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Two independent targeting reagents and wild-type rescue will distinguish a DCTN1-dependent response from off-target effects, stress, and culture changes. The ResearchHub Foundation RFP attributes the starting observation to Sun, Park, DeTomaso et al., bioRxiv (2026), Fig. 4L. The RFP-linked preprint and source data require independent review. This study tests transcriptional reproducibility—not cholesterol transport, synthesis flux, or disease causality. DCTN1 reduction is not equivalent to a patient variant. Study design: from neuronal preparation to confirmation The experiment has two stages. First, establish a DCTN1 reduction that neurons tolerate and identify when to measure the response. Then repeat the selected condition in new differentiations. Use targeted RT-qPCR and sister-well imaging/protein assays rather than RNA-seq or lipidomics. 1. Start with a defined neuronal platform Use one authenticated human iPSC background carrying doxycycline-inducible NGN2 and a stable, constitutively expressed dCas9-KRAB CRISPRi system. These are platform specifications, not a claim that a particular laboratory already holds this line. Obtain the line identifier, genotype, construct maps, permissions and prior differentiation QC before selection. If no suitable existing platform is available, revise costs and timing before proceeding. NGN2-induced cortical-like neurons provide a relatively uniform population for this first transcriptional test. They are not midbrain dopaminergic neurons and do not model Perry syndrome directly. Test mycoplasma status and document neuronal yield, maturity and DCTN1 expression before committing the full study. 2. Differentiate and mature the neurons before targeting DCTN1 Use the protocols.io method “iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 2,” V.1, June 6, 2023 as the candidate differentiation backbone. Its indexed record describes stable tetracycline-inducible NGN2, induction during days 0–3, maturation-medium stages at days 4/7 and days 10–28, and cortical-marker enrichment. Full-text retrieval failed during this revision; these indexed details are not a verified executable SOP. Obtain and approve the complete version before choosing exact coating, density, media recipes and feeding instructions. Plan to mature cultures through differentiation day 28 before DCTN1 targeting. Record medium and supplement lots, including lipid-containing components, and keep feeding and collection intervals identical across groups. Do not introduce a lipid-depletion challenge into the primary experiment. At day 28, assess MAP2-positive neuronal fraction, morphology, viability and baseline stress in sentinel wells. Proposed qualification gates are at least 90% MAP2-positive cells among counted nuclei and no major baseline stress or neurite deterioration. These are study-design targets, not thresholds established by the cited protocol; validate and freeze them with the receiving laboratory. If cultures fail, improve differentiation rather than adjusting the primary analysis for poor composition. 3. Introduce DCTN1 targeting only after maturation Deliver either of two independent DCTN1 promoter-targeting guides or a non-targeting guide to the mature neurons. Use guide delivery—not a second doxycycline switch—to start CRISPRi. This separates DCTN1 reduction from NGN2 induction. Match delivery reagent, vector burden and handling across groups, and verify guide-delivery coverage so bulk RNA does not conceal a mostly unperturbed population. For rescue groups, introduce wild-type DCTN1 cDNA from a heterologous promoter before targeting and verify expression at the targeting baseline. Promoter-directed CRISPRi should spare this construct, but verify escape experimentally. Match total DCTN1 near control levels and include non-targeting-plus-rescue controls to detect overexpression effects. Rescue dose and lead time are selected during qualification. 4. Use a small pilot to choose the dose and sampling day Across two independent differentiations, test two guide-delivery levels and collect at 3, 7 and 10 days after targeting. These are proposed pilot times, not published DCTN1 kinetics. At each time, measure DCTN1 RNA/protein, live/dead staining, cell counts, neuronal morphology and ATF4/CHOP stress. Run separate pathway-response and health-assay controls to demonstrate assay sensitivity. Aim initially for a moderate 30–60% reduction in DCTN1 protein. Select the earliest time with reproducible engagement for both guides, at least 90% viability relative to matched controls, and acceptable identity, stress and neurite measures. Freeze quantitative acceptable-change bounds after assay qualification; absence of statistical significance is not evidence of cellular health. Do not choose the time producing the largest sterol response. If no interpretable window exists, stop or redesign. 5. Repeat the selected condition in independent differentiations Budget for six new differentiations in the same genetic background. Use the pilot variance and a prespecified meaningful effect to confirm whether six provides adequate power and precision. Increase replication and revise costs if needed; do not present six as guaranteed sufficient. Each differentiation contains the following conditions, randomized across plate positions: Group Purpose Non-targeting guide + empty vector Primary matched control DCTN1 guide A or B + empty vector Two independent tests of DCTN1 reduction DCTN1 guide A or B + wild-type rescue Test perturbation specificity Non-targeting guide + rescue Detect rescue-expression effects Sham delivery Detect delivery-associated injury Non-targeting guide + qualified pathway-response control Demonstrate sterol-transcript assay responsiveness At the selected time, collect separate sister wells for RNA, protein and imaging. This gives 8 conditions × 6 differentiations = 48 confirmatory RNA samples and a minimum of 144 confirmatory culture wells. Reserve the remaining capacity within the 96-sample RNA allowance for pilot/rescue qualification. Final plate maps must include any additional protein-yield wells and controls; do not count sister wells as biological replicates. 6. Measure the response and interpret it Measure HMGCR, HMGCS1, MVK, MVD, FDFT1 and SQLE by RT-qPCR, alongside DCTN1 and validated reference genes. This is a proposed pathway panel, pending source review—not an asserted exact replication panel. Validate primer specificity/efficiency, include no-template and no-RT controls, and freeze the reference-gene and normalization rules before confirmation. Define the primary score as the mean normalized log2 expression across the six sterol genes, with each gene weighted equally. Compare DCTN1 targeting with the matched non-targeting control within differentiation, report guide-specific effects, and prespecify the combined primary contrast. Assess rescue attenuation separately. Report individual genes, HMGCR/SQLE proteins, DCTN1 engagement and blinded health/identity measurements alongside the score. Support requires concordant induction from both guides, attenuation by qualified rescue and acceptable neuronal health. A precise null challenges the premise only when engagement and assay responsiveness are valid and confidence bounds exclude the prespecified meaningful induction. Toxicity, failed rescue/engagement, discordant guides or inadequate precision are inconclusive. Rescue shows perturbation specificity, not cholesterol mediation. Preregister the score, sampling time, meaningful-effect bound, exclusions, missingness and multiplicity plan before confirmation. Analyze at the differentiation level with preparation blocking and report effects with 95% confidence intervals. Keep pilot data separate. Share the plate maps, raw data, QC, code and negative results. Before execution, attach the complete protocols.io version and document every adaptation; the DCTN1 targeting, rescue and sampling scheme above are proposed study-specific additions, not steps validated by that differentiation protocol. Itemized budget — $44,220 USD All amounts are explicit planning allowances, not verified supplier prices or quotes. Quantities cover qualification and the planned confirmation. Existing incubators, microscopy, qPCR and immunoblot equipment are required; access fees are included, but new capital equipment and new cell-line engineering are excluded. Obtain institutional/vendor quotes before purchasing. Item / reagents Quantity × estimated allowance USD Existing neuronal/CRISPRi line access and initial QC 1 package × $1,500 $1,500 Basal medium, supplements and neuronal differentiation/maturation factors 8 preparation allocations × $375 $3,000 Poly-D-lysine/laminin, culture plates, tips and consumables 1 study lot × $1,500 $1,500 Two DCTN1 guides and non-targeting guide: cloning/validation 1 set × $1,000 $1,000 Guide-resistant wild-type DCTN1 rescue and empty vector Synthesis/cloning/validation package × $2,500 $2,500 Delivery reagents/core vector preparation; doxycycline and pathway/stress controls Delivery $1,500 + control reagent set $300 $1,800 RNA extraction with DNase; reverse transcription reagents 2 × 50-prep kits at $600; 100 RT reactions at $600 $1,800 qPCR primers, master mix, plates and seals Primer design/validation $1,000 + assay supplies $1,200 $2,200 Antibodies: DCTN1, HMGCR, SQLE, MAP2, ATF4/CHOP, secondary/loading controls 1 panel allowance × $2,100 $2,100 Immunoblot gels/membranes/detection; live/dead and nuclear stains Blot supplies $600 + stains $400 $1,000 Cell identity/authentication and mycoplasma testing 1 study QC allocation × $600 $600 Research staff: culture, assays and analysis 360 hours × $50 loaded rate $18,000 Microscopy/qPCR core access and analysis support 40 hours × $60 $2,400 Shipping, data storage and permitted administration 1 allocation × $800 $800 Direct costs Subtotal $40,200 Contingency 10% of direct costs $4,020 TOTAL REQUEST Qualification + confirmation $44,220 Stage funding: $16,000 for setup/qualification; $28,220 released after assay gates and power review. These are portions of the total, not additional costs. Add any required institutional overhead beyond the administration allowance before commitment. Optional independent-background replication: separately estimated $12,000–$18,000, subject to quotes and power analysis; excluded from the base request. The RFP’s $1,000 shared pool is not assumed awarded or deducted. Timeline, execution gates and outputs Months 1–2: source review, quotes, permissions, reagent/rescue setup and qualification. Months 3–5: finish qualification, preregister and run confirmation if gates pass. Months 6–7: analysis and reporting. An established neuronal/CRISPRi platform is essential to this schedule; failed qualification stops or redirects work. The receiving laboratory must approve the protocol and demonstrate culture, molecular and imaging capability. Secure applicable human-cell/material, stem-cell and biosafety permissions before execution. Share protocols, QC, data, code and negative/inconclusive results subject to legitimate restrictions. Final effect bounds and sample size must be frozen before confirmation; this budget is an efficient planning estimate, not a guarantee of power or an execution authorization.8b:T2d78,DCTN1 Reduction: Testing the Early Neuronal Sterol Response Candidate design #1 | Budget request: $44,220 USD | Duration: approximately 5–7 months after access and approvals Objective Determine whether titrated DCTN1 reduction induces a coordinated sterol-biosynthesis transcriptional response before neuronal deterioration. Two independent targeting reagents and wild-type rescue will distinguish a DCTN1-dependent response from off-target effects, stress, and culture changes. The ResearchHub Foundation RFP attributes the starting observation to Sun, Park, DeTomaso et al., bioRxiv (2026), Fig. 4L. The RFP-linked preprint and source data require independent review. This study tests transcriptional reproducibility—not cholesterol transport, synthesis flux, or disease causality. DCTN1 reduction is not equivalent to a patient variant. Study design: from neuronal preparation to confirmation The experiment has two stages. First, establish a DCTN1 reduction that neurons tolerate and identify when to measure the response. Then repeat the selected condition in new differentiations. Use targeted RT-qPCR and sister-well imaging/protein assays rather than RNA-seq or lipidomics. 1. Start with a defined neuronal platform Use one authenticated human iPSC background carrying doxycycline-inducible NGN2 and a stable, constitutively expressed dCas9-KRAB CRISPRi system. These are platform specifications, not a claim that a particular laboratory already holds this line. Obtain the line identifier, genotype, construct maps, permissions and prior differentiation QC before selection. If no suitable existing platform is available, revise costs and timing before proceeding. NGN2-induced cortical-like neurons provide a relatively uniform population for this first transcriptional test. They are not midbrain dopaminergic neurons and do not model Perry syndrome directly. Test mycoplasma status and document neuronal yield, maturity and DCTN1 expression before committing the full study. 