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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present the acute genetic manipulation of sliced human cortical organoids by electroporation. These cortical organoid models are particularly amenable to injection as ventricle-like structures can be readily identified after slicing, enabling the functional investigation of human cortical development, neurodevelopmental disorders, and cortical evolution.

Abstract

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

Introduction

The neocortex refers to the outer covering of the cerebral hemispheres and is a structure that is unique to mammals. The neocortex represents the seat of higher cognitive functions1,2,3,4,5. During development, neural stem and progenitor cells give rise to neurons in a process termed neurogenesis. Functional studies investigating human neocortex development provide the basis for elucidating the mechanisms underlying human neural stem cell regulation, neural pathologies, and human brain evolution2,6,7.

Historically, studies of human brain development relied on descriptive histological approaches using post-mortem tissue, with more recent free-floating tissue culture systems enabling functional investigations with human fetal tissue8,9. Additionally, human fetal brain tissue was shown to have the capacity to self-organize into long-term expanding organoids10. Fetal tissue research has provided important insights into human development11,12, yet restricted tissue availability and ethical considerations limit its widespread application for mechanistic studies of human brain development. In the past decade, protocols have been developed that allow the generation of three-dimensional neural organoids from human pluripotent stem cells (hPSC), including human induced PSC (hiPSC)13,14.

Cerebral organoids display important features of the developing brain, such as the formation of ventricle-like structures, apicobasal polarity, cortical cytoarchitecture, interkinetic nuclear migration during radial glia division, and neuronal migration. Importantly, these new human organoid models recapitulate characteristics of the human developing brain that are not modeled well in the mouse, including evolutionarily relevant key neural progenitor types, in particular basal radial glia (or outer radial glia)7,14,15. Several limitations of early cerebral organoid protocols - such as issues with organoid heterogeneity, limited nutrient supply to the inner core, and varied regional identity-have been addressed in recent protocol advancements and further improvements can be expected in the coming years15,16,17,18,19. Human cerebral and cortical organoids have quickly become key models to study human cortical development20, neurological disorders21,22,23,24 and brain evolution25,26,27,28,29, and to perform large-scale screening approaches30,31,32.

For acute genetic manipulation, two methods have primarily been used in animal models of neocortex development: viral delivery by infection of target cells33 and in utero or in vitro electroporation34,35. Injection of DNA - and, more recently, CRISPR/Cas9 ribonucleoprotein (RNP) complexes36- into the lateral ventricles, followed by electroporation, provides the advantage that specific regions of the brain can be targeted based on the orientation of the electroporation electrodes. Electroporation involves brief electric pulses that temporarily increase cell membrane permeability, allowing the introduction of DNA and other charged molecules into cells. In utero electroporation was first performed in the mouse37, where it rapidly became a widely applied methodology for developmental neurobiology. The method was subsequently also applied to other species, such as the rat38,39 and the ferret40,41,42, a gyrencephalic species used to study neocortex expansion and cortical folding3,43,44,45.

Electroporation has also become an important method in human brain organoid research46. In cerebral organoids, electroporation has been applied to visualize cell morphology and neuronal axons14,47, to deliver gene knockdown reagents22,48,49, and for investigation of gene function by overexpression50. The method is not restricted to human models but has also been applied for genetic modification of primate cerebral organoids50,51. Moreover, the electroporation of cortical organoids generated in a Spin Ω spinning bioreactor has been described52.

In this protocol, we outline the electroporation of sliced human cortical organoids15 for studies of gene function in cortical development. Brain region-specific organoids increase reproducibility and consistency, which are critical for the success of quantitative analysis, for example, in disease modeling. In the sliced human cortical organoid protocol15, potent patterning cues are applied during iPSC differentiation to obtain a homogeneous population of dorsal forebrain progenitors, which is followed by the application of culture media that promote tissue growth with fewer instructive signals53,54. Slicing of cortical organoids has been shown to reduce cell death resulting from reduced availability of nutrients and oxygen in the organoid core15. Moreover, slicing supports the development of ventricle-like structures containing abundant basal radial glia, with sustained neurogenesis leading to the formation of an expanded cortical plate-like region15. The repeated slicing in this protocol also makes these cortical organoids particularly suitable to electroporation, as the ventricle lumens can be easily identified and targeted by injection. Electroporation of sliced cortical organoids has been applied to study gene regulatory regions and cortical evolution26,55.

