactions or behaviors , also indicating evidence of validity of the test concept.There were moderate floor effects for both the 3- and 5-item LFS, but the 3-item version had a higher mean value than the 5-item version. The low severity scores may have been due to our reduction of the rating scale from 0 to 10 to 1–10. However, other studies on the LFS have also reported floor effects , so thisThe 3-item LFS was not biased by diagnosis, as indicated by the lack of DIF and similar response patterns across two different patient groups. Combined with the results of prior studies among patients with cancer and HIV , 14, thiOne challenge resulting from the use of different short versions of the LFS is how to compare scores from versions containing different items. One solution might be to use Rasch analysis to generate a stable and disease-generic item hierarchy that can be used to select subsets of items for specific studies and still generate comparable measures through conversion tables or computer-adaptive testing (CAT) procedures. Even though some similarities in the item hierarchies from our earlier studies occur in relation to these findings , more inBased on the findings from this study, the idea may arise that a single “perfect” fatigue item could perform as well as multi-item versions assessing subtly different aspects of fatigue. Although this could be explored in this sample and others where the LFS has been evaluated using Rasch analysis , 14, anoThe translation of any PROM requires the use of stringent procedures to ensure conceptual equivalence in the new translation compared with the original language version . ConceptThe strengths of this secondary analysis included the relatively large samples from two diverse patient populations and the thorough evaluation of the psychometric properties. However, a significant limitation is that this study evaluated only five of the original 13 LFS items, so it remains unclear whether the three items retained in this analysis represent the best three items for inclusion in a brief fatigue severity PROM. It would therefore be interesting to determine whether the 3-item version generated in this analysis outperforms other potential short versions, particularly the 5-item short form developed in the prior study of women living with HIV .In addition, this study evaluated a Norwegian version of the LFS. Translation of the concept of fatigue into Norwegian and perhaps other languages is difficult, since some English words and phrases do not have direct equivalents in Norwegian or other languages. Thus, generalization of the findings from this Norwegian study to other populations must be done with caution.Finally, the mode of data collection was not identical in the stroke and osteoarthritis samples. The stroke sample was interviewed in person, while the osteoarthritis sample completed the questionnaire independently. Thus, the patients with stroke may have received help in understanding the meaning of the different items, whereas the patients with osteoarthritis were left to interpret the items on their own. Although no DIF was found based on diagnostic group, the differing data collection mode for the two samples may have introduced bias in the interpretation of the items and, thus, may have influenced the results.The results of this study indicate that a 3-item version of the Lee Fatigue Scale has acceptable psychometric properties and is sufficiently generic for use as a PROM for fatigue severity with patients post-stroke and patients living with osteoarthritis. Future research should be conducted to evaluate the validity of the 3-item version for use among other clinical populations.


Here, we describe a set of CRISPR-based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signaling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signaling. However, the role of such proteins in regulating cellular processes  PIs havskeleton . PIs als mammals . Phosphaosophila  and a copolarity . Dysregupolarity  and PIK3polarity ; mutatio cancers . Mutatio cancers . Thus, tGiven their importance in a large number of important subcellular processes, PI signaling is tightly regulated. The seven PIs are generated in cells by the activity of a set of evolutionarily conserved lipid kinases that are specific for the substrate they will use, as well as the position on the inositol at which they will phosphorylate . In turnin vivo function challenging.Although PI signaling is broadly a conserved feature of all eukaryotic cells, some aspects of this pathway are found uniquely in metazoa and play important roles in animal development and tissue architecture. However, there remain key challenges in the analysis of PI signaling in metazoan development. For example, PIs may regulate the same subcellular process (e.g endocytosis) but may be co-opted to control the trafficking of unique cargoes in different tissues. Second, through their ability to control the secretion of intercellular signaling morphogens and hormones that mediate intercellular signaling, PIs can regulate tissue development in a non-cell autonomous manner. Third, given their key functions in cell biology, whole body knockouts of these genes often result in organismal lethality. Finally, in many cases, a given biochemical activity is underpinned by multiple genes in mammalian genomes and gene redundancy usually makes analysis of Drosophila offers a powerful alternative to overcome the challenges of studying PI function in cell and developmental processes. Many developmental mechanisms are conserved between Drosophila and other metazoan models. A detailed annotation of the PI signaling genes in the Drosophila genome  that was to be used to generate dgRNA transgenic flies were sequenced (sequencing data is available for S2R+ cells on hyperlink and for isogenized attP40 stock on hyperlink). Sequences thus obtained were aligned against the reference genome and also against each other. When aligned against the reference genome, both the isogenized parent fly stock and the S2R+ cell genome sequences showed more than 600,000 single-nucleotide polymorphisms (SNPs) , nos-Cas9 II  and nos-Cas9 III . Only 