2. Differentiate and mature the neurons before targeting DCTN1 Use the protocols.io method “iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 2,” V.1, June 6, 2023 as the candidate differentiation backbone. Its indexed record describes stable tetracycline-inducible NGN2, induction during days 0–3, maturation-medium stages at days 4/7 and days 10–28, and cortical-marker enrichment. Full-text retrieval failed during this revision; these indexed details are not a verified executable SOP. Obtain and approve the complete version before choosing exact coating, density, media recipes and feeding instructions. Plan to mature cultures through differentiation day 28 before DCTN1 targeting. Record medium and supplement lots, including lipid-containing components, and keep feeding and collection intervals identical across groups. Do not introduce a lipid-depletion challenge into the primary experiment. At day 28, assess MAP2-positive neuronal fraction, morphology, viability and baseline stress in sentinel wells. Proposed qualification gates are at least 90% MAP2-positive cells among counted nuclei and no major baseline stress or neurite deterioration. These are study-design targets, not thresholds established by the cited protocol; validate and freeze them with the receiving laboratory. If cultures fail, improve differentiation rather than adjusting the primary analysis for poor composition. 3. Introduce DCTN1 targeting only after maturation Deliver either of two independent DCTN1 promoter-targeting guides or a non-targeting guide to the mature neurons. Use guide delivery—not a second doxycycline switch—to start CRISPRi. This separates DCTN1 reduction from NGN2 induction. Match delivery reagent, vector burden and handling across groups, and verify guide-delivery coverage so bulk RNA does not conceal a mostly unperturbed population. For rescue groups, introduce wild-type DCTN1 cDNA from a heterologous promoter before targeting and verify expression at the targeting baseline. Promoter-directed CRISPRi should spare this construct, but verify escape experimentally. Match total DCTN1 near control levels and include non-targeting-plus-rescue controls to detect overexpression effects. Rescue dose and lead time are selected during qualification. 4. Use a small pilot to choose the dose and sampling day Across two independent differentiations, test two guide-delivery levels and collect at 3, 7 and 10 days after targeting. These are proposed pilot times, not published DCTN1 kinetics. At each time, measure DCTN1 RNA/protein, live/dead staining, cell counts, neuronal morphology and ATF4/CHOP stress. Run separate pathway-response and health-assay controls to demonstrate assay sensitivity. Aim initially for a moderate 30–60% reduction in DCTN1 protein. Select the earliest time with reproducible engagement for both guides, at least 90% viability relative to matched controls, and acceptable identity, stress and neurite measures. Freeze quantitative acceptable-change bounds after assay qualification; absence of statistical significance is not evidence of cellular health. Do not choose the time producing the largest sterol response. If no interpretable window exists, stop or redesign. 5. Repeat the selected condition in independent differentiations Budget for six new differentiations in the same genetic background. Use the pilot variance and a prespecified meaningful effect to confirm whether six provides adequate power and precision. Increase replication and revise costs if needed; do not present six as guaranteed sufficient. Each differentiation contains the following conditions, randomized across plate positions: GroupPurposeNon-targeting guide + empty vectorPrimary matched controlDCTN1 guide A or B + empty vectorTwo independent tests of DCTN1 reductionDCTN1 guide A or B + wild-type rescueTest perturbation specificityNon-targeting guide + rescueDetect rescue-expression effectsSham deliveryDetect delivery-associated injuryNon-targeting guide + qualified pathway-response controlDemonstrate sterol-transcript assay responsiveness At the selected time, collect separate sister wells for RNA, protein and imaging. This gives 8 conditions × 6 differentiations = 48 confirmatory RNA samples and a minimum of 144 confirmatory culture wells. Reserve the remaining capacity within the 96-sample RNA allowance for pilot/rescue qualification. Final plate maps must include any additional protein-yield wells and controls; do not count sister wells as biological replicates. 6. Measure the response and interpret it Measure HMGCR, HMGCS1, MVK, MVD, FDFT1 and SQLE by RT-qPCR, alongside DCTN1 and validated reference genes. This is a proposed pathway panel, pending source review—not an asserted exact replication panel. Validate primer specificity/efficiency, include no-template and no-RT controls, and freeze the reference-gene and normalization rules before confirmation. Define the primary score as the mean normalized log2 expression across the six sterol genes, with each gene weighted equally. Compare DCTN1 targeting with the matched non-targeting control within differentiation, report guide-specific effects, and prespecify the combined primary contrast. Assess rescue attenuation separately. Report individual genes, HMGCR/SQLE proteins, DCTN1 engagement and blinded health/identity measurements alongside the score. Support requires concordant induction from both guides, attenuation by qualified rescue and acceptable neuronal health. A precise null challenges the premise only when engagement and assay responsiveness are valid and confidence bounds exclude the prespecified meaningful induction. Toxicity, failed rescue/engagement, discordant guides or inadequate precision are inconclusive. Rescue shows perturbation specificity, not cholesterol mediation. Preregister the score, sampling time, meaningful-effect bound, exclusions, missingness and multiplicity plan before confirmation. Analyze at the differentiation level with preparation blocking and report effects with 95% confidence intervals. Keep pilot data separate. Share the plate maps, raw data, QC, code and negative results. Before execution, attach the complete protocols.io version and document every adaptation; the DCTN1 targeting, rescue and sampling scheme above are proposed study-specific additions, not steps validated by that differentiation protocol. Itemized budget — $44,220 USD All amounts are explicit planning allowances, not verified supplier prices or quotes. Quantities cover qualification and the planned confirmation. Existing incubators, microscopy, qPCR and immunoblot equipment are required; access fees are included, but new capital equipment and new cell-line engineering are excluded. Obtain institutional/vendor quotes before purchasing. Item / reagentsQuantity × estimated allowanceUSDExisting neuronal/CRISPRi line access and initial QC1 package × $1,500$1,500Basal medium, supplements and neuronal differentiation/maturation factors8 preparation allocations × $375$3,000Poly-D-lysine/laminin, culture plates, tips and consumables1 study lot × $1,500$1,500Two DCTN1 guides and non-targeting guide: cloning/validation1 set × $1,000$1,000Guide-resistant wild-type DCTN1 rescue and empty vectorSynthesis/cloning/validation package × $2,500$2,500Delivery reagents/core vector preparation; doxycycline and pathway/stress controlsDelivery $1,500 + control reagent set $300$1,800RNA extraction with DNase; reverse transcription reagents2 × 50-prep kits at $600; 100 RT reactions at $600$1,800qPCR primers, master mix, plates and sealsPrimer design/validation $1,000 + assay supplies $1,200$2,200Antibodies: DCTN1, HMGCR, SQLE, MAP2, ATF4/CHOP, secondary/loading controls1 panel allowance × $2,100$2,100Immunoblot gels/membranes/detection; live/dead and nuclear stainsBlot supplies $600 + stains $400$1,000Cell identity/authentication and mycoplasma testing1 study QC allocation × $600$600Research staff: culture, assays and analysis360 hours × $50 loaded rate$18,000Microscopy/qPCR core access and analysis support40 hours × $60$2,400Shipping, data storage and permitted administration1 allocation × $800$800Direct costsSubtotal$40,200Contingency10% of direct costs$4,020TOTAL REQUESTQualification + confirmation$44,220 Stage funding: $16,000 for setup/qualification; $28,220 released after assay gates and power review. These are portions of the total, not additional costs. Add any required institutional overhead beyond the administration allowance before commitment. Optional independent-background replication: separately estimated $12,000–$18,000, subject to quotes and power analysis; excluded from the base request. The RFP’s $1,000 shared pool is not assumed awarded or deducted. Timeline, execution gates and outputs Months 1–2: source review, quotes, permissions, reagent/rescue setup and qualification. Months 3–5: finish qualification, preregister and run confirmation if gates pass. Months 6–7: analysis and reporting. An established neuronal/CRISPRi platform is essential to this schedule; failed qualification stops or redirects work. The receiving laboratory must approve the protocol and demonstrate culture, molecular and imaging capability. Secure applicable human-cell/material, stem-cell and biosafety permissions before execution. Share protocols, QC, data, code and negative/inconclusive results subject to legitimate restrictions. 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First, establish a DCTN1 reduction that neurons tolerate and identify when to measure the response. Then repeat the selected condition in new differentiations. Use targeted RT-qPCR and sister-well imaging/protein assays rather than RNA-seq or lipidomics."}]}, {"type": "heading", "attrs": {"id": "77607ac1-ff24-4df0-943b-b51a9ab03256", "data-toc-id": "77607ac1-ff24-4df0-943b-b51a9ab03256", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "1. Start with a defined neuronal platform"}]}, {"type": "paragraph", "attrs": {"id": "2d4a78a6-54af-4c0c-9c19-f1ef58918d5f", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Use one authenticated human iPSC background carrying doxycycline-inducible NGN2 and a stable, constitutively expressed dCas9-KRAB CRISPRi system. These are platform specifications, not a claim that a particular laboratory already holds this line. Obtain the line identifier, genotype, construct maps, permissions and prior differentiation QC before selection. If no suitable existing platform is available, revise costs and timing before proceeding."}]}, {"type": "paragraph", "attrs": {"id": "f588f8a5-4dce-42f3-aacd-1194c00e35ef", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "NGN2-induced cortical-like neurons provide a relatively uniform population for this first transcriptional test. They are not midbrain dopaminergic neurons and do not model Perry syndrome directly. Test mycoplasma status and document neuronal yield, maturity and DCTN1 expression before committing the full study."}]}, {"type": "heading", "attrs": {"id": "b1df5153-1f68-47ed-89fa-e03863c32a97", "data-toc-id": "b1df5153-1f68-47ed-89fa-e03863c32a97", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "2. Differentiate and mature the neurons before targeting DCTN1"}]}, {"type": "paragraph", "attrs": {"id": "4ebcac7c-290c-4ee1-8894-03a532b5d289", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Use the protocols.io method "}, {"type": "text", "marks": [{"type": "link", "attrs": {"href": "https://www.protocols.io/view/indi-transcription-factor-ngn2-differentiation-of-bzchp2t6", "target": "_blank", "rel": "noopener noreferrer nofollow", "class": "link", "title": null}}], "text": "\u201ciNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 2,\u201d V.1, June 6, 2023"}, {"type": "text", "text": " as the candidate differentiation backbone. Its indexed record describes stable tetracycline-inducible NGN2, induction during days 0\u20133, maturation-medium stages at days 4/7 and days 10\u201328, and cortical-marker enrichment. Full-text retrieval failed during this revision; these indexed details are not a verified executable SOP. Obtain and approve the complete version before choosing exact coating, density, media recipes and feeding instructions."}]}, {"type": "paragraph", "attrs": {"id": "995f513b-b3f5-4329-98ba-44ddf9769c49", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Plan to mature cultures through differentiation day 28 before DCTN1 targeting. Record medium and supplement lots, including lipid-containing components, and keep feeding and collection intervals identical across groups. Do not introduce a lipid-depletion challenge into the primary experiment. At day 28, assess MAP2-positive neuronal fraction, morphology, viability and baseline stress in sentinel wells."}]}, {"type": "paragraph", "attrs": {"id": "c3e5b4f9-8aed-4f91-a13d-2782a7754cab", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Proposed qualification gates are at least 90% MAP2-positive cells among counted nuclei and no major baseline stress or neurite deterioration. These are study-design targets, not thresholds established by the cited protocol; validate and freeze them with the receiving laboratory. If cultures fail, improve differentiation rather than adjusting the primary analysis for poor composition."}]}, {"type": "heading", "attrs": {"id": "c80646a7-3d80-4b17-94f2-8aed0426bfa2", "data-toc-id": "c80646a7-3d80-4b17-94f2-8aed0426bfa2", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "3. Introduce DCTN1 targeting only after maturation"}]}, {"type": "paragraph", "attrs": {"id": "e6e47268-7fb8-4177-a94d-f92eeb8543b1", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Deliver either of two independent DCTN1 promoter-targeting guides or a non-targeting guide to the mature neurons. Use guide delivery\u2014not a second doxycycline switch\u2014to start CRISPRi. This separates DCTN1 reduction from NGN2 induction. Match delivery reagent, vector burden and handling across groups, and verify guide-delivery coverage so bulk RNA does not conceal a mostly unperturbed population."