During the electroporation procedure, the injection mix is delivered to the lumen of ventricle-like structures. Upon application of a pulsed electric field, the mix is taken up by apical radial glia that line the ventricle. As apical radial glia divide and give rise to more committed cell types4, the electroporated agents are passed on to basal progenitor cells and neurons. Electroporated progeny are distributed in the ventricular zone (VZ) and subventricular zone (SVZ) after 3 days and span most of the cortical wall, including the cortical plate (CP)-like region, at 7 days post-electroporation during mid-neurogenic stages26,55.

Here, we describe CRISPR/Cas9-mediated gene disruption56 by electroporation in sliced human cortical organoids26. In addition, the electroporation method can also be applied for gene overexpression, visualization of cell morphology and cellular processes by expression of fluorescent proteins, delivery of plasmid libraries for Massive Parallel Reporter Assays (MPRA), delivery of epigenome editing tools and labeling of cells for live imaging.

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Protocol

All experiments involving human induced pluripotent stem cells (hiPSC) were performed in accordance with the ethical standards outlined in the 1964 Helsinki Declaration and approved by the Dresden University Hospital Ethical Review Committee (IRB00001473; IORG0001076; ethical approval number SR-EK-456092021).

1. Design of guide RNAs for acute CRISPR/Cas9-mediated gene knockout

  1. Design a pair of guide RNAs (gRNAs) targeting the same exon with a distance of ideally 7-11 bp (avoiding multiples of 3 bp) between the protospacer adjacent motifs (PAMs) to increase the likelihood of protein disruption using software or online tools.
    NOTE: Ideally, exons that encode functional domains should be targeted. Alternatively, the first exons of the transcript can be chosen (Figure 1A). Select the guide RNAs based on activity score (close to 1)57 and specificity score (close to 100 %)58.

2. Culture of human induced pluripotent stem cells

  1. To generate human cortical organoids and to validate gRNA function, use the hiPSC line CRTDi004-A59 which is derived from a healthy donor.
  2. Culture the iPSCs as previously described60 on basal membrane matrix coated 6-well plates in stem cell medium under standard conditions (37 °C, 5% CO2). Induced PSCs should be passaged at 80% confluency using an enzyme for single-cell dissociation with supplementation of ROCK pathway inhibitor (1:1,000) during the first 24 h.
    NOTE: Other iPSC lines may be used for organoid generation and gRNA validation.

3. Extraction of genomic DNA from human induced pluripotent stem cells

  1. Use 2 wells of 80%-90% confluent hiPSC of a 6-well plate for genomic DNA extraction. Dissociate the cells from the plate with an enzyme to make a single-cell suspension. Take the cells up in 1 mL of stem cell medium (total volume), then spin for 10 min at 600 x g and remove the supernatant.
  2. Resuspend the cell pellet in 250 µL of lysis buffer (50 mM TrisHCl (pH 7.5), 100 mM NaCl, 0.5 % SDS, 5 mM EDTA in H2O) and 12.5 µL of Proteinase K (10 mg/mL stock). Incubate for 2 h at 55 °C and 250 rpm.
  3. Mix in 100 µL of 5 mM NaCl. Spin for 10 min at 18,000 x g at 4 °C and transfer the supernatant into new 1.5 mL reaction tubes.
  4. Add 250 µL of isopropanol, invert the tubes several times, and spin for 15 min at 18,000 x g at 4 °C.
  5. Remove the supernatant, wash with 500 µL of 70 % EtOH (molecular biology grade), invert the tubes, and spin for 10 min at 18,000 x g at 4 °C.
  6. Finally, completely remove the supernatant by carefully pipetting it off and pressing the open reaction tubes on paper towels.
  7. Let the pellet air dry for 10 min at RT, then dissolve the pellet in 100 µL of nuclease-free water for 30 min at 37 °C.
  8. Measure the concentration of the DNA with a spectrometer. Isolated genomic DNA is stable at -20 °C for several months.

4. Verification of template recognition by gRNA (in vitro)

NOTE: As the first approach to validate successful template recognition by gRNAs, perform an in vitro Cas9 reaction where double-stranded DNA is cleaved into two fragments that can be distinguished by agarose gel electrophoresis40,61,62.