1 gRNA target (targeting CG10426), out of the 206 generated, seemed to have one base mismatch marked in red  , 4. Whens (SNPs) , 4. Whild in red , while tCG3682 has upto six splice variants with four putative start sites). In cases where two or more putative genes are annotated on the same locus, sgRNA target regions were chosen such that the coding sequences of neighboring genes are not disrupted . Once a set of two ~ 100 bp regions were identified for each gene keeping in mind the above-mentioned criteria, we designed the sgRNAs using an online tool (http://targetfinder.flycrispr.neuro.brown.edu) with zero predicted off-targets. During the course of this study, an additional online tool  became available. Thereafter, this tool was used to obtain a predicted efficiency score. The most efficient sgRNAs in the region were then checked in our sequence database for presence of any mismatches in either S2R+ cells or injection fly stocks compared to the reference genome sequence. The best sgRNA sequences predicted to efficiently target specific genes with no predicted off-targets and absence of any mismatches were chosen for synthesis. Any sgRNA sequences that did not qualify these criteria were discarded and new sgRNAs designed to fit all the above criteria. A full list of genes for which dgRNAs were designed is listed in For each gene, two regions were identified wherein an sgRNA would be designed to target each region. The first sgRNA would be designed to target the first coding exon and the second sgRNA to target the exon with the stop codon . In case+ cells. The sgRNA pairs that led to the deletion of the gene, as tested by PCR and sequencing of the target gene loci, were chosen for dual-gRNA (dgRNA) construction. In case a particular pair of sgRNAs failed to delete the target gene, a new set of sgRNAs were designed for both the first and the last coding exon and different combinations of sgRNAs were tested until the most optimal pair capable of deleting the target gene was identified.The first sgRNAs targeting a region close to the start codon for each gene were cloned into the pBFv6.2 vector and the second sgRNAs targeting a region close to the stop codon into the pBFv6.2B vector . Both these vectors ubiquitously express the gRNAs under the U6.2 promoter . The two+ cells to generate gene deletions for cell culture-based experiments. For genes that are present on the second chromosome, the dgRNA construct was inserted on the third chromosome (Act5c-Cas9 (BDSC 545901 M{Act5C-Cas9.P}ZH-2A w*: y) flies and larval progeny were tested for deletions by PCR and sequencing. Out of 103 genes, 102 gave the desired deletion product, while one did not give a band of the expected length . These constructs were then microinjected to generate dgRNA transgenic flies for each of the 103 PI signaling genes . This dgromosome . For alld length . This coocrl (KOocrl) generated in this study (see sections below) that removes the open-reading frame of ocrl. In western blots from wild-type animals, a band of 97 kDa was detected that was absent in KOocrl  in nephrocytes  expresses at lower levels thereby reducing the cytotoxic effects of the otherwise highly expressing Cas9.C  into S2R+ cells constitutively expressing Tubulin-Gal4. As a control, the cells were parallelly co-transfected with the pBS-hsp70-Cas9 plasmid (Addgene Plasmid #46294) and the dgRNA against CG5734. Forty-eight hours post-transfection the cells were harvested. Half the cells were used for protein extraction and western blotting. Western blotting showed that Cas9 was being expressed from the UAS-Cas9-T2A-eGFP construct. The molecular size of Cas9 from this construct was similar to Cas9 expressed from pBS-hsp70-Cas9. Detection of a band corresponding to the molecular weight of free eGFP in cells transfected with UAS-Cas9-T2A-eGFP suggested that the T2A sequence was working efficiently to generate Cas9 and eGFP as two independent proteins from a single mRNA  flies. The progeny flies were dissected, salivary glands stained for Cas9 and imaged. All salivary gland cells expressed eGFP and were also stained positive for Cas9 thus demonstrating that the UAS-Cas9-T2A-eGFP construct can be used as expected to drive Cas9 expression and to mark the Cas9-expressing cells with eGFP  with the added advantage of eGFP expression to mark Cas9-expressing cells. These UAS-Cas9-T2A-eGFP flies were used for all experiments described further.After verification in S2Rith eGFP . The eGFDrosophila eye serves as an excellent model to perform genetic screens as it is not essential for viability and offers a phenotype that can be easily scored. Previously, influential studies have used Drosophila eyes to screen for genes regulating cell growth and proliferation  1:50, a+ cells were harvested by repeated flushing of the media and pelleted down in a 1.7 ml Eppendorf vial by centrifugation at a speed of 800 rpm for 10 min. The pelleted cells were lysed by repeated pipetting in Laemli loading buffer. Following this, the samples were heated at 95°C for 5 min and loaded onto an SDS-PAGE. The proteins were then transferred onto a nitrocellulose membrane and incubated overnight at 4°C with indicated antibodies. Primary antibody concentrations used were anti-dCas9 (Takara 632628) 1:2000; anti-GFP (Santa Cruz sc9996) 1:2000; anti-RDGB , incubated at 37°C for 30 min and 95°C for 10 min. One μl of the supernatant was used to perform PCR with an outer set of primers . In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Drosophila. The reagents generated in this study will be a useful and versatile genetic tool for researchers in the field.Phosphoinositide (PI) signaling is important for various biological processes in all metazoan organisms. The authors generated a CRISPR-based genome engineering toolkit to make tissue-specific mutations in π signaling genes in Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Utpal Banerjee as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Three reviewers suggested major criticisms on validations of the new tool and generated stocks. Therefore, the revised manuscript should demonstrate efficient knockout events by expression analysis