}]}, {"type": "paragraph", "attrs": {"id": "2a8522fd-b91d-4901-a2f8-8ba09b68e599", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "For rescue groups, introduce wild-type DCTN1 cDNA from a heterologous promoter before targeting and verify expression at the targeting baseline. Promoter-directed CRISPRi should spare this construct, but verify escape experimentally. Match total DCTN1 near control levels and include non-targeting-plus-rescue controls to detect overexpression effects. Rescue dose and lead time are selected during qualification."}]}, {"type": "heading", "attrs": {"id": "85aa3b03-a64d-45ef-9978-f615b2a6d52d", "data-toc-id": "85aa3b03-a64d-45ef-9978-f615b2a6d52d", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "4. Use a small pilot to choose the dose and sampling day"}]}, {"type": "paragraph", "attrs": {"id": "be374eaf-6922-4522-969b-7ef8cf9ed9f5", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Across two independent differentiations, test two guide-delivery levels and collect at 3, 7 and 10 days after targeting. These are proposed pilot times, not published DCTN1 kinetics. At each time, measure DCTN1 RNA/protein, live/dead staining, cell counts, neuronal morphology and ATF4/CHOP stress. Run separate pathway-response and health-assay controls to demonstrate assay sensitivity."}]}, {"type": "paragraph", "attrs": {"id": "a66be6c7-a4eb-4775-bc01-ccf6a7742e13", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Aim initially for a moderate 30\u201360% reduction in DCTN1 protein. Select the earliest time with reproducible engagement for both guides, at least 90% viability relative to matched controls, and acceptable identity, stress and neurite measures. Freeze quantitative acceptable-change bounds after assay qualification; absence of statistical significance is not evidence of cellular health. Do not choose the time producing the largest sterol response. If no interpretable window exists, stop or redesign."}]}, {"type": "heading", "attrs": {"id": "4c331a32-bd8a-4a47-ba27-6218e12740eb", "data-toc-id": "4c331a32-bd8a-4a47-ba27-6218e12740eb", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "5. Repeat the selected condition in independent differentiations"}]}, {"type": "paragraph", "attrs": {"id": "e6ef1bf6-b147-4dd3-a54b-0a202374bdaa", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Budget for six new differentiations in the same genetic background. Use the pilot variance and a prespecified meaningful effect to confirm whether six provides adequate power and precision. Increase replication and revise costs if needed; do not present six as guaranteed sufficient. Each differentiation contains the following conditions, randomized across plate positions:"}]}, {"type": "table", "attrs": {"id": "f78db6ce-9666-4f36-bf40-b83625952c65"}, "content": [{"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "dd63d2d2-2d0b-4bf9-a412-b400912fe074", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Group"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "310bb685-b8ba-4f06-988d-78350696bf94", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Purpose"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "8ce68500-9828-4d0f-a7da-701dd5921c8d", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Non-targeting guide + empty vector"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "7a85bd47-7e88-49a4-aaac-eda15028452f", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Primary matched control"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "fff9b2ba-9097-45e2-ac05-515db3bcc431", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "DCTN1 guide A or B + empty vector"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "9b0fb2e2-e497-4b3b-ae88-c361afbb784a", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Two independent tests of DCTN1 reduction"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "0aed9c19-dbfa-4d91-8a77-a997a880802b", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "DCTN1 guide A or B + wild-type rescue"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "625ea505-b1e5-43d5-994e-d3dc6bf03db7", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Test perturbation specificity"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "a98a61c5-7dce-4cd5-866f-d9e62701cefe", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Non-targeting guide + rescue"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "c7eec004-1294-49a3-94da-d48a5dc1b8dc", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Detect rescue-expression effects"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "9faba7ca-a329-49ad-9514-d1e6a8eaafb0", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Sham delivery"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "ea063c0d-f657-4844-b9cb-823bb2d422e9", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Detect delivery-associated injury"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "f6eb6335-d620-4bd9-9fae-c861a71aa32e", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Non-targeting guide + qualified pathway-response control"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "c65ad965-b234-4478-93d5-22bd1917b919", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Demonstrate sterol-transcript assay responsiveness"}]}]}]}]}, {"type": "paragraph", "attrs": {"id": "a7848fe1-241a-4c3b-97c5-7aab3c648f9e", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "At the selected time, collect separate sister wells for RNA, protein and imaging. This gives 8 conditions \u00d7 6 differentiations = 48 confirmatory RNA samples and a minimum of 144 confirmatory culture wells. Reserve the remaining capacity within the 96-sample RNA allowance for pilot/rescue qualification. Final plate maps must include any additional protein-yield wells and controls; do not count sister wells as biological replicates."}]}, {"type": "heading", "attrs": {"id": "2fc8ebed-fd9d-4043-8944-29db781558d2", "data-toc-id": "2fc8ebed-fd9d-4043-8944-29db781558d2", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "6. Measure the response and interpret it"}]}, {"type": "paragraph", "attrs": {"id": "1314cd39-4ba5-4553-9be0-7866268f8a4b", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Measure HMGCR, HMGCS1, MVK, MVD, FDFT1 and SQLE by RT-qPCR, alongside DCTN1 and validated reference genes. This is a proposed pathway panel, pending source review\u2014not an asserted exact replication panel. Validate primer specificity/efficiency, include no-template and no-RT controls, and freeze the reference-gene and normalization rules before confirmation."}]}, {"type": "paragraph", "attrs": {"id": "0da1e460-2672-4b69-b34f-3f0dc5a898b6", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Define the primary score as the mean normalized log2 expression across the six sterol genes, with each gene weighted equally. Compare DCTN1 targeting with the matched non-targeting control within differentiation, report guide-specific effects, and prespecify the combined primary contrast. Assess rescue attenuation separately. Report individual genes, HMGCR/SQLE proteins, DCTN1 engagement and blinded health/identity measurements alongside the score."}]}, {"type": "paragraph", "attrs": {"id": "6b967740-d2e1-40b5-8bb3-c08b4fad5c07", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Support requires concordant induction from both guides, attenuation by qualified rescue and acceptable neuronal health. A precise null challenges the premise only when engagement and assay responsiveness are valid and confidence bounds exclude the prespecified meaningful induction. Toxicity, failed rescue/engagement, discordant guides or inadequate precision are inconclusive. Rescue shows perturbation specificity, not cholesterol mediation."}]}, {"type": "paragraph", "attrs": {"id": "d300c9aa-a623-402a-96bb-ba5f81d674e9", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Preregister the score, sampling time, meaningful-effect bound, exclusions, missingness and multiplicity plan before confirmation. Analyze at the differentiation level with preparation blocking and report effects with 95% confidence intervals. Keep pilot data separate. Share the plate maps, raw data, QC, code and negative results. 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{"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "f993c4c1-0725-4b52-b2d8-f2f78270be94", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Contingency"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "9733c86d-f11b-428b-8337-d27338b57129", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "10% of direct costs"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "bb2b0827-c9fd-4e8f-ad9a-9ff26343c9be", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "$4,020"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "996b9436-0fa2-4d37-ba6b-e3f40bb56416", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "TOTAL REQUEST"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "9540c4ca-35df-4353-88f6-33fa08f05b15", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Qualification + confirmation"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "a6de7eb5-4dd0-498d-b749-5c9374fa09f4", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "$44,220"}]}]}]}]}, {"type": "paragraph", "attrs": {"id": "4b150cf7-f509-40a3-8e48-1db61318f056", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Stage funding: $16,000 for setup/qualification; $28,220 released after assay gates and power review. These are portions of the total, not additional costs. Add any required institutional overhead beyond the administration allowance before commitment. Optional independent-background replication: separately estimated $12,000\u2013$18,000, subject to quotes and power analysis; excluded from the base request. The RFP\u2019s $1,000 shared pool is not assumed awarded or deducted."}]}, {"type": "heading", "attrs": {"id": "9f1c0fb2-ebb4-4a51-8566-9fa04c414e42", "data-toc-id": "9f1c0fb2-ebb4-4a51-8566-9fa04c414e42", "textAlign": null, "level": 2}, "content": [{"type": "text", "text": "Timeline, execution gates and outputs"}]}, {"type": "paragraph", "attrs": {"id": "c7eacc19-d8e8-4ded-a50a-d55ef4d4218c", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Months 1\u20132: source review, quotes, permissions, reagent/rescue setup and qualification. "}, {"type": "hardBreak"}, {"type": "text", "text": "Months 3\u20135: finish qualification, preregister and run confirmation if gates pass. "}, {"type": "hardBreak"}, {"type": "text", "text": "Months 6\u20137: analysis and reporting. An established neuronal/CRISPRi platform is essential to this schedule; failed qualification stops or redirects work."}]}, {"type": "paragraph", "attrs": {"id": "7e493e07-da3f-40e1-8199-edb7e63dfd60", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "The receiving laboratory must approve the protocol and demonstrate culture, molecular and imaging capability. Secure applicable human-cell/material, stem-cell and biosafety permissions before execution. Share protocols, QC, data, code and negative/inconclusive results subject to legitimate restrictions. Final effect bounds and sample size must be frozen before confirmation; this budget is an efficient planning estimate, not a guarantee of power or an execution authorization."}]}]}8d:T2d78,DCTN1 Reduction: Testing the Early Neuronal Sterol Response Candidate design #1 | Budget request: $44,220 USD | Duration: approximately 5–7 months after access and approvals Objective Determine whether titrated DCTN1 reduction induces a coordinated sterol-biosynthesis transcriptional response before neuronal deterioration. Two independent targeting reagents and wild-type rescue will distinguish a DCTN1-dependent response from off-target effects, stress, and culture changes. The ResearchHub Foundation RFP attributes the starting observation to Sun, Park, DeTomaso et al., bioRxiv (2026), Fig. 4L. The RFP-linked preprint and source data require independent review. This study tests transcriptional reproducibility—not cholesterol transport, synthesis flux, or disease causality. DCTN1 reduction is not equivalent to a patient variant. Study design: from neuronal preparation to confirmation The experiment has two stages. First, establish a DCTN1 reduction that neurons tolerate and identify when to measure the response. Then repeat the selected condition in new differentiations. Use targeted RT-qPCR and sister-well imaging/protein assays rather than RNA-seq or lipidomics. 1. Start with a defined neuronal platform Use one authenticated human iPSC background carrying doxycycline-inducible NGN2 and a stable, constitutively expressed dCas9-KRAB CRISPRi system. These are platform specifications, not a claim that a particular laboratory already holds this line. Obtain the line identifier, genotype, construct maps, permissions and prior differentiation QC before selection. If no suitable existing platform is available, revise costs and timing before proceeding. NGN2-induced cortical-like neurons provide a relatively uniform population for this first transcriptional test. They are not midbrain dopaminergic neurons and do not model Perry syndrome directly. Test mycoplasma status and document neuronal yield, maturity and DCTN1 expression before committing the full study. 