  1. To generate the template, perform a polymerase chain reaction (PCR) with a high-fidelity PCR master mix and 10 µM primers on genomic DNA isolated from hiPSC (section 3) using the following cycling conditions: initial denaturation at 98 °C for 30 s, 40 cycles [98 °C, 10 s; 60 °C, 30 s; 72 °C, 1 min] and final elongation at 72 °C for 5 min.
    1. Adjust the annealing temperature according to the PCR primers. Ensure the amplicon is 800 bp to 1.5 kb in size with asymmetric localization of the gRNA binding sites.
    2. Resolve the PCR product by gel electrophoresis on a 1.5% agarose gel, cut the corresponding band from the gel and extract the DNA26.
    3. Measure the DNA concentration of the extracted PCR product with a spectrometer.
  2. Assemble the functional gRNA by combining 10 µM of CRISPR RNA (crRNA, custom sequence, 100 µM stock) and 10 µM tracrRNA (100 µM stock) in nuclease-free duplex buffer (5 µL total volume). Incubate the mix for 5 min at 95 °C, then allow it to cool down to room temperature (RT).
  3. Create the ribonucleoprotein (RNP) complex by combining 10 µM of the gRNA from step 4.2 and 1 µM Cas9 enzyme (from Streptococcus pyogenes, 62 µM stock) in PBS for 15 min at RT (25 µL total volume).
  4. To perform the in vitro digestion reaction, mix 1 µM of the RNP complex (from step 4.3) with 0.1 µM DNA template (PCR product from step 4.1) and 1x Cas9 nuclease reaction buffer (containing 200 mM HEPES, 1 M NaCl, 50 mM MgCl2, 1 mM EDTA; pH 6.5; can be stored in aliquots at -20 °C).
    1. Fill up to a final volume of 10 µL with nuclease-free water.
    2. Incubate the reaction at 37 °C for 1 h.
    3. Add 2 mg/mL Proteinase K and incubate for another 10 min at 56 °C to release the DNA substrate from the Cas9 enzyme. Store the digested reaction mix at 4 °C or -20 °C until final analysis.
      NOTE: Using a 10:1 molar ratio of Cas9-RNP:DNA substrate is recommended to obtain the best cleavage efficiency. Dilute the PCR product in nuclease-free water to the required concentration, if necessary. As a negative control, a reaction mix lacking either the gRNA or Cas9 enzyme may be taken along.
  5. Visualize cleaved products by agarose gel electrophoresis on a 2% agarose gel.
    NOTE: Ideally, two differently sized bands should be visible and the template band should have disappeared or be strongly reduced for efficient gRNAs (Figure 1B).

5. Verification of gRNA function in human induced pluripotent stem cells

NOTE: It is recommended to test the efficiency of gRNAs targeting the same cell line from which cortical organoids will be generated26. For this, RNP complexes containing the relevant gRNAs are co-transfected with a GFP expression plasmid into hiPSCs, the cells are cultured for 3 days, and genomic DNA is subsequently isolated for sequencing analysis. For each gRNA test, roughly 200,000 cells are required. Cells should not be more than 70%-80% confluent and should have been passaged at least twice after thawing. It is important to take a negative control (gLACZ)36,63 and a mock condition (nucleofection solution without RNP) along.