and by extensive molecular analysis of targeted mutations for many more genes rather than a few. Essential revisions are summarized as below and full reviewers' comments are attached for your reference.Summary:Drosophila and offers germline or tissue-specific knockouts of Drosophila π signaling genes. There have been several independent efforts to generate systematic knockout tools in this study, and it will be a valuable toolkit for the fly community working on π signaling in Drosophila cells and whole animals. However, this report shows a few successful cases of gene knockouts and insufficient assessments for the new tool, and therefore, the manuscript requires additional extensive molecular and genetic evaluation of the presented data.This study describes the generation of a CRISPR/Cas-9 mediated gene editing toolkit in Essential revisions:1) Confirm DNA lesions in many more cases:i) The authors designed dgRNA constructs to generate gene deletion. Although they claimed that the targeted deletions were confirmed in S2R+ cells, it is unclear whether the mutations were confirmed in flies.According to subsection “Investigating the role of π signalling in eye development using the dgRNA toolkit”, a few representative ey>Cas9;dgRNA lines were tested for genomic deletion. Then, it appears that the majority of 103 transgenic lines were not tested for molecular lesions. Table 1 shows a list of genes with the size of the genomic deletion. If in fact only a few lines were tested, then it would be difficult to properly evaluate the data in Table 1. It is important to know how many lines were tested for deletion and how many actually showed an expected size of deletions.2) Better validation of the efficacy:i) The authors verified the efficiency and usefulness of the tool by targeting π genes in the eye. Most of the known target genes resulted in desirable phenotypes in the eye; however, some did not. In this case, it could be a matter of protein stability as the authors explained, but it also could be due to the efficiency or the variability of the new tool in the eye. The tool requires a better validation on a cell-by-cell basis. For example, with the use of in situ hybridization or an antibody against the protein , expressions of target genes could be measured. If the phenotype/expression level could be quantified, efficiencies of whole body mutant, tissue-specific RNAi, and tissue-specific deletion need to be compared.ii) In Figure 4A, the authors have shown a truncated band of CG5734 without showing its full-length gene. Even with the presence of truncated size, the intact gene may be still viable in some eye cells. It again will be important to show how efficient it is to use the new tool at a tissue level.iii) In addition to ey-gal4, the tool requires validations with additional tissue-specific drivers. This will not only verify its effectiveness but enhance the broader applications.iv) In subsection “Generation of whole fly knockouts and using dgRNA transgenics”, it seems that they tried germline knockout for several genes, but the procedure is not mentioned and the data are described very ambiguously. "The deletions obtained in this manner are highly efficient (between 4-100% efficiency in F1 flies and between 3-70% efficiency in F2 flies) in generating germline knockouts with deletions ranging in size between 400bp and 38kb." From this description, it is difficult to know whether the deletions are highly efficient as claimed. The procedure for making germline knockout and the results need to be described in full detail. In Table 1, a number of tissue-specific knockouts show no phenotype. It is possible that some of them may not have expected mutations.v) There is no data showing the loss of protein expression in eye discs from eye-specific knockout lines. Crosses with ey-Gal4 will generate mosaic tissues in developing eye discs. It is important to know how efficient is a somatic knockout in the region of ey-Gal4 expression.3) Compare SNPs in other strains:Drosophila and S2R+ cells and match the reference genome, that they should work in other genetic backgrounds. However, these backgrounds don't sample the full range of nucleotide variation in laboratory stocks. Can the authors compare their gRNA sequences with genomes of other common lab strains ?i) The authors make the argument that because their dgRNA constructs work in both their BL25709 ii) Authors have carefully selected dgRNA sequences by comparing genomic sequences of S2R+ cells, parent fly stocks, and reference fly genome. This will be a useful resource for researchers who would refer to this study in the future. Therefore, detailed descriptions of SNPs found  need to be added in addition to the diagram shown in Figure 2.4) Separate methods from results:i) Since the manuscript describes methods in the result, it does not separate the methods from the main text. However, it should either be more specific in the text or include the methods part for further details. As one example, UAS-Cas9-eGFP transgenic flies newly generated in this study require detail information for those who will order stocks.Reviewer #1:In this manuscript, Gaghu and colleagues have designed a set of CRISPR based genome editing tools and constructed reagents by which 103 phosphoinositide (PI)-related genes can be individually deleted. With the use of the reagents, authors have identified novel π genes that control eye development and verified the effectiveness of the tools.This study will potentially benefit the fly community by providing the new tissue-specific CRISPR tool. However, as a resource paper, the manuscript requires better validations on the technique itself and focus on describing the validity of the tool.1) Authors have carefully selected dgRNA sequences by comparing genomic sequences of S2R+ cells, parent fly stocks, and reference fly genome. This will be a useful resource for researchers who would refer to this study in the future. Therefore, detailed descriptions of SNPs found  need to be added in addition to the diagram shown in Figure 2.a) In the same vein, if SNPs in different backgrounds are critical for the CRISPR efficiency, giving