2. Differentiate and mature the neurons before targeting DCTN1 Use the protocols.io method “iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 2,” V.1, June 6, 2023 as the candidate differentiation backbone. Its indexed record describes stable tetracycline-inducible NGN2, induction during days 0–3, maturation-medium stages at days 4/7 and days 10–28, and cortical-marker enrichment. Full-text retrieval failed during this revision; these indexed details are not a verified executable SOP. Obtain and approve the complete version before choosing exact coating, density, media recipes and feeding instructions. Plan to mature cultures through differentiation day 28 before DCTN1 targeting. Record medium and supplement lots, including lipid-containing components, and keep feeding and collection intervals identical across groups. Do not introduce a lipid-depletion challenge into the primary experiment. At day 28, assess MAP2-positive neuronal fraction, morphology, viability and baseline stress in sentinel wells. Proposed qualification gates are at least 90% MAP2-positive cells among counted nuclei and no major baseline stress or neurite deterioration. These are study-design targets, not thresholds established by the cited protocol; validate and freeze them with the receiving laboratory. If cultures fail, improve differentiation rather than adjusting the primary analysis for poor composition. 3. Introduce DCTN1 targeting only after maturation Deliver either of two independent DCTN1 promoter-targeting guides or a non-targeting guide to the mature neurons. Use guide delivery—not a second doxycycline switch—to start CRISPRi. This separates DCTN1 reduction from NGN2 induction. Match delivery reagent, vector burden and handling across groups, and verify guide-delivery coverage so bulk RNA does not conceal a mostly unperturbed population. For rescue groups, introduce wild-type DCTN1 cDNA from a heterologous promoter before targeting and verify expression at the targeting baseline. Promoter-directed CRISPRi should spare this construct, but verify escape experimentally. Match total DCTN1 near control levels and include non-targeting-plus-rescue controls to detect overexpression effects. Rescue dose and lead time are selected during qualification. 4. Use a small pilot to choose the dose and sampling day Across two independent differentiations, test two guide-delivery levels and collect at 3, 7 and 10 days after targeting. These are proposed pilot times, not published DCTN1 kinetics. At each time, measure DCTN1 RNA/protein, live/dead staining, cell counts, neuronal morphology and ATF4/CHOP stress. Run separate pathway-response and health-assay controls to demonstrate assay sensitivity. Aim initially for a moderate 30–60% reduction in DCTN1 protein. Select the earliest time with reproducible engagement for both guides, at least 90% viability relative to matched controls, and acceptable identity, stress and neurite measures. Freeze quantitative acceptable-change bounds after assay qualification; absence of statistical significance is not evidence of cellular health. Do not choose the time producing the largest sterol response. If no interpretable window exists, stop or redesign. 5. Repeat the selected condition in independent differentiations Budget for six new differentiations in the same genetic background. Use the pilot variance and a prespecified meaningful effect to confirm whether six provides adequate power and precision. Increase replication and revise costs if needed; do not present six as guaranteed sufficient. Each differentiation contains the following conditions, randomized across plate positions: GroupPurposeNon-targeting guide + empty vectorPrimary matched controlDCTN1 guide A or B + empty vectorTwo independent tests of DCTN1 reductionDCTN1 guide A or B + wild-type rescueTest perturbation specificityNon-targeting guide + rescueDetect rescue-expression effectsSham deliveryDetect delivery-associated injuryNon-targeting guide + qualified pathway-response controlDemonstrate sterol-transcript assay responsiveness At the selected time, collect separate sister wells for RNA, protein and imaging. This gives 8 conditions × 6 differentiations = 48 confirmatory RNA samples and a minimum of 144 confirmatory culture wells. Reserve the remaining capacity within the 96-sample RNA allowance for pilot/rescue qualification. Final plate maps must include any additional protein-yield wells and controls; do not count sister wells as biological replicates. 6. Measure the response and interpret it Measure HMGCR, HMGCS1, MVK, MVD, FDFT1 and SQLE by RT-qPCR, alongside DCTN1 and validated reference genes. This is a proposed pathway panel, pending source review—not an asserted exact replication panel. Validate primer specificity/efficiency, include no-template and no-RT controls, and freeze the reference-gene and normalization rules before confirmation. Define the primary score as the mean normalized log2 expression across the six sterol genes, with each gene weighted equally. Compare DCTN1 targeting with the matched non-targeting control within differentiation, report guide-specific effects, and prespecify the combined primary contrast. Assess rescue attenuation separately. Report individual genes, HMGCR/SQLE proteins, DCTN1 engagement and blinded health/identity measurements alongside the score. Support requires concordant induction from both guides, attenuation by qualified rescue and acceptable neuronal health. A precise null challenges the premise only when engagement and assay responsiveness are valid and confidence bounds exclude the prespecified meaningful induction. Toxicity, failed rescue/engagement, discordant guides or inadequate precision are inconclusive. Rescue shows perturbation specificity, not cholesterol mediation. Preregister the score, sampling time, meaningful-effect bound, exclusions, missingness and multiplicity plan before confirmation. Analyze at the differentiation level with preparation blocking and report effects with 95% confidence intervals. Keep pilot data separate. Share the plate maps, raw data, QC, code and negative results. Before execution, attach the complete protocols.io version and document every adaptation; the DCTN1 targeting, rescue and sampling scheme above are proposed study-specific additions, not steps validated by that differentiation protocol. Itemized budget — $44,220 USD All amounts are explicit planning allowances, not verified supplier prices or quotes. Quantities cover qualification and the planned confirmation. Existing incubators, microscopy, qPCR and immunoblot equipment are required; access fees are included, but new capital equipment and new cell-line engineering are excluded. Obtain institutional/vendor quotes before purchasing. Item / reagentsQuantity × estimated allowanceUSDExisting neuronal/CRISPRi line access and initial QC1 package × $1,500$1,500Basal medium, supplements and neuronal differentiation/maturation factors8 preparation allocations × $375$3,000Poly-D-lysine/laminin, culture plates, tips and consumables1 study lot × $1,500$1,500Two DCTN1 guides and non-targeting guide: cloning/validation1 set × $1,000$1,000Guide-resistant wild-type DCTN1 rescue and empty vectorSynthesis/cloning/validation package × $2,500$2,500Delivery reagents/core vector preparation; doxycycline and pathway/stress controlsDelivery $1,500 + control reagent set $300$1,800RNA extraction with DNase; reverse transcription reagents2 × 50-prep kits at $600; 100 RT reactions at $600$1,800qPCR primers, master mix, plates and sealsPrimer design/validation $1,000 + assay supplies $1,200$2,200Antibodies: DCTN1, HMGCR, SQLE, MAP2, ATF4/CHOP, secondary/loading controls1 panel allowance × $2,100$2,100Immunoblot gels/membranes/detection; live/dead and nuclear stainsBlot supplies $600 + stains $400$1,000Cell identity/authentication and mycoplasma testing1 study QC allocation × $600$600Research staff: culture, assays and analysis360 hours × $50 loaded rate$18,000Microscopy/qPCR core access and analysis support40 hours × $60$2,400Shipping, data storage and permitted administration1 allocation × $800$800Direct costsSubtotal$40,200Contingency10% of direct costs$4,020TOTAL REQUESTQualification + confirmation$44,220 Stage funding: $16,000 for setup/qualification; $28,220 released after assay gates and power review. These are portions of the total, not additional costs. Add any required institutional overhead beyond the administration allowance before commitment. Optional independent-background replication: separately estimated $12,000–$18,000, subject to quotes and power analysis; excluded from the base request. The RFP’s $1,000 shared pool is not assumed awarded or deducted. Timeline, execution gates and outputs Months 1–2: source review, quotes, permissions, reagent/rescue setup and qualification. Months 3–5: finish qualification, preregister and run confirmation if gates pass. Months 6–7: analysis and reporting. An established neuronal/CRISPRi platform is essential to this schedule; failed qualification stops or redirects work. The receiving laboratory must approve the protocol and demonstrate culture, molecular and imaging capability. Secure applicable human-cell/material, stem-cell and biosafety permissions before execution. Share protocols, QC, data, code and negative/inconclusive results subject to legitimate restrictions. Final effect bounds and sample size must be frozen before confirmation; this budget is an efficient planning estimate, not a guarantee of power or an execution authorization.8e:T9301,{"type": "doc", "content": [{"type": "heading", "attrs": {"id": "9f7e1198-861d-4dc8-80e3-a9726d377d2f", "data-toc-id": "9f7e1198-861d-4dc8-80e3-a9726d377d2f", "textAlign": null, "level": 1}, "content": [{"type": "text", "text": "DCTN1 Reduction: Testing the Early Neuronal Sterol Response"}]}, {"type": "paragraph", "attrs": {"id": "7c600945-edbe-4944-9807-96407fefb1de", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Candidate design #1 | Budget request: $44,220 USD | Duration: approximately 5\u20137 months after access and approvals"}]}, {"type": "heading", "attrs": {"id": "acc67ca3-c996-4549-a1e9-ba8963af6888", "data-toc-id": "acc67ca3-c996-4549-a1e9-ba8963af6888", "textAlign": null, "level": 2}, "content": [{"type": "text", "text": "Objective"}]}, {"type": "paragraph", "attrs": {"id": "91a4aa28-78bf-4d61-99c6-b8450a46788b", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Determine whether titrated DCTN1 reduction induces a coordinated sterol-biosynthesis transcriptional response before neuronal deterioration. Two independent targeting reagents and wild-type rescue will distinguish a DCTN1-dependent response from off-target effects, stress, and culture changes."}]}, {"type": "paragraph", "attrs": {"id": "cdf63b3c-5918-4c1d-87d5-24b5f12b7912", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "The "}, {"type": "text", "marks": [{"type": "link", "attrs": {"href": "https://www.researchhub.com/grant/33104/testing-the-dctn1cholesterol-hypothesis-in-the-wet-lab", "target": "_blank", "rel": "noopener noreferrer nofollow", "class": "link", "title": null}}], "text": "ResearchHub Foundation RFP"}, {"type": "text", "text": " attributes the starting observation to Sun, Park, DeTomaso et al., bioRxiv (2026), Fig. 4L. The "}, {"type": "text", "marks": [{"type": "link", "attrs": {"href": "https://doi.org/10.64898/2026.10.15.759436", "target": "_blank", "rel": "noopener noreferrer nofollow", "class": "link", "title": null}}], "text": "RFP-linked preprint"}, {"type": "text", "text": " and source data require independent review. This study tests transcriptional reproducibility\u2014not cholesterol transport, synthesis flux, or disease causality. DCTN1 reduction is not equivalent to a patient variant."}]}, {"type": "heading", "attrs": {"id": "74ca0021-63d7-4a9f-9cf5-ec20a4586f65", "data-toc-id": "74ca0021-63d7-4a9f-9cf5-ec20a4586f65", "textAlign": null, "level": 2}, "content": [{"type": "text", "text": "Study design: from neuronal preparation to confirmation"}]}, {"type": "paragraph", "attrs": {"id": "b17d146e-71eb-46b0-ac39-ff021351f7d9", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "The experiment has two stages. First, establish a DCTN1 reduction that neurons tolerate and identify when to measure the response. Then repeat the selected condition in new differentiations. Use targeted RT-qPCR and sister-well imaging/protein assays rather than RNA-seq or lipidomics."}]}, {"type": "heading", "attrs": {"id": "77607ac1-ff24-4df0-943b-b51a9ab03256", "data-toc-id": "77607ac1-ff24-4df0-943b-b51a9ab03256", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "1. Start with a defined neuronal platform"}]}, {"type": "paragraph", "attrs": {"id": "2d4a78a6-54af-4c0c-9c19-f1ef58918d5f", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Use one authenticated human iPSC background carrying doxycycline-inducible NGN2 and a stable, constitutively expressed dCas9-KRAB CRISPRi system. These are platform specifications, not a claim that a particular laboratory already holds this line. Obtain the line identifier, genotype, construct maps, permissions and prior differentiation QC before selection. If no suitable existing platform is available, revise costs and timing before proceeding."}]}, {"type": "paragraph", "attrs": {"id": "f588f8a5-4dce-42f3-aacd-1194c00e35ef", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "NGN2-induced cortical-like neurons provide a relatively uniform population for this first transcriptional test. They are not midbrain dopaminergic neurons and do not model Perry syndrome directly. Test mycoplasma status and document neuronal yield, maturity and DCTN1 expression before committing the full study."