  1. Change the medium of the hiPSC to include ROCK inhibitor (1:1,000) 2 h prior to the experiment to increase the viability of the cells in a cell culture hood.
  2. Coat the appropriate number of wells of a 6-well plate with a basal membrane matrix.
  3. Start the nucleofector unit ("X unit" for 16-well strips) and select the program "ES cells, H9" as pre-programmed (pulse CB150). Select the appropriate number of wells of the strip.
  4. Prepare the RNP complexes by combining 24 µM crRNA and 24 µM tracrRNA in Duplex Buffer.
    1. Incubate at 95 °C for 5 min, then let cool down to RT, as described in step 4.2.
    2. In the meantime, dilute the Cas9 enzyme to 8 µM in PBS.
    3. Combine 3 µL of gRNA and 3 µL of Cas9, and incubate 15 min at RT to assemble the RNP.
      NOTE: A molar ratio of 3:1 gRNA:Cas9 gives optimal cleavage efficiency. To test gRNAs as a pair, prepare each RNP separately and combine 1.5 µL of each RNP at the end.
  5. During the 15 min RNP incubation, prepare the hiPSC for nucleofection under a cell culture hood.
    1. Replace the basal membrane coating with 1.5 mL of stem cell medium supplemented with ROCK inhibitor (1:1,000) and 1x Penicillin/Streptomycin per well.
    2. Prepare the nucleofection solution as a master mix on ice following manufacturer's instructions.
    3. Detach hiPSCs from the plate to make a single cell suspension (as described in section 2) and count the cells with Trypan Blue staining in a Neubauer counting chamber.
    4. Pellet 200,000 live cells per gRNA pair at 600 x g for 5 min.
  6. In the cell culture hood, on ice, add 3 µL of RNP to 20 µL of nucleofection solution plus 0.2 µg/µL pCAG-GFP plasmid.
  7. Resuspend the pelleted cells in the 23 µL of the final nucleofection mix and transfer them into 1 well of a 16-well nucleofection strip on ice.
  8. Once all conditions are in the strip, place it in the nucleofector machine X unit outside of the hood and press the Start button to initiate the nucleofection.
  9. Subsequently, transport the nucleofection strip back under the cell culture hood and transfer nucleofected cells to the prepared 6-well plate by rinsing out the strip-wells with some medium from the plate.
  10. Culture the hiPSC for 3 days, changing medium containing 1x Penicillin/Streptomycin daily. After 24 h, the ROCK inhibitor is no longer needed.
  11. Check for GFP signal under a conventional fluorescent microscope (Figure 1C).
    1. On day 3, sort and collect GFP-positive cells by fluorescent activated cell sorting (FACS).
    2. Prepare the sorting buffer with 2% FBS in PBS and keep at 4 °C.
    3. Cool down a table-top centrifuge to 4 °C. Prepare 1.5 mL reaction tubes with 50 µL sorting buffer containing ROCK inhibitor (1:1,000) for collection of cells after FACS on ice and 5 mL polystyrene round-bottom tubes with cell-strainer caps on ice.
    4. Make single-cell suspensions of the nucleofected cells.
    5. Resuspend the cells in 1 mL of stem cell medium containing ROCK inhibitor (1:1,000) per sample and centrifuge at 600 x g for 5 min.
    6. Remove the supernatant and resuspend the cells in 200 µL of sorting buffer with ROCK inhibitor (1:1,000), then add 0.4 µL of DAPI (2 ng/µL) to identify live cells by FACS.
    7. Pipet the cell solution through the cell-strainer cap of the cooled round-bottom tubes to remove any residual cell clumps.
      NOTE: DAPI may compromise cell viability over time, and thus, it is best to add fresh to each sample before sorting.
    8. Sort 10,000 live GFP-positive living (DAPI-negative) cells into the previously prepared 1.5 mL reaction tubes with 50 µL of sorting buffer containing ROCK inhibitor (on ice).
  12. After FACS, directly extract genomic DNA from sorted cells.
    1. Spin down the collected, sorted cells in the sorting buffer at 600 x g for 10 min.
    2. Remove the supernatant and take up the cell pellet in 250 µL of lysis buffer plus 12.5 µL of Proteinase K per sample.
    3. Continue as described in section 3.
    4. Finally, dissolve the DNA pellet in 50 µL of nuclease-free water.
  13. Perform a PCR to generate a template for Sanger sequencing as described in step 4.1. As a control, use the DNA from the gLACZ condition and the primers targeting the gene of interest (same primers as used in section 4). Extract the appropriate bands from the agarose gel electrophoresis, purify it over a column, and send it for Sanger sequencing.
  14. Align Sanger sequencing results using a pairwise, global alignment.
    NOTE: Successful targeting can be identified by superimposed signals or incomplete sequences resulting from random insertions/deletions around the gRNA binding site (Figure 1D).

6. Generation of sliced human cortical organoids

NOTE: Human cortical organoids (hCO) are generated according to the sliced neocortical organoid (SNO) method, as previously described in detail15,60.

  1. Detach small hiPSC colonies from 1 well of a 6-well plate with 1 mg/mL collagenase by incubating for 30-40 min at 37 °C in an incubator. In the meantime, coat a new 6-well plate with the basal membrane matrix.
    1. Stop the enzyme with 1 mL of stem cell medium and collect the colonies with a wide-bore tip in 5 mL of stem cell medium in a 15 mL conical tube.
      NOTE: Any steps mentioning "wide-bore pipet tips" can also be carried out with tips cut to an opening of at least 2 mm in diameter.
    2. Once the colonies have settled to the bottom, remove the old medium and replace it with fresh stem cell medium.
    3. Using a wide-bore tip, distribute the colonies evenly on the coated 6-well plate with 1.5 mL of stem cell medium per well.
    4. Culture the hiPSC for 2-3 days under standard conditions.
  2. Once the hiPSC colonies have reached roughly 1.5 mm in diameter, detach all colonies again with 1 mg/mL collagenase by incubating for up to 1 h at 37 °C.
    1. Stop the enzyme with 6 mL of forebrain medium 115 and collect detached colonies in a 15 mL conical tube using wide-bore tips.
    2. Once the colonies have settled, remove the old medium and wash with 5 mL of forebrain medium 1.
    3. Finally, transfer the colonies to an ultra-low attachment 6-well plate containing 3 mL of forebrain medium 1 per well with wide-bore tips and let them form embryoid bodies (EBs). This step indicates day 0 of the human cortical organoid protocol. From here on, follow the protocol as published15.
  3. On day 7, embed EBs in a basal membrane matrix, with about 20 EBs per "cookie" and culture for 1 week in forebrain medium 2 15. At day 14, mechanically release organoids from the matrix and continue culturing in 6-well ultra-low attachment plates in forebrain medium 315 on an orbital shaker at 120 rpm.
  4. From day 35, 1% v/v basal membrane matrix is supplemented to the medium.
  5. At week 6, embed organoids in 3% low-melting point agarose and cut on a vibratome into 500-µm-thick slices (Figure 2A, B), which supports oxygen and nutrient supply throughout the organoids. If necessary, repeat the slicing of organoids every 4 weeks.
  6. From day 70, culture organoids in forebrain medium 415.