a few examples of such cases would enhance the importance of resources provided.2) Since the manuscript describes methods in the result, it does not separate the methods from the main text. However, it should either be more specific in the text or include the methods part fur further details. As one example, UAS-Cas9-eGFP transgenic flies newly generated in this study require detail information for those who will order stocks.3) The authors verified the efficiency and usefulness of the tool by targeting π genes in the eye. Most of the known target genes resulted in desirable phenotypes in the eye; however, some did not. In this case, it could be a matter of protein stability as the authors explained, but it also could be due to the efficiency or the variability of the new tool in the eye. The tool requires a better validation in a cell-by-cell basis. For example, with the use of in situ hybridization or an antibody against the protein, expressions of target genes could be measured. If the phenotype/expression level could be quantified, efficiencies of whole body mutant, tissue-specific RNAi, and tissue-specific deletion need to be compared.a) In Figure 4A, the authors have shown a truncated band of CG5734 without showing its full-length gene. Even with the presence of truncated size, the intact gene may be still viable in some eye cells. It again will be important to show how efficient it is to use the new tool at a tissue level.4) In addition to ey-gal4, the tool requires validations with additional tissue-specific drivers. This will not only verify its effectiveness but enhance the broader applications.Reviewer #2:Drosophila π signaling genes. With additional constructs, they could also be used to generate new alleles or tagged proteins. This is a useful toolkit for the growing community working on signalling, transport and metabolism of π in Drosophila cells and whole animals. The lines target two sites in each gene, are robust to some target genome variation, and in some cases are validated using RNAi or known mutant phenotypes. This is a very useful collection of well-designed lines, with good supporting validation.This study offers a library of 103 gRNA lines to generate germline or tissue-specific knockouts of Drosophila and S2R+ cells and match the reference genome, that they should work in other genetic backgrounds. However, these backgrounds don't sample the full range of nucleotide variation in laboratory stocks. Can the authors compare their gRNA sequences with genomes of other common lab strains ?The authors make the argument that because their dgRNA constructs work in both their BL25709 Reviewer #3:Drosophila. There have been several independent efforts to generate Cas9-based systematic gene knockout tools in culture cells and flies. This study attempts to demonstrate the feasibility of constructing guide RNAi library targeted to a relatively small set the genes involved in phosphoinositides (PI) metabolism and signaling. This report shows a few successful cases of gene knockout and a list of 103 targeted genes. Therefore, in principle, the genetic tools generated in this study can be valuable resources in the field of π signaling. However, I feel that the general strategy used in this study is not particularly novel, and more importantly, analysis of their library appears to be rather limited and preliminary for proper evaluation of the presented data.This manuscript describes the generation of a genetic tool kit for tissue-specific and whole animal knockout using CRISPR/Cas9-mediated gene editing in Merits of this work:1) They sequenced S2R+ genome and an isogenic fly genome. After comparing these sequences and the reference genome, gRNA targets were carefully selected to avoid potential mismatches or off-targets.2) This study used double guide RNAs targeted to 1st and last coding exons to generate null mutations by deletion. The use of T2A-mediated eGFP expression also helps to identify Cas9-expressing cells.3) Cas9 mutagenesis targeted to a full set of PI-related genes is a new attempt.Weaknesses:Overall, their strategy for tissue-specific knockout is similar to two recent papers . Hence, novelty of this manuscript is somewhat diminished. In addition, there are major issues related to their methods and data, as listed below.1) Authors designed dgRNA constructs to generate gene deletion. Although they claimed that the targeted deletions were confirmed in S2R+ cells, it is unclear whether the mutations were confirmed in flies.According to subsection “Investigating the role of π signalling in eye development using the dgRNA toolkit”, a few representative ey>Cas9;dgRNA lines were tested for genomic deletion. Then, it appears that the majority of 103 transgenic lines were not tested for molecular lesions. Table 1 shows a list of genes with the size of the genomic deletion. If in fact only a few lines were tested, then it would be difficult to properly evaluate the data in Table 1. It is important to know how many lines were tested for deletion and how many actually showed expected size of deletions.2) In subsection “Generation of whole fly knockouts and using dgRNA transgenics”, it seems that they tried germline knockout for several genes, but the procedure is not mentioned and the data are described very ambiguously. "The deletions obtained in this manner are highly efficient (between 4-100% efficiency in F1 flies and between 3-70% efficiency in F2 flies) in generating germline knockouts with deletions ranging in size between 400bp and 38kb." From this description, it is difficult to know whether the deletions are highly efficient as claimed. The procedure for making germline knockout and the results need to be described in full detail. In Table 1, a number of tissue-specific knockout show no phenotype. It is possible that some of them may not have expected mutations.3) There is no data showing the loss of protein expression in eye discs from eye-specific knockout lines. Crosses with ey-Gal4 will generate mosaic tissues in developing eye discs. It is important to know how efficient is somatic knockout in the region of ey-Gal4 expression.eLife. Your revised article