}]}, {"type": "heading", "attrs": {"id": "b1df5153-1f68-47ed-89fa-e03863c32a97", "data-toc-id": "b1df5153-1f68-47ed-89fa-e03863c32a97", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "2. Differentiate and mature the neurons before targeting DCTN1"}]}, {"type": "paragraph", "attrs": {"id": "4ebcac7c-290c-4ee1-8894-03a532b5d289", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Use the protocols.io method "}, {"type": "text", "marks": [{"type": "link", "attrs": {"href": "https://www.protocols.io/view/indi-transcription-factor-ngn2-differentiation-of-bzchp2t6", "target": "_blank", "rel": "noopener noreferrer nofollow", "class": "link", "title": null}}], "text": "\u201ciNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 2,\u201d V.1, June 6, 2023"}, {"type": "text", "text": " as the candidate differentiation backbone. Its indexed record describes stable tetracycline-inducible NGN2, induction during days 0\u20133, maturation-medium stages at days 4/7 and days 10\u201328, and cortical-marker enrichment. Full-text retrieval failed during this revision; these indexed details are not a verified executable SOP. Obtain and approve the complete version before choosing exact coating, density, media recipes and feeding instructions."}]}, {"type": "paragraph", "attrs": {"id": "995f513b-b3f5-4329-98ba-44ddf9769c49", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Plan to mature cultures through differentiation day 28 before DCTN1 targeting. Record medium and supplement lots, including lipid-containing components, and keep feeding and collection intervals identical across groups. Do not introduce a lipid-depletion challenge into the primary experiment. At day 28, assess MAP2-positive neuronal fraction, morphology, viability and baseline stress in sentinel wells."}]}, {"type": "paragraph", "attrs": {"id": "c3e5b4f9-8aed-4f91-a13d-2782a7754cab", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Proposed qualification gates are at least 90% MAP2-positive cells among counted nuclei and no major baseline stress or neurite deterioration. These are study-design targets, not thresholds established by the cited protocol; validate and freeze them with the receiving laboratory. If cultures fail, improve differentiation rather than adjusting the primary analysis for poor composition."}]}, {"type": "heading", "attrs": {"id": "c80646a7-3d80-4b17-94f2-8aed0426bfa2", "data-toc-id": "c80646a7-3d80-4b17-94f2-8aed0426bfa2", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "3. Introduce DCTN1 targeting only after maturation"}]}, {"type": "paragraph", "attrs": {"id": "e6e47268-7fb8-4177-a94d-f92eeb8543b1", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Deliver either of two independent DCTN1 promoter-targeting guides or a non-targeting guide to the mature neurons. Use guide delivery\u2014not a second doxycycline switch\u2014to start CRISPRi. This separates DCTN1 reduction from NGN2 induction. Match delivery reagent, vector burden and handling across groups, and verify guide-delivery coverage so bulk RNA does not conceal a mostly unperturbed population."}]}, {"type": "paragraph", "attrs": {"id": "2a8522fd-b91d-4901-a2f8-8ba09b68e599", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "For rescue groups, introduce wild-type DCTN1 cDNA from a heterologous promoter before targeting and verify expression at the targeting baseline. Promoter-directed CRISPRi should spare this construct, but verify escape experimentally. Match total DCTN1 near control levels and include non-targeting-plus-rescue controls to detect overexpression effects. Rescue dose and lead time are selected during qualification."}]}, {"type": "heading", "attrs": {"id": "85aa3b03-a64d-45ef-9978-f615b2a6d52d", "data-toc-id": "85aa3b03-a64d-45ef-9978-f615b2a6d52d", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "4. Use a small pilot to choose the dose and sampling day"}]}, {"type": "paragraph", "attrs": {"id": "be374eaf-6922-4522-969b-7ef8cf9ed9f5", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Across two independent differentiations, test two guide-delivery levels and collect at 3, 7 and 10 days after targeting. These are proposed pilot times, not published DCTN1 kinetics. At each time, measure DCTN1 RNA/protein, live/dead staining, cell counts, neuronal morphology and ATF4/CHOP stress. Run separate pathway-response and health-assay controls to demonstrate assay sensitivity."}]}, {"type": "paragraph", "attrs": {"id": "a66be6c7-a4eb-4775-bc01-ccf6a7742e13", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Aim initially for a moderate 30\u201360% reduction in DCTN1 protein. Select the earliest time with reproducible engagement for both guides, at least 90% viability relative to matched controls, and acceptable identity, stress and neurite measures. Freeze quantitative acceptable-change bounds after assay qualification; absence of statistical significance is not evidence of cellular health. Do not choose the time producing the largest sterol response. If no interpretable window exists, stop or redesign."}]}, {"type": "heading", "attrs": {"id": "4c331a32-bd8a-4a47-ba27-6218e12740eb", "data-toc-id": "4c331a32-bd8a-4a47-ba27-6218e12740eb", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "5. Repeat the selected condition in independent differentiations"}]}, {"type": "paragraph", "attrs": {"id": "e6ef1bf6-b147-4dd3-a54b-0a202374bdaa", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Budget for six new differentiations in the same genetic background. Use the pilot variance and a prespecified meaningful effect to confirm whether six provides adequate power and precision. Increase replication and revise costs if needed; do not present six as guaranteed sufficient. Each differentiation contains the following conditions, randomized across plate positions:"}]}, {"type": "table", "attrs": {"id": "f78db6ce-9666-4f36-bf40-b83625952c65"}, "content": [{"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "dd63d2d2-2d0b-4bf9-a412-b400912fe074", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Group"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "310bb685-b8ba-4f06-988d-78350696bf94", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Purpose"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "8ce68500-9828-4d0f-a7da-701dd5921c8d", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Non-targeting guide + empty vector"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "7a85bd47-7e88-49a4-aaac-eda15028452f", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Primary matched control"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "fff9b2ba-9097-45e2-ac05-515db3bcc431", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "DCTN1 guide A or B + empty vector"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "9b0fb2e2-e497-4b3b-ae88-c361afbb784a", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Two independent tests of DCTN1 reduction"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "0aed9c19-dbfa-4d91-8a77-a997a880802b", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "DCTN1 guide A or B + wild-type rescue"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "625ea505-b1e5-43d5-994e-d3dc6bf03db7", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Test perturbation specificity"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "a98a61c5-7dce-4cd5-866f-d9e62701cefe", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Non-targeting guide + rescue"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "c7eec004-1294-49a3-94da-d48a5dc1b8dc", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Detect rescue-expression effects"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "9faba7ca-a329-49ad-9514-d1e6a8eaafb0", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Sham delivery"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "ea063c0d-f657-4844-b9cb-823bb2d422e9", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Detect delivery-associated injury"}]}]}]}, {"type": "tableRow", "content": [{"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "f6eb6335-d620-4bd9-9fae-c861a71aa32e", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Non-targeting guide + qualified pathway-response control"}]}]}, {"type": "tableCell", "attrs": {"colspan": 1, "rowspan": 1, "colwidth": null, "style": null}, "content": [{"type": "paragraph", "attrs": {"id": "c65ad965-b234-4478-93d5-22bd1917b919", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Demonstrate sterol-transcript assay responsiveness"}]}]}]}]}, {"type": "paragraph", "attrs": {"id": "a7848fe1-241a-4c3b-97c5-7aab3c648f9e", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "At the selected time, collect separate sister wells for RNA, protein and imaging. This gives 8 conditions \u00d7 6 differentiations = 48 confirmatory RNA samples and a minimum of 144 confirmatory culture wells. Reserve the remaining capacity within the 96-sample RNA allowance for pilot/rescue qualification. Final plate maps must include any additional protein-yield wells and controls; do not count sister wells as biological replicates."}]}, {"type": "heading", "attrs": {"id": "2fc8ebed-fd9d-4043-8944-29db781558d2", "data-toc-id": "2fc8ebed-fd9d-4043-8944-29db781558d2", "textAlign": null, "level": 3}, "content": [{"type": "text", "text": "6. Measure the response and interpret it"}]}, {"type": "paragraph", "attrs": {"id": "1314cd39-4ba5-4553-9be0-7866268f8a4b", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Measure HMGCR, HMGCS1, MVK, MVD, FDFT1 and SQLE by RT-qPCR, alongside DCTN1 and validated reference genes. This is a proposed pathway panel, pending source review\u2014not an asserted exact replication panel. Validate primer specificity/efficiency, include no-template and no-RT controls, and freeze the reference-gene and normalization rules before confirmation."}]}, {"type": "paragraph", "attrs": {"id": "0da1e460-2672-4b69-b34f-3f0dc5a898b6", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Define the primary score as the mean normalized log2 expression across the six sterol genes, with each gene weighted equally. Compare DCTN1 targeting with the matched non-targeting control within differentiation, report guide-specific effects, and prespecify the combined primary contrast. Assess rescue attenuation separately. Report individual genes, HMGCR/SQLE proteins, DCTN1 engagement and blinded health/identity measurements alongside the score."}]}, {"type": "paragraph", "attrs": {"id": "6b967740-d2e1-40b5-8bb3-c08b4fad5c07", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Support requires concordant induction from both guides, attenuation by qualified rescue and acceptable neuronal health. A precise null challenges the premise only when engagement and assay responsiveness are valid and confidence bounds exclude the prespecified meaningful induction. Toxicity, failed rescue/engagement, discordant guides or inadequate precision are inconclusive. Rescue shows perturbation specificity, not cholesterol mediation."}]}, {"type": "paragraph", "attrs": {"id": "d300c9aa-a623-402a-96bb-ba5f81d674e9", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Preregister the score, sampling time, meaningful-effect bound, exclusions, missingness and multiplicity plan before confirmation. Analyze at the differentiation level with preparation blocking and report effects with 95% confidence intervals. Keep pilot data separate. Share the plate maps, raw data, QC, code and negative results. Before execution, attach the complete protocols.io version and document every adaptation; the DCTN1 targeting, rescue and sampling scheme above are proposed study-specific additions, not steps validated by that differentiation protocol."}]}, {"type": "heading", "attrs": {"id": "719c1d2e-d27e-4661-b7a6-f452cb83eee7", "data-toc-id": "719c1d2e-d27e-4661-b7a6-f452cb83eee7", "textAlign": null, "level": 2}, "content": [{"type": "text", "text": "Itemized budget \u2014 $44,220 USD"}]}, {"type": "paragraph", "attrs": {"id": "27efb578-7c56-4414-9348-8d739ad4023c", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "All amounts are explicit planning allowances, not verified supplier prices or quotes. Quantities cover qualification and the planned confirmation. Existing incubators, microscopy, qPCR and immunoblot equipment are required; access fees are included, but new capital equipment and new cell-line engineering are excluded. 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These are portions of the total, not additional costs. Add any required institutional overhead beyond the administration allowance before commitment. Optional independent-background replication: separately estimated $12,000\u2013$18,000, subject to quotes and power analysis; excluded from the base request. The RFP\u2019s $1,000 shared pool is not assumed awarded or deducted."}]}, {"type": "heading", "attrs": {"id": "9f1c0fb2-ebb4-4a51-8566-9fa04c414e42", "data-toc-id": "9f1c0fb2-ebb4-4a51-8566-9fa04c414e42", "textAlign": null, "level": 2}, "content": [{"type": "text", "text": "Timeline, execution gates and outputs"}]}, {"type": "paragraph", "attrs": {"id": "c7eacc19-d8e8-4ded-a50a-d55ef4d4218c", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "Months 1\u20132: source review, quotes, permissions, reagent/rescue setup and qualification. "}, {"type": "hardBreak"}, {"type": "text", "text": "Months 3\u20135: finish qualification, preregister and run confirmation if gates pass. "}, {"type": "hardBreak"}, {"type": "text", "text": "Months 6\u20137: analysis and reporting. An established neuronal/CRISPRi platform is essential to this schedule; failed qualification stops or redirects work."}]}, {"type": "paragraph", "attrs": {"id": "7e493e07-da3f-40e1-8199-edb7e63dfd60", "class": null, "textAlign": null}, "content": [{"type": "text", "text": "The receiving laboratory must approve the protocol and demonstrate culture, molecular and imaging capability. Secure applicable human-cell/material, stem-cell and biosafety permissions before execution. Share protocols, QC, data, code and negative/inconclusive results subject to legitimate restrictions. Final effect bounds and sample size must be frozen before confirmation; this budget is an efficient planning estimate, not a guarantee of power or an execution authorization."}]}]}8f:T54cc,