7. Electroporation of sliced human cortical organoids

NOTE: Human cortical organoids can be injected and electroporated as soon as ventricle-like structures are visible, roughly from week 2 onwards. For later-stage organoids (week 5 and older), the electroporation is most efficient and the viability of cells is best when the procedure is performed 2 days after slicing of organoids26,55. After slicing, ventricle-like structures are easy to identify, which aids the injection procedure (Figure 2C, D). Moreover, ventricle-like structures remain partially open for some time after slicing, allowing excess injection solution to escape, which protects the integrity of the adherens junction belt lining the ventricles. The slicing schedule can be shifted to match the experimental timeline. Slicing should be repeated every 3-4 weeks for long-term cultures.

  1. Pull the borosilicate glass capillaries for injections at a microcapillary puller with the following settings: P 500, heat 600, pull 30, velocity 40, time 1. Store the capillaries in a large Petri dish using tape for fixation.
  2. On the day of electroporation, freshly prepare the CRISPRR/Cas9 RNP complex as described in step 5.4 with a final concentration of 24 µM RNP. Co-electroporation of 0.1 µg/µL pCAG-GFP plasmid (or of any other fluorescent reporter) supports the identification of electroporated cells. In addition, add 0.1% Fast Green solution in water to the final injection mix, which enables confirmation of the successful delivery of the mix.
    NOTE: Make separate mixes for each gene of interest and the gLACZ control.
  3. Prewarm Tyrode's solution (one pack Tyrode's salt, 1 g of NaHCO3, 13 mL of 1 M HEPES pH 7.25 in 1 L of dH2O) to 37 °C. Store the remaining Tyrode's solution at 4 °C for several months.
  4. Select hCO with well-developed ventricle-like structures (Figure 2E) and discard any organoids that do not show ventricle-like structures (Figure 2F). Transfer up to 10 organoids to a 6 cm Petri dish containing Tyrode's solution and keep the remaining hCOs in the incubator.
  5. Fill a glass microcapillary with 10 µL of the injection solution with the help of a microloader pipette tip and open the capillary by pinching off the thin end with tweezers. Check if the capillary is open by releasing some injection mix into the Tyrode's solution. Carry out the injections with a microinjector in a continuous setting with controlled gating via a foot switch.
  6. Insert the capillary into the center of ventricle-like structures (Figure 2C) and inject 0.2-0.5 µL of the mix by pressing the foot switch.
    NOTE: Up to 5 ventricles may be injected per hCO with a total of 7-8 hCOs processed per replicate. Ideally, ventricles facing the outside of the hCO should be targeted as these are generally the most well-developed (Figure 2G, H).
  7. Use fine forceps to push the organoid against to restrict movement. Do not actually grab the organoids (Figure 2C).
  8. After injection, use a wide-bore pipet tip to transfer 1-2 organoids into an electroporation chamber containing Tyrode's solution (Figure 2C, D).
  9. Orient the injected organoids towards the electrodes so that apical radial glia in ventricle-like regions facing the outside of the organoid are targeted.
  10. Attach the cables to the electroporation chamber with the injected side of the organoid facing the positive pole (anode; Figure 2D).
  11. Press the Pulse button of the electroporator machine to electroporate with five pulses of 38 V for 50 ms each at intervals of 1 s.
    NOTE: The electroporation settings may depend on the available electroporator and may need to be optimized.
  12. Repeat the injection and electroporation steps until the desired number of organoids is processed.
  13. Collect the electroporated organoids in a medium at RT until all samples for the same condition have been processed. Ensure that they are not outside of the incubator for more than 30 min in total.
  14. Subsequently, return the electroporated hCOs into the appropriate culture medium and culture them without shaking for the first 24 h.
    NOTE: After 2-3 days, the GFP signal should be visible in electroporated hCOs under a fluorescent stereoscope (Figure 2H). The time point of analysis after electroporation should be chosen depending on the biological question to be answered. After 3-7 days, GFP-positive cells are located across the VZ, SVZ, and CP15,60.