has been evaluated by Utpal Banerjee (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:All the reviewers agreed that the authors added a substantial amount of new data and appropriately addressed most of the concerns.Reviewer #1:In the revised manuscript, Trivedi et al. provide a significantly improved version of their work with additional analyses and in vivo validations and addressed most of the concerns raised by the reviewers. Two additional points need to be modified to clarify the results.1) In Figure 2B, the authors validated the efficiency of ocrl deletion by immunohistochemistry and showed that ocrl knockout reduces the anti-OCRL staining in the wing disc. However, staining patterns shown in controls are too low, and it is hard to tell whether the antibody used was working in the disc. Providing a reference for the antibody that shows an expression pattern in a representative organ and/or highlighting higher mag images will be critical to reassure the data. If this antibody was first used in an IHC experiment, proper confirmations for the antibody would be needed.2) In Figure 3C, gel images do not have proper labels.3) If possible, it will be best to organize the deposition of generated flies to major stock centers before publication and describe it in the text.Reviewer #2:The authors have added substantial new validation, particularly on the areas of SNP variation, the lesions obtained, and homozygous phenotypes. This now gives the reader greater confidence in the usefulness of their resource – for somatic knockouts one can often find a localized phenotype but not always, perhaps due to perdurance of a stable gene product; and most of their gRNA stocks can be used to generate germline knockouts, and they have established mutant stocks for about half of them.Taken together, I think this collection will be a valuable resource targeting many genes of great interest for the fly signaling and lipid communities. The paper now gives a much better sense of the approach's strengths and limitations, and there is enough information to make many lines of wide use immediately and to develop further mutant stocks for the remainder. To make these lines accessible to the community, I hope that both the gRNA and mutant stocks will be offered promptly to the major stock centers, if the authors haven't done so already.Reviewer #3:1) The authors have revised the manuscript with additional experiments and clarifications. The revised manuscript appears to be significantly improved by extensive molecular analysis of targeted mutations in fly tissues. Phenotype analysis with additional GAL4 drivers also supports the effectiveness of the CRISPR/Cas9 tools.2) Considering that the majority of targeted genes by ey-GAL4 do not show phenotypes, it is still possible that targeting might not have caused sufficient loss of protein expression in many genes. In this regard, checking the expression level for only one gene (Ocrl) is a weak point. However, the new data showing deletions in most targeted genes seems to compensate for the weakness. In summary, the revised manuscript seems to be acceptable, but a few mistakes listed below should be corrected.3) Some genotypes in figures are not italicized. Those genotypes need to be fixed.4) Anti-Ocrl staining appears to be nuclear staining to me, although it is too weak to be certain. A better way to show the mutation effect is to use a different Gal4 such as en-Gal4 to show an internal control. It would be good to show a test for antibody specificity. Essential revisions:1) Confirm DNA lesions in many more cases:i) The authors designed dgRNA constructs to generate gene deletion. Although they claimed that the targeted deletions were confirmed in S2R+ cells, it is unclear whether the mutations were confirmed in flies.According to subsection “Investigating the role of π signalling in eye development using the dgRNA toolkit”, a few representative ey>Cas9;dgRNA lines were tested for genomic deletion. Then, it appears that the majority of 103 transgenic lines were not tested for molecular lesions. Table 1 shows a list of genes with the size of the genomic deletion. If in fact only a few lines were tested, then it would be difficult to properly evaluate the data in Table 1. It is important to know how many lines were tested for deletion and how many actually showed an expected size of deletions.To address the above comment following has been done:a) dgRNA for all the 103 genes were tested for deletions in flies after crossing with Act5c-Cas9. 102 of these seem to show deletion in flies. Thus, in addition to our previous experiments  where we showed deletions generated by all of the dgRNAs in S2R+ cells, we have now included new data showing that 102/103 dgRNAs also work in fly tissues (Supplementary file 1).b) The exact size of deletion for each dgRNA is now added in Supplementary file 1 according to the results obtained in flies after sequencing the deletion.2) Better validation of the efficacy:i) The authors verified the efficiency and usefulness of the tool by targeting π genes in the eye. Most of the known target genes resulted in desirable phenotypes in the eye; however, some did not. In this case, it could be a matter of protein stability as the authors explained, but it also could be due to the efficiency or the variability of the new tool in the eye. The tool requires a better validation on a cell-by-cell basis. For example, with the use of in situ hybridization or an antibody against the protein , expressions of target genes could be measured. If the phenotype/expression level could be quantified, efficiencies of whole body mutant, tissue-specific RNAi, and tissue-specific deletion need to be compared.ocrl was expressed, there was a reduction in OCRL protein expression . Quantification of the staining intensity across discs  showed that the reduction in OCRL protein levels was quite uniform suggested minimal cell to cell variability in OCRL protein depletion with these reagents.We agree with this point. It has been a challenge to obtain appropriate antibodies during the current pandemic with many labs shut or difficult shipping conditions. Therefore, we