DCTN1 Reduction: Testing the Early Neuronal Sterol Response

Candidate design #1 | Budget request: $44,220 USD | Duration: approximately 5–7 months after access and approvals

Objective

Determine whether titrated DCTN1 reduction induces a coordinated sterol-biosynthesis transcriptional response before neuronal deterioration. Two independent targeting reagents and wild-type rescue will distinguish a DCTN1-dependent response from off-target effects, stress, and culture changes.

The ResearchHub Foundation RFP attributes the starting observation to Sun, Park, DeTomaso et al., bioRxiv (2026), Fig. 4L. The RFP-linked preprint and source data require independent review. This study tests transcriptional reproducibility—not cholesterol transport, synthesis flux, or disease causality. DCTN1 reduction is not equivalent to a patient variant.

Study design: from neuronal preparation to confirmation

The experiment has two stages. First, establish a DCTN1 reduction that neurons tolerate and identify when to measure the response. Then repeat the selected condition in new differentiations. Use targeted RT-qPCR and sister-well imaging/protein assays rather than RNA-seq or lipidomics.

1. Start with a defined neuronal platform

Use one authenticated human iPSC background carrying doxycycline-inducible NGN2 and a stable, constitutively expressed dCas9-KRAB CRISPRi system. These are platform specifications, not a claim that a particular laboratory already holds this line. Obtain the line identifier, genotype, construct maps, permissions and prior differentiation QC before selection. If no suitable existing platform is available, revise costs and timing before proceeding.

NGN2-induced cortical-like neurons provide a relatively uniform population for this first transcriptional test. They are not midbrain dopaminergic neurons and do not model Perry syndrome directly. Test mycoplasma status and document neuronal yield, maturity and DCTN1 expression before committing the full study.

2. Differentiate and mature the neurons before targeting DCTN1

Use the protocols.io method “iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 2,” V.1, June 6, 2023 as the candidate differentiation backbone. Its indexed record describes stable tetracycline-inducible NGN2, induction during days 0–3, maturation-medium stages at days 4/7 and days 10–28, and cortical-marker enrichment. Full-text retrieval failed during this revision; these indexed details are not a verified executable SOP. Obtain and approve the complete version before choosing exact coating, density, media recipes and feeding instructions.

Plan to mature cultures through differentiation day 28 before DCTN1 targeting. Record medium and supplement lots, including lipid-containing components, and keep feeding and collection intervals identical across groups. Do not introduce a lipid-depletion challenge into the primary experiment. At day 28, assess MAP2-positive neuronal fraction, morphology, viability and baseline stress in sentinel wells.

Proposed qualification gates are at least 90% MAP2-positive cells among counted nuclei and no major baseline stress or neurite deterioration. These are study-design targets, not thresholds established by the cited protocol; validate and freeze them with the receiving laboratory. If cultures fail, improve differentiation rather than adjusting the primary analysis for poor composition.

3. Introduce DCTN1 targeting only after maturation

Deliver either of two independent DCTN1 promoter-targeting guides or a non-targeting guide to the mature neurons. Use guide delivery—not a second doxycycline switch—to start CRISPRi. This separates DCTN1 reduction from NGN2 induction. Match delivery reagent, vector burden and handling across groups, and verify guide-delivery coverage so bulk RNA does not conceal a mostly unperturbed population.

For rescue groups, introduce wild-type DCTN1 cDNA from a heterologous promoter before targeting and verify expression at the targeting baseline. Promoter-directed CRISPRi should spare this construct, but verify escape experimentally. Match total DCTN1 near control levels and include non-targeting-plus-rescue controls to detect overexpression effects. Rescue dose and lead time are selected during qualification.

4. Use a small pilot to choose the dose and sampling day

Across two independent differentiations, test two guide-delivery levels and collect at 3, 7 and 10 days after targeting. These are proposed pilot times, not published DCTN1 kinetics. At each time, measure DCTN1 RNA/protein, live/dead staining, cell counts, neuronal morphology and ATF4/CHOP stress. Run separate pathway-response and health-assay controls to demonstrate assay sensitivity.

Aim initially for a moderate 30–60% reduction in DCTN1 protein. Select the earliest time with reproducible engagement for both guides, at least 90% viability relative to matched controls, and acceptable identity, stress and neurite measures. Freeze quantitative acceptable-change bounds after assay qualification; absence of statistical significance is not evidence of cellular health. Do not choose the time producing the largest sterol response. If no interpretable window exists, stop or redesign.