8. Fixation and cryosectioning of electroporated human cortical organoids

  1. Fix electroporated organoids in 4% paraformaldehyde (PFA) for 30 min at RT in a 2 mL reaction tube.
    CAUTION: PFA is toxic and carcinogenic. Perform any steps involving PFA under a fume hood. The usage of nitrile gloves and safety goggles is strongly recommended.
  2. Wash once in PBS for 5 min and store in sterile-filtered 30% sucrose in PBS at 4 °C until the tissue is fully saturated with sucrose.
  3. Embed fixed organoids in an embedding medium for frozen tissue specimen plus 15% sucrose in PBS in blocks on dry ice.
  4. Prepare 20-30 µm sections at a cryostat and collect the tissue on adhesive slides26,64.
    NOTE: The protocol can be paused either once the fixed organoids are in sucrose (stable at 4 °C for several months) or embedded in an embedding medium (frozen blocks can be stored in air-tight bags at -20 °C for several months to years).

9. Immunohistochemical analysis of electroporated human cortical organoids

  1. For immunohistochemical analysis26,64, perform antigen retrieval in citrate buffer (10 mM sodium citrate, 0.05% polysorbate 20 in dH2O, pH 6.0) for 1 h at 70 °C in a water bath.
  2. Wash once in PBS for 5 min, then dry slides briefly and circle the sections with a wax pen.
  3. Quench using 0.1 M glycine in PBS (pH 7.4) for 30 min at RT and subsequently wash it twice with PBS for 5 min each.
  4. Cover the sections in blocking buffer (10% horse serum, 0.1% Octoxinol 9 in PBS) and incubate for 30 min at RT.
  5. Prepare the primary antibodies in blocking buffer and incubate on sections overnight at 4 °C.
  6. On the next day, wash three times with PBS for 5 min each, incubate with secondary antibodies and DAPI in blocking buffer for 1 h at RT, and wash three times again with PBS for 5 min each.
  7. Mount the slides in the mounting medium and image at a fluorescent microscope (Figure 3).
    NOTE: It is recommended to include an antibody against the fluorophore used in the injection mix to identify targeted cells since the native signal is often quenched after fixation. Antibodies and dyes used for immunofluorescence staining, as represented in Figure 3, are listed in Table 1.

10. Verification of CRISPR/Cas9-mediated knockout at protein level

  1. To verify the knockout at the protein level, perform immunohistochemistry for the target protein on the electroporated organoids (Figure 3I).
  2. Image both the gLACZ control and the targeted organoids using the same settings to allow comparison of fluorescence intensity (Figure 3I, J). Do this in a zone-specific manner to compare the effect on the different cell types of the developing neocortex. Count cells in ImageJ/Fiji (plugin Cell Counter) or by segmenting using the StarDist plugin65.

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Results

CRISPR/Cas9-mediated gene ablation requires the design of gRNAs. Generally, targeting one of the first exons of a gene of interest with a pair of gRNAs spaced 7-11 bp apart (avoiding multiples of 3 bp) works well (Figure 1A). As a first test, the efficiency of template recognition by gRNAs may be interrogated in vitro using a PCR template26,40,62. Efficient targeting and Cas9-mediated cutti...

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Discussion

Human cerebral and cortical organoids have become key models to investigate human neocortex development7,16,17. For modeling of human disorders, isogenic hiPSC lines and organoids have become the gold standard68. However, since the generation of novel iPSC lines is time-consuming and costly, the acute manipulation of cortical organoid models for studies of gene function has been widely applied.

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Disclosures

The authors declare that they have no conflicts of interest.

Acknowledgements

We are grateful to the facilities of the CRTD and Dresden Concept partners for the outstanding support provided, notably K. Neumann and her team at the Stem Cell Engineering Facility, H. Hartmann and her team at the Light Microscopy Facility, A. Gompf and her team at the Flow Cytometry Facility and Hartmut Wolf of the MPI-CBG workshop for the construction of the electroporation chambers. We thank Joshua Schmidt for his feedback on the manuscript. MA acknowledges funding from the Center for Regenerative Therapies TU Dresden, the DFG (Emmy Noether, AL 2231/1-1), and the Schram Foundation.