were only able to perform a limited number of experiments. A well-validated antibody for OCRL was available. We were able to use this and show that in discs where the dgRNA for ii) In Figure 4A, the authors have shown a truncated band of CG5734 without showing its full-length gene. Even with the presence of truncated size, the intact gene may be still viable in some eye cells. It again will be important to show how efficient it is to use the new tool at a tissue level.This has been tested for several genes. Most do not show wildtype bands either because of high efficiency of deletion, or more likely because smaller deletion products are amplified favorably in PCR cycles. However, we have added a Figure 4—figure supplement 1 with an example where the WT product was amplified.iii) In addition to ey-gal4, the tool requires validations with additional tissue-specific drivers. This will not only verify its effectiveness but enhance the broader applications.dgRNApTEN resulted in a larger wing blade while deletion of dgRNApi3K92Eand dgRNAaktresulted in substantial reductions in wing blade size. These results are now presented in Figure 4E.We also performed additional deletions with tissue specific GAL4 lines. We expressed three different guideRNAs for the PI3K signalling pathway using nub-GAL4 that expresses in a specific domain of the developing wing imaginal disc. Each of these dgRNAs , when expressed in the developing eye disc with UAS-Cas9 had resulted in changes in tissue growth resulting in altered eye size. Likewise, when expressed using nub-GAL4, they resulted in equivalent effects in the phenotype of the adult wing. Thus, deletion of Overall, during the course of this study, we have shown that our dgRNAs when expressed using four tissue specific drivers  produce deletions and phenotypes. In addition, we have also demonstrated its ability to produce deletions with the ubiquitous Act5C-Gal4 that expresses in all tissues.iv) In subsection “Generation of whole fly knockouts and using dgRNA transgenics”, it seems that they tried germline knockout for several genes, but the procedure is not mentioned and the data are described very ambiguously. "The deletions obtained in this manner are highly efficient (between 4-100% efficiency in F1 flies and between 3-70% efficiency in F2 flies) in generating germline knockouts with deletions ranging in size between 400bp and 38kb." From this description, it is difficult to know whether the deletions are highly efficient as claimed. The procedure for making germline knockout and the results need to be described in full detail.The absolute numbers have now been added to Supplementary file 1.In Table 1, a number of tissue-specific knockouts show no phenotype. It is possible that some of them may not have expected mutations.We have a number of examples where there was no phenotype, but testing for deletions by PCR in these showed that a deletion had indeed taken place. This has now been added as Figure 4—figure supplement 2. It is possible that in some of these cases (i) the gene in question does not have a role in eye development (ii) some amount of residual pre-deletion RNA/protein perdures in the cells and is sufficient to support normal function.v) There is no data showing the loss of protein expression in eye discs from eye-specific knockout lines. Crosses with ey-Gal4 will generate mosaic tissues in developing eye discs. It is important to know how efficient is a somatic knockout in the region of ey-Gal4 expression.dgRNAocrl/+ and Act5c-Cas9/+ due to limitation of antibody availability.Due to the challenge of obtaining appropriate antibodies during the current pandemic with many labs shut or difficult shipping conditions, we were only able to perform a limited number of experiments. A well- validated antibody for OCRL was available. We were able to use this and show that in discs where the dgRNA for ocrl was expressed there was a reduction in OCRL protein expression . Quantification of the staining intensity across discs  showed that the reduction in OCRL protein levels was quite uniform. Please note that we were able to do this experiment with only 2 genotypes- Act5c-Cas9/+; 3) Compare SNPs in other strains:Drosophila and S2R+ cells and match the reference genome, that they should work in other genetic backgrounds. However, these backgrounds don't sample the full range of nucleotide variation in laboratory stocks. Can the authors compare their gRNA sequences with genomes of other common lab strains ?i) The authors make the argument that because their dgRNA constructs work in both their BL25709 We have compared the sequences obtained using NGS with WT genomic seq from Flybase. In addition, the designed gRNAs have now been matched to recently added genomic sequences of reference genome r_6, vasa-Cas9 (BDSC 51324), nos-Cas9 II (BDSC 78781) and nos-Cas9 III (BDSC 78782). Only 1 gRNA target (targeting CG10426), out of the 206 generated, seemed to have one base mismatch (Supplementary file 1- the variant base marked in red ), while the other 205 targets matched between all the genomes. These gRNA can hence be used in many genetic backgrounds.ii) Authors have carefully selected dgRNA sequences by comparing genomic sequences of S2R+ cells, parent fly stocks, and reference fly genome. This will be a useful resource for researchers who would refer to this study in the future. Therefore, detailed descriptions of SNPs found  need to be added in addition to the diagram shown in Figure 2.The detailed description of all SNP’s found have been added as Figure 2; Supplementary file 3 and 4.4) Separate methods from results:i) Since the manuscript describes methods in the result, it does not separate the methods from the main text. However, it should either be more specific in the text or include the methods part for further details. As one example, UAS-Cas9-eGFP transgenic flies newly generated in this study require detail information for those who will order stocks.A separate section of Materials and methods has now been added to detail all the experimental protocols.Reviewer #1:In this manuscript, Gaghu and colleagues have designed a set of CRISPR based genome