5. Repeat the selected condition in independent differentiations

Budget for six new differentiations in the same genetic background. Use the pilot variance and a prespecified meaningful effect to confirm whether six provides adequate power and precision. Increase replication and revise costs if needed; do not present six as guaranteed sufficient. Each differentiation contains the following conditions, randomized across plate positions:

Group	Purpose
Non-targeting guide + empty vector	Primary matched control
DCTN1 guide A or B + empty vector	Two independent tests of DCTN1 reduction
DCTN1 guide A or B + wild-type rescue	Test perturbation specificity
Non-targeting guide + rescue	Detect rescue-expression effects
Sham delivery	Detect delivery-associated injury
Non-targeting guide + qualified pathway-response control	Demonstrate sterol-transcript assay responsiveness

At the selected time, collect separate sister wells for RNA, protein and imaging. This gives 8 conditions × 6 differentiations = 48 confirmatory RNA samples and a minimum of 144 confirmatory culture wells. Reserve the remaining capacity within the 96-sample RNA allowance for pilot/rescue qualification. Final plate maps must include any additional protein-yield wells and controls; do not count sister wells as biological replicates.

6. Measure the response and interpret it

Measure HMGCR, HMGCS1, MVK, MVD, FDFT1 and SQLE by RT-qPCR, alongside DCTN1 and validated reference genes. This is a proposed pathway panel, pending source review—not an asserted exact replication panel. Validate primer specificity/efficiency, include no-template and no-RT controls, and freeze the reference-gene and normalization rules before confirmation.

Define the primary score as the mean normalized log2 expression across the six sterol genes, with each gene weighted equally. Compare DCTN1 targeting with the matched non-targeting control within differentiation, report guide-specific effects, and prespecify the combined primary contrast. Assess rescue attenuation separately. Report individual genes, HMGCR/SQLE proteins, DCTN1 engagement and blinded health/identity measurements alongside the score.

Support requires concordant induction from both guides, attenuation by qualified rescue and acceptable neuronal health. A precise null challenges the premise only when engagement and assay responsiveness are valid and confidence bounds exclude the prespecified meaningful induction. Toxicity, failed rescue/engagement, discordant guides or inadequate precision are inconclusive. Rescue shows perturbation specificity, not cholesterol mediation.

Preregister the score, sampling time, meaningful-effect bound, exclusions, missingness and multiplicity plan before confirmation. Analyze at the differentiation level with preparation blocking and report effects with 95% confidence intervals. Keep pilot data separate. Share the plate maps, raw data, QC, code and negative results. Before execution, attach the complete protocols.io version and document every adaptation; the DCTN1 targeting, rescue and sampling scheme above are proposed study-specific additions, not steps validated by that differentiation protocol.

Itemized budget — $44,220 USD

All amounts are explicit planning allowances, not verified supplier prices or quotes. Quantities cover qualification and the planned confirmation. Existing incubators, microscopy, qPCR and immunoblot equipment are required; access fees are included, but new capital equipment and new cell-line engineering are excluded. Obtain institutional/vendor quotes before purchasing.

Item / reagents	Quantity × estimated allowance	USD
Existing neuronal/CRISPRi line access and initial QC	1 package × $1,500	$1,500
Basal medium, supplements and neuronal differentiation/maturation factors	8 preparation allocations × $375	$3,000
Poly-D-lysine/laminin, culture plates, tips and consumables	1 study lot × $1,500	$1,500
Two DCTN1 guides and non-targeting guide: cloning/validation	1 set × $1,000	$1,000
Guide-resistant wild-type DCTN1 rescue and empty vector	Synthesis/cloning/validation package × $2,500	$2,500
Delivery reagents/core vector preparation; doxycycline and pathway/stress controls	Delivery $1,500 + control reagent set $300	$1,800
RNA extraction with DNase; reverse transcription reagents	2 × 50-prep kits at $600; 100 RT reactions at $600	$1,800
qPCR primers, master mix, plates and seals	Primer design/validation $1,000 + assay supplies $1,200	$2,200
Antibodies: DCTN1, HMGCR, SQLE, MAP2, ATF4/CHOP, secondary/loading controls	1 panel allowance × $2,100	$2,100
Immunoblot gels/membranes/detection; live/dead and nuclear stains	Blot supplies $600 + stains $400	$1,000
Cell identity/authentication and mycoplasma testing	1 study QC allocation × $600	$600
Research staff: culture, assays and analysis	360 hours × $50 loaded rate	$18,000
Microscopy/qPCR core access and analysis support	40 hours × $60	$2,400
Shipping, data storage and permitted administration	1 allocation × $800	$800
Direct costs	Subtotal	$40,200
Contingency	10% of direct costs	$4,020
TOTAL REQUEST	Qualification + confirmation	$44,220

Stage funding: $16,000 for setup/qualification; $28,220 released after assay gates and power review. These are portions of the total, not additional costs. Add any required institutional overhead beyond the administration allowance before commitment. Optional independent-background replication: separately estimated $12,000–$18,000, subject to quotes and power analysis; excluded from the base request. The RFP’s $1,000 shared pool is not assumed awarded or deducted.

Timeline, execution gates and outputs

Months 1–2: source review, quotes, permissions, reagent/rescue setup and qualification.
Months 3–5: finish qualification, preregister and run confirmation if gates pass.
Months 6–7: analysis and reporting. An established neuronal/CRISPRi platform is essential to this schedule; failed qualification stops or redirects work.

The receiving laboratory must approve the protocol and demonstrate culture, molecular and imaging capability. Secure applicable human-cell/material, stem-cell and biosafety permissions before execution. Share protocols, QC, data, code and negative/inconclusive results subject to legitimate restrictions. Final effect bounds and sample size must be frozen before confirmation; this budget is an efficient planning estimate, not a guarantee of power or an execution authorization.

90:T54cc,

DCTN1 Reduction: Testing the Early Neuronal Sterol Response

Candidate design #1 | Budget request: $44,220 USD | Duration: approximately 5–7 months after access and approvals

Objective

Study design: from neuronal preparation to confirmation

1. Start with a defined neuronal platform

2. Differentiate and mature the neurons before targeting DCTN1

3. Introduce DCTN1 targeting only after maturation

4. Use a small pilot to choose the dose and sampling day

5. Repeat the selected condition in independent differentiations

Group	Purpose
Non-targeting guide + empty vector	Primary matched control
DCTN1 guide A or B + empty vector	Two independent tests of DCTN1 reduction
DCTN1 guide A or B + wild-type rescue	Test perturbation specificity
Non-targeting guide + rescue	Detect rescue-expression effects
Sham delivery	Detect delivery-associated injury
Non-targeting guide + qualified pathway-response control	Demonstrate sterol-transcript assay responsiveness

6. Measure the response and interpret it

Itemized budget — $44,220 USD

Item / reagents	Quantity × estimated allowance	USD
Existing neuronal/CRISPRi line access and initial QC	1 package × $1,500	$1,500
Basal medium, supplements and neuronal differentiation/maturation factors	8 preparation allocations × $375	$3,000
Poly-D-lysine/laminin, culture plates, tips and consumables	1 study lot × $1,500	$1,500
Two DCTN1 guides and non-targeting guide: cloning/validation	1 set × $1,000	$1,000
Guide-resistant wild-type DCTN1 rescue and empty vector	Synthesis/cloning/validation package × $2,500	$2,500
Delivery reagents/core vector preparation; doxycycline and pathway/stress controls	Delivery $1,500 + control reagent set $300	$1,800
RNA extraction with DNase; reverse transcription reagents	2 × 50-prep kits at $600; 100 RT reactions at $600	$1,800
qPCR primers, master mix, plates and seals	Primer design/validation $1,000 + assay supplies $1,200	$2,200
Antibodies: DCTN1, HMGCR, SQLE, MAP2, ATF4/CHOP, secondary/loading controls	1 panel allowance × $2,100	$2,100
Immunoblot gels/membranes/detection; live/dead and nuclear stains	Blot supplies $600 + stains $400	$1,000
Cell identity/authentication and mycoplasma testing	1 study QC allocation × $600	$600
Research staff: culture, assays and analysis	360 hours × $50 loaded rate	$18,000
Microscopy/qPCR core access and analysis support	40 hours × $60	$2,400
Shipping, data storage and permitted administration	1 allocation × $800	$800
Direct costs	Subtotal	$40,200
Contingency	10% of direct costs	$4,020
TOTAL REQUEST	Qualification + confirmation	$44,220