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Materials

NameCompanyCatalog NumberComments
15 mL PP Centrifuge Tubes, conicalCorning430791
4D-Nucleofector Core UnitLonza
4D-Nucleofector X UnitLonza
5 mL polystyrene round-bottom tube with cell-strainer capCorningFalcon 352235
6-well plate, nunclon treatedThermo Fisher140675For hiPSC culture
6-well plate, ultra low attachmentCorning3471For organoid culture
A83-01STEMCELL Technologies72022Forebrain medium 1 (https://doi.org/10.1016/j.stem.2020.02.002)15
Air compressorAerotec
Alexa Fluor 488 Donkey Anti-Chicken IgY (IgG) (H+L)Jackson Immuno Research703-545-155Secondary antibody, RRID: AB_2340375; dilution 1:1000
Alexa Fluor 555 Donkey Anti-Rabbit IgG (H+L)InvitrogenA-31572Secondary antibody, RRID: AB_162543; dilution 1:1000
Alt-R CRISPR-Cas9 crRNA, 2 nmolIntegrated DNA technologiesCustom design (order as 2 nmol)
Alt-R CRISPR-Cas9 tracrRNAIntegrated DNA technologies10725325 nmol
Alt-R S.p. HiFi Cas9 Nuclease V3Integrated DNA technologies1081060100 µg, from Streptococcus pyogenes
Amphotericin B (Fungizone)Gibco15290018Forebrain medium 2, 3, and 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
Anti-GFP primary antibody (chicken, polyclonal)Abcamab13970RRID: AB_300798; dilution 1:2000
Anti-PCGF4 primary antibody (mouse, monoclonal)Millipore05-637 RRID: AB_309865; dilution 1:300
ApoTome fluorescent microscopeZeiss
Ascorbic AcidSigma Aldrich1043003Forebrain medium 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
B27-supplement (+ vitamin A)Gibco17504044Forebrain medium 3, 4   (https://doi.org/10.1016/j.stem.2020.02.002)15
Banana to Micrograbber Cable KitHarvard Apparatus BTX45-0216
Biometra TRIO-Thermocycler (PCR machine)Analytik Jena846-2-070-723
Capillaries, borosilicate glass with filament, OD 1.2 mm; ID 0.69 mm; 10 cm lengthScience ProductsBF120-69-10Need to be pulled to specific thickness
CHIR-99021STEMCELL Technologies72052Forebrain medium 2  (https://doi.org/10.1016/j.stem.2020.02.002)15
Collagenase Type IVGibco17104019
CRTDi004-AStem Cell Engineering Facility at CMCB DDhttps://hpscreg.eu/cell-line/CRTDi004-ASingle-cell adapted hiPSC line from healthy donor
DAPIRoche102362760011 mg/mL stock, use 1:1000 diluted for IF
Dibutyryl-cAMPSTEMCELL Technologies73884Forebrain medium 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
Dimethyl sulfoxide (DMSO)Sigma AldrichD2650
DMEM/F-12, HEPESFisher Scientific31330095Forebrain medium 1, 2, and 3  (https://doi.org/10.1016/j.stem.2020.02.002)15
DorsomorphinSTEMCELL Technologies72102Forebrain medium 1  (https://doi.org/10.1016/j.stem.2020.02.002)15
Dumont #55 Forceps, straigth, 11 cmFine Science Tools11295-51
ECM 830 Square Wave Electroporation SystemHarvard Apparatus BTX45-2052
EthanolSigma Aldrich32205-1L-M
Ethylenediaminetetraacetic acid (EDTA)Sigma AldrichEDS-500G
Fast Green FCFSigma AldrichF7252
Fetal Bovine Serum (FBS)GE Healthcare SH30070.03 
Flaming/Brown Micropipette PullerSutter Instrument Co.P-97For pulling of microcapillaries
Geneious PrimeGraphPad Software LLC d.b.a GeneiousSoftware version 2024.0.5
gLACZKalebic et al., 2016; Platt et al., 20145'-TGCGAATACGCCCACGCGATCGG; underlined nucleotides = PAM
GlutaMAXGibco 35050038Forebrain medium 1, 2, 3, and 4 (https://doi.org/10.1016/j.stem.2020.02.002)15
GlycineSigma AldrichG8898
gPCGF4 KO1this paper5'-TGAACTTGGACATCACAAATAGG (corresponds to the KO images in Figure 3I+J)
gPCGF4 KO2this paper5'-ACAAATAGGACAATACTTGCTGG (corresponds to the KO images in Figure 3I+J)
HEPESmade in house1 M stock
Horse Serum, heat-inactivatedGibco26050088