editing tools and constructed reagents by which 103 phosphoinositide (PI)-related genes can be individually deleted. With the use of the reagents, authors have identified novel π genes that control eye development and verified the effectiveness of the tools.This study will potentially benefit the fly community by providing the new tissue-specific CRISPR tool. However, as a resource paper, the manuscript requires better validations on the technique itself and focus on describing the validity of the tool.1) Authors have carefully selected dgRNA sequences by comparing genomic sequences of S2R+ cells, parent fly stocks, and reference fly genome. This will be a useful resource for researchers who would refer to this study in the future. Therefore, detailed descriptions of SNPs found  need to be added in addition to the diagram shown in Figure 2.This has been done. Discussed in Essential Revisions.a) In the same vein, if SNPs in different backgrounds are critical for the CRISPR efficiency, giving a few examples of such cases would enhance the importance of resources provided.We found only one example of a guide that had a mismatch due to a strain specific SNP. This has been explained in the text.2) Since the manuscript describes methods in the result, it does not separate the methods from the main text. However, it should either be more specific in the text or include the methods part fur further details. As one example, UAS-Cas9-eGFP transgenic flies newly generated in this study require detail information for those who will order stocks.A separate Materials and methods section has been added.3) The authors verified the efficiency and usefulness of the tool by targeting π genes in the eye. Most of the known target genes resulted in desirable phenotypes in the eye; however, some did not. In this case, it could be a matter of protein stability as the authors explained, but it also could be due to the efficiency or the variability of the new tool in the eye. The tool requires a better validation in a cell-by-cell basis. For example, with the use of in situ hybridization or an antibody against the protein, expressions of target genes could be measured. If the phenotype/expression level could be quantified, efficiencies of whole body mutant, tissue-specific RNAi, and tissue-specific deletion need to be compared.a) In Figure 4A, the authors have shown a truncated band of CG5734 without showing its full-length gene. Even with the presence of truncated size, the intact gene may be still viable in some eye cells. It again will be important to show how efficient it is to use the new tool at a tissue level.4) In addition to ey-gal4, the tool requires validations with additional tissue-specific drivers. This will not only verify its effectiveness but enhance the broader applications.Reviewer #3:Drosophila. There have been several independent efforts to generate Cas9-based systematic gene knockout tools in culture cells and flies. This study attempts to demonstrate the feasibility of constructing guide RNAi library targeted to a relatively small set the genes involved in phosphoinositides (PI) metabolism and signaling. This report shows a few successful cases of gene knockout and a list of 103 targeted genes. Therefore, in principle, the genetic tools generated in this study can be valuable resources in the field of π signaling. However, I feel that the general strategy used in this study is not particularly novel, and more importantly, analysis of their library appears to be rather limited and preliminary for proper evaluation of the presented data.This manuscript describes the generation of a genetic tool kit for tissue-specific and whole animal knockout using CRISPR/Cas9-mediated gene editing in Merits of this work:1) They sequenced S2R+ genome and an isogenic fly genome. After comparing these sequences and the reference genome, gRNA targets were carefully selected to avoid potential mismatches or off-targets.2) This study used double guide RNAs targeted to 1st and last coding exons to generate null mutations by deletion. The use of T2A-mediated eGFP expression also helps to identify Cas9-expressing cells.3) Cas9 mutagenesis targeted to a full set of PI-related genes is a new attempt.Weaknesses:Overall, their strategy for tissue-specific knockout is similar to two recent papers . Hence, novelty of this manuscript is somewhat diminished. In addition, there are major issues related to their methods and data, as listed below.1) Authors designed dgRNA constructs to generate gene deletion. Although they claimed that the targeted deletions were confirmed in S2R+ cells, it is unclear whether the mutations were confirmed in flies.According to subsection “Investigating the role of π signalling in eye development using the dgRNA toolkit”, a few representative ey>Cas9;dgRNA lines were tested for genomic deletion. Then, it appears that the majority of 103 transgenic lines were not tested for molecular lesions. Table 1 shows a list of genes with the size of the genomic deletion. If in fact only a few lines were tested, then it would be difficult to properly evaluate the data in Table 1. It is important to know how many lines were tested for deletion and how many actually showed expected size of deletions.This has been done and added to Supplementary file 1.2) In subsection “Generation of whole fly knockouts and using dgRNA transgenics”, it seems that they tried germline knockout for several genes, but the procedure is not mentioned and the data are described very ambiguously. "The deletions obtained in this manner are highly efficient (between 4-100% efficiency in F1 flies and between 3-70% efficiency in F2 flies) in generating germline knockouts with deletions ranging in size between 400bp and 38kb." From this description, it is difficult to know whether the deletions are highly efficient as claimed. The procedure for making germline knockout and the results need to be described in full detail. In Table 1, a number of tissue-specific knockout show no phenotype. It is possible that some of them may not have expected mutations.The process for making germ line knockouts has been described under Materials and