Timeline, execution gates and outputs

91:T2edf,DCTN1 Reduction: Testing the Early Neuronal Sterol Response Candidate design #1 | Budget request: $44,220 USD | Duration: approximately 5–7 months after access and approvals Objective Determine whether titrated DCTN1 reduction induces a coordinated sterol-biosynthesis transcriptional response before neuronal deterioration. Two independent targeting reagents and wild-type rescue will distinguish a DCTN1-dependent response from off-target effects, stress, and culture changes. The ResearchHub Foundation RFP attributes the starting observation to Sun, Park, DeTomaso et al., bioRxiv (2026), Fig. 4L. The RFP-linked preprint and source data require independent review. This study tests transcriptional reproducibility—not cholesterol transport, synthesis flux, or disease causality. DCTN1 reduction is not equivalent to a patient variant. Study design: from neuronal preparation to confirmation The experiment has two stages. First, establish a DCTN1 reduction that neurons tolerate and identify when to measure the response. Then repeat the selected condition in new differentiations. Use targeted RT-qPCR and sister-well imaging/protein assays rather than RNA-seq or lipidomics. 1. Start with a defined neuronal platform Use one authenticated human iPSC background carrying doxycycline-inducible NGN2 and a stable, constitutively expressed dCas9-KRAB CRISPRi system. These are platform specifications, not a claim that a particular laboratory already holds this line. Obtain the line identifier, genotype, construct maps, permissions and prior differentiation QC before selection. If no suitable existing platform is available, revise costs and timing before proceeding. NGN2-induced cortical-like neurons provide a relatively uniform population for this first transcriptional test. They are not midbrain dopaminergic neurons and do not model Perry syndrome directly. Test mycoplasma status and document neuronal yield, maturity and DCTN1 expression before committing the full study. 2. Differentiate and mature the neurons before targeting DCTN1 Use the protocols.io method “iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 2,” V.1, June 6, 2023 as the candidate differentiation backbone. Its indexed record describes stable tetracycline-inducible NGN2, induction during days 0–3, maturation-medium stages at days 4/7 and days 10–28, and cortical-marker enrichment. Full-text retrieval failed during this revision; these indexed details are not a verified executable SOP. Obtain and approve the complete version before choosing exact coating, density, media recipes and feeding instructions. Plan to mature cultures through differentiation day 28 before DCTN1 targeting. Record medium and supplement lots, including lipid-containing components, and keep feeding and collection intervals identical across groups. Do not introduce a lipid-depletion challenge into the primary experiment. At day 28, assess MAP2-positive neuronal fraction, morphology, viability and baseline stress in sentinel wells. Proposed qualification gates are at least 90% MAP2-positive cells among counted nuclei and no major baseline stress or neurite deterioration. These are study-design targets, not thresholds established by the cited protocol; validate and freeze them with the receiving laboratory. If cultures fail, improve differentiation rather than adjusting the primary analysis for poor composition. 3. Introduce DCTN1 targeting only after maturation Deliver either of two independent DCTN1 promoter-targeting guides or a non-targeting guide to the mature neurons. Use guide delivery—not a second doxycycline switch—to start CRISPRi. This separates DCTN1 reduction from NGN2 induction. Match delivery reagent, vector burden and handling across groups, and verify guide-delivery coverage so bulk RNA does not conceal a mostly unperturbed population. For rescue groups, introduce wild-type DCTN1 cDNA from a heterologous promoter before targeting and verify expression at the targeting baseline. Promoter-directed CRISPRi should spare this construct, but verify escape experimentally. Match total DCTN1 near control levels and include non-targeting-plus-rescue controls to detect overexpression effects. Rescue dose and lead time are selected during qualification. 4. Use a small pilot to choose the dose and sampling day Across two independent differentiations, test two guide-delivery levels and collect at 3, 7 and 10 days after targeting. These are proposed pilot times, not published DCTN1 kinetics. At each time, measure DCTN1 RNA/protein, live/dead staining, cell counts, neuronal morphology and ATF4/CHOP stress. Run separate pathway-response and health-assay controls to demonstrate assay sensitivity. Aim initially for a moderate 30–60% reduction in DCTN1 protein. Select the earliest time with reproducible engagement for both guides, at least 90% viability relative to matched controls, and acceptable identity, stress and neurite measures. Freeze quantitative acceptable-change bounds after assay qualification; absence of statistical significance is not evidence of cellular health. Do not choose the time producing the largest sterol response. If no interpretable window exists, stop or redesign. 5. Repeat the selected condition in independent differentiations Budget for six new differentiations in the same genetic background. Use the pilot variance and a prespecified meaningful effect to confirm whether six provides adequate power and precision. Increase replication and revise costs if needed; do not present six as guaranteed sufficient. Each differentiation contains the following conditions, randomized across plate positions: Group Purpose Non-targeting guide + empty vector Primary matched control DCTN1 guide A or B + empty vector Two independent tests of DCTN1 reduction DCTN1 guide A or B + wild-type rescue Test perturbation specificity Non-targeting guide + rescue Detect rescue-expression effects Sham delivery Detect delivery-associated injury Non-targeting guide + qualified pathway-response control Demonstrate sterol-transcript assay responsiveness At the selected time, collect separate sister wells for RNA, protein and imaging. This gives 8 conditions × 6 differentiations = 48 confirmatory RNA samples and a minimum of 144 confirmatory culture wells. Reserve the remaining capacity within the 96-sample RNA allowance for pilot/rescue qualification. Final plate maps must include any additional protein-yield wells and controls; do not count sister wells as biological replicates. 6. Measure the response and interpret it Measure HMGCR, HMGCS1, MVK, MVD, FDFT1 and SQLE by RT-qPCR, alongside DCTN1 and validated reference genes. This is a proposed pathway panel, pending source review—not an asserted exact replication panel. Validate primer specificity/efficiency, include no-template and no-RT controls, and freeze the reference-gene and normalization rules before confirmation. Define the primary score as the mean normalized log2 expression across the six sterol genes, with each gene weighted equally. Compare DCTN1 targeting with the matched non-targeting control within differentiation, report guide-specific effects, and prespecify the combined primary contrast. Assess rescue attenuation separately. Report individual genes, HMGCR/SQLE proteins, DCTN1 engagement and blinded health/identity measurements alongside the score. Support requires concordant induction from both guides, attenuation by qualified rescue and acceptable neuronal health. A precise null challenges the premise only when engagement and assay responsiveness are valid and confidence bounds exclude the prespecified meaningful induction. Toxicity, failed rescue/engagement, discordant guides or inadequate precision are inconclusive. Rescue shows perturbation specificity, not cholesterol mediation. Preregister the score, sampling time, meaningful-effect bound, exclusions, missingness and multiplicity plan before confirmation. Analyze at the differentiation level with preparation blocking and report effects with 95% confidence intervals. Keep pilot data separate. Share the plate maps, raw data, QC, code and negative results. Before execution, attach the complete protocols.io version and document every adaptation; the DCTN1 targeting, rescue and sampling scheme above are proposed study-specific additions, not steps validated by that differentiation protocol. Itemized budget — $44,220 USD All amounts are explicit planning allowances, not verified supplier prices or quotes. Quantities cover qualification and the planned confirmation. Existing incubators, microscopy, qPCR and immunoblot equipment are required; access fees are included, but new capital equipment and new cell-line engineering are excluded. Obtain institutional/vendor quotes before purchasing. Item / reagents Quantity × estimated allowance USD Existing neuronal/CRISPRi line access and initial QC 1 package × $1,500 $1,500 Basal medium, supplements and neuronal differentiation/maturation factors 8 preparation allocations × $375 $3,000 Poly-D-lysine/laminin, culture plates, tips and consumables 1 study lot × $1,500 $1,500 Two DCTN1 guides and non-targeting guide: cloning/validation 1 set × $1,000 $1,000 Guide-resistant wild-type DCTN1 rescue and empty vector Synthesis/cloning/validation package × $2,500 $2,500 Delivery reagents/core vector preparation; doxycycline and pathway/stress controls Delivery $1,500 + control reagent set $300 $1,800 RNA extraction with DNase; reverse transcription reagents 2 × 50-prep kits at $600; 100 RT reactions at $600 $1,800 qPCR primers, master mix, plates and seals Primer design/validation $1,000 + assay supplies $1,200 $2,200 Antibodies: DCTN1, HMGCR, SQLE, MAP2, ATF4/CHOP, secondary/loading controls 1 panel allowance × $2,100 $2,100 Immunoblot gels/membranes/detection; live/dead and nuclear stains Blot supplies $600 + stains $400 $1,000 Cell identity/authentication and mycoplasma testing 1 study QC allocation × $600 $600 Research staff: culture, assays and analysis 360 hours × $50 loaded rate $18,000 Microscopy/qPCR core access and analysis support 40 hours × $60 $2,400 Shipping, data storage and permitted administration 1 allocation × $800 $800 Direct costs Subtotal $40,200 Contingency 10% of direct costs $4,020 TOTAL REQUEST Qualification + confirmation $44,220 Stage funding: $16,000 for setup/qualification; $28,220 released after assay gates and power review. These are portions of the total, not additional costs. Add any required institutional overhead beyond the administration allowance before commitment. Optional independent-background replication: separately estimated $12,000–$18,000, subject to quotes and power analysis; excluded from the base request. The RFP’s $1,000 shared pool is not assumed awarded or deducted. Timeline, execution gates and outputs Months 1–2: source review, quotes, permissions, reagent/rescue setup and qualification. Months 3–5: finish qualification, preregister and run confirmation if gates pass. Months 6–7: analysis and reporting. An established neuronal/CRISPRi platform is essential to this schedule; failed qualification stops or redirects work. The receiving laboratory must approve the protocol and demonstrate culture, molecular and imaging capability. Secure applicable human-cell/material, stem-cell and biosafety permissions before execution. Share protocols, QC, data, code and negative/inconclusive results subject to legitimate restrictions. 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99:T36a8,

Candidate design #1 | Budget request: $44,220 USD | Duration: approximately 5–7 months after access and approvals

Objective

Study design: from neuronal preparation to confirmation

1. Start with a defined neuronal platform

2. Differentiate and mature the neurons before targeting DCTN1

3. Introduce DCTN1 targeting only after maturation

4. Use a small pilot to choose the dose and sampling day

5. Repeat the selected condition in independent differentiations

Group	Purpose
Non-targeting guide + empty vector	Primary matched control
DCTN1 guide A or B + empty vector	Two independent tests of DCTN1 reduction
DCTN1 guide A or B + wild-type rescue	Test perturbation specificity
Non-targeting guide + rescue	Detect rescue-expression effects
Sham delivery	Detect delivery-associated injury
Non-targeting guide + qualified pathway-response control	Demonstrate sterol-transcript assay responsiveness

6. Measure the response and interpret it

Itemized budget — $44,220 USD

Item / reagents	Quantity × estimated allowance	USD
Existing neuronal/CRISPRi line access and initial QC	1 package × $1,500	$1,500
Basal medium, supplements and neuronal differentiation/maturation factors	8 preparation allocations × $375	$3,000
Poly-D-lysine/laminin, culture plates, tips and consumables	1 study lot × $1,500	$1,500
Two DCTN1 guides and non-targeting guide: cloning/validation	1 set × $1,000	$1,000
Guide-resistant wild-type DCTN1 rescue and empty vector	Synthesis/cloning/validation package × $2,500	$2,500
Delivery reagents/core vector preparation; doxycycline and pathway/stress controls	Delivery $1,500 + control reagent set $300	$1,800
RNA extraction with DNase; reverse transcription reagents	2 × 50-prep kits at $600; 100 RT reactions at $600	$1,800
qPCR primers, master mix, plates and seals	Primer design/validation $1,000 + assay supplies $1,200	$2,200
Antibodies: DCTN1, HMGCR, SQLE, MAP2, ATF4/CHOP, secondary/loading controls	1 panel allowance × $2,100	$2,100
Immunoblot gels/membranes/detection; live/dead and nuclear stains	Blot supplies $600 + stains $400	$1,000
Cell identity/authentication and mycoplasma testing	1 study QC allocation × $600	$600
Research staff: culture, assays and analysis	360 hours × $50 loaded rate	$18,000
Microscopy/qPCR core access and analysis support	40 hours × $60	$2,400
Shipping, data storage and permitted administration	1 allocation × $800	$800
Direct costs	Subtotal	$40,200
Contingency	10% of direct costs	$4,020
TOTAL REQUEST	Qualification + confirmation	$44,220

Timeline, execution gates and outputs

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Drafts

DCTN1 Reduction: Testing the Early Neuronal Sterol Response

Drafts

Drafts

DCTN1 Reduction: Testing the Early Neuronal Sterol Response

Reviewed by1

Key insightsBeta

Reviewed by1

Key insightsBeta