Human GDNF Recombinant ProteinThermo Fisher450-10Forebrain medium 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
Human/Mouse/Rat BDNF Recombinant ProteinThermo Fisher450-02Forebrain medium 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
Hydrochloric acid (HCl)Sigma Aldrich258148To make TrisHCl
ImmEdge (wax) PenVector LaboraoriesH-4000
Insulin solution humanSigma Aldrich I9278Forebrain medium 3  (https://doi.org/10.1016/j.stem.2020.02.002)15
IsopropanolFisher ScientificBP2618-1
Knockout Serum ReplacementGibco 10828-010Forebrain medium 1  (https://doi.org/10.1016/j.stem.2020.02.002)15
Laser Scanning Confocal 980 MicroscopeZeiss
Low Melting Point Agarose, ultra pureThermo Fisher16520100for embedding of hCOs during vibratome sectioning
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-freeCorning354230For organoid culture
Matrigel hESC-Qualified Matrix, LDEV-freeCorning354277For hiPSC culture
MgCl2, 1 MThermo FisherAM9530G
Microinjector PicoPump + Foot SwitchWorld Precision InstrumentsSYS-PV820
Microloader Pipette Tips 0.5 to 20 µLEppendorf5242956003For loading of glass capillaries
Mowiol 4-88Sigma Aldrich81381
mTeSR 1STEMCELL Technologies85850Stem cell medium
N-2 supplementGibco17502048Forebrain medium 2, and 3  (https://doi.org/10.1016/j.stem.2020.02.002)15
NaClSigma AldrichS5886
NaHCO3Sigma AldrichS5761
NEB Next High Fidelity 2x PCR Mastermix New England BiolabsM0541S
Neubauer counting chamberBrand718605
Neurobasal mediumGibco21103049Forebrain medium 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
Non-Essential Amino AcidsGibco 11140050Forebrain medium 1, 2, 3, and 4 (https://doi.org/10.1016/j.stem.2020.02.002)15
Nuclease-Free Duplex BufferIntegrated DNA technologies1072570
O. C. T. CompoundTissue-Tek4583Embedding medium for frozen tissue specimen
P3 Primary Cell 4D-Nucleofector X Kit SLonzaV4XP-3032
ParaformaldehydeFisher ChemicalP/0840/53
pCAG-GFPAddgene11150
Peel-A-Way Embedding Mold TruncatedT12Polysciences Inc.18986-1For embedding of hCOs for vibratome and cryo-sectioning
Penicillin-Streptomycin (10,000 U/mL) Gibco15140122Forebrain medium 1, 2, 3, and 4  (https://doi.org/10.1016/j.stem.2020.02.002)15 and for stem cell medium after nucleofection
Petri Dish 60 mm x 15 mm, No Vent, SterileCorningBP50-02
Petri Dish Platinum Electrode Chamber, 5 mm gapHarvard Apparatus BTX45-0504
Phosphate buffered saline (PBS)made in house
Proteinase KSigma AldrichP2308From Titrachium album; 10 mg/mL stock
QIAquick Gel Extraction KitQiagen28706
RHO/ROCK pathway inhibitor (Y-27632)STEMCELL Technologies72308
SB-431542STEMCELL Technologies72232Forebrain medium 2  (https://doi.org/10.1016/j.stem.2020.02.002)15
Sodium Citrate DihydrateSigma AldrichW302600-1KG-K
Sodium Dodecyl Sulfate (SDS)Sigma AldrichL3771
Sucrose Sigma AldrichS7903
SuperFrost Plus adhesion slidesFisher Scientific10149870
SZX10 with a KL 300 LEDOlympusSZX10Or alternative stereoscope
Thermo ShakerGrant bioPSC24N
Triton-XSigma AldrichT9284a.k.a. Octoxinol 9
Trizma baseSigma AldrichT1503To make TrisHCl
Trypan Blue Solution, 0.4%Thermo Fisher15250061
TrypLE Express Enzyme (1x)Thermo Fisher12604021Dissociation enzmye to make single-cell suspensions
Tween 20Sigma AldrichP1379a.k.a. Polysorbate 20
Tyrode's saltSigma AldrichT2145-10x1l
Vibrating Microtome (vibratome)LeicaVT1200 S
Wide-Bore 1000 µL Universal Fit Filter TipsCorningTF-1005-WB-R-S
β-MercaptoethanolGibco 21985-023Forebrain medium 1, 3, and 4  (https://doi.org/10.1016/j.stem.2020.02.002)15

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