methods Figure 4—figure supplement 2 now included showing examples where a gene gave no phenotype but PCR analysis clearly detected a deletion3) There is no data showing the loss of protein expression in eye discs from eye-specific knockout lines. Crosses with ey-Gal4 will generate mosaic tissues in developing eye discs. It is important to know how efficient is somatic knockout in the region of ey-Gal4 expression.ocrl where inducing a deletion resulted in depletion of protein. Depletion has been quantified .We have now included protein expression data for an example-Reviewer #1:In the revised manuscript, Trivedi et al. provide a significantly improved version of their work with additional analyses and in vivo validations and addressed most of the concerns raised by the reviewers. Two additional points need to be modified to clarify the results.1) In Figure 2B, the authors validated the efficiency of ocrl deletion by immunohistochemistry and showed that ocrl knockout reduces the anti-OCRL staining in the wing disc. However, staining patterns shown in controls are too low, and it is hard to tell whether the antibody used was working in the disc. Providing a reference for the antibody that shows an expression pattern in a representative organ and/or highlighting higher mag images will be critical to reassure the data. If this antibody was first used in an IHC experiment, proper confirmations for the antibody would be needed.Under the present circumstances, it has proved very, very difficult to locate suitable antibodies and procure them for these experiments. Sometimes, when antibodies are described in the literature and apparently readily available, labs are closed/not-accessible or there are shipping restrictions in place. This is a genuine and serious problem limiting our ability to carry out additional experiments. We request all reviewers and editors to please understand the situation.We happened to have some of the ocrl antibody previously procured for another project and have used what was left for a single experiment. Each point on the graph is a single disc.However, to address the scientific question being addressed and reassure reviewers that the staining being presented in Figure 2B.C is both specific and reproducible, we present data previously generated using these same reagents that should answer the questions being posed by the reviewers.Drosophila, we have been working with both a germline knockout of ocrl as well as a nephrocyte specific knockout generated using these same reagents. carried out more detailed analysis using these reagents. This is part of another manuscript under process right now. We present it here to reassure the reviewer that the ocrl antibody and gRNA reagents do reproducibly result in protein depletion when used for tissue specific depletion and that the result shown in the paper with the disc. These data are part of a separate manuscript and presented here in As part of a project on the renal phenotypes of ocrl, modelled in 2) In Figure 3C, gel images do not have proper labels.Labelling has now been done.3) If possible, it will be best to organize the deposition of generated flies to major stock centers before publication and describe it in the text.Drosophila Stock centre. This is now mentioned in the manuscript text.We are already in touch with the DGRC to deposit the gRNA plasmids in this repository. Logistics are in progress. With regard to the transgenic flies we are in touch to deposit these 105 fly stocks flies at the Bloomington Reviewer #2:The authors have added substantial new validation, particularly on the areas of SNP variation, the lesions obtained, and homozygous phenotypes. This now gives the reader greater confidence in the usefulness of their resource – for somatic knockouts one can often find a localized phenotype but not always, perhaps due to perdurance of a stable gene product; and most of their gRNA stocks can be used to generate germline knockouts, and they have established mutant stocks for about half of them.Taken together, I think this collection will be a valuable resource targeting many genes of great interest for the fly signaling and lipid communities. The paper now gives a much better sense of the approach's strengths and limitations, and there is enough information to make many lines of wide use immediately and to develop further mutant stocks for the remainder. To make these lines accessible to the community, I hope that both the gRNA and mutant stocks will be offered promptly to the major stock centers, if the authors haven't done so already.Drosophila Stock centre. This is now mentioned in the manuscript text and the paperwork and logistics is underway.We are already in touch with the DGRC to deposit the gRNA plasmids in this repository. Logistics are in progress. With regard to the transgenic flies we are in touch to deposit these 105 fly stocks flies at the Bloomington Reviewer #3:1) The authors have revised the manuscript with additional experiments and clarifications. The revised manuscript appears to be significantly improved by extensive molecular analysis of targeted mutations in fly tissues. Phenotype analysis with additional GAL4 drivers also supports the effectiveness of the CRISPR/Cas9 tools.2) Considering that the majority of targeted genes by ey-GAL4 do not show phenotypes, it is still possible that targeting might not have caused sufficient loss of protein expression in many genes. In this regard, checking the expression level for only one gene (Ocrl) is a weak point. However, the new data showing deletions in most targeted genes seems to compensate for the weakness. In summary, the revised manuscript seems to be acceptable, but a few mistakes listed below should be corrected.3) Some genotypes in figures are not italicized. Those genotypes need to be fixed.This has been fixed.4) Anti-Ocrl staining appears to be nuclear staining to me, although it is too weak to be certain. A better way to show the mutation effect is to use a different Gal4 such as en-Gal4 to show an internal control. It would be good to show a test for antibody specificity.This point is addressed in detail in response to reviewer #1.