Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

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 functions^1,2,3,4,5. 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 evolution

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 o....

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 template^26,40,62. Efficient targeting and Cas9-mediated cutti.......

Discussion

Human cerebral and cortical organoids have become key models to investigate human neocortex development^7,16,17. 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL PP Centrifuge Tubes, conical	Corning	430791
4D-Nucleofector Core Unit	Lonza
4D-Nucleofector X Unit	Lonza
5 mL polystyrene round-bottom tube with cell-strainer cap	Corning	Falcon 352235
6-well plate, nunclon treated	Thermo Fisher	140675	For hiPSC culture
6-well plate, ultra low attachment	Corning	3471	For organoid culture
A83-01	STEMCELL Technologies	72022	Forebrain medium 1 (https://doi.org/10.1016/j.stem.2020.02.002)15
Air compressor	Aerotec
Alexa Fluor 488 Donkey Anti-Chicken IgY (IgG) (H+L)	Jackson Immuno Research	703-545-155	Secondary antibody, RRID: AB_2340375; dilution 1:1000
Alexa Fluor 555 Donkey Anti-Rabbit IgG (H+L)	Invitrogen	A-31572	Secondary antibody, RRID: AB_162543; dilution 1:1000
Alt-R CRISPR-Cas9 crRNA, 2 nmol	Integrated DNA technologies		Custom design (order as 2 nmol)
Alt-R CRISPR-Cas9 tracrRNA	Integrated DNA technologies	1072532	5 nmol
Alt-R S.p. HiFi Cas9 Nuclease V3	Integrated DNA technologies	1081060	100 µg, from Streptococcus pyogenes
Amphotericin B (Fungizone)	Gibco	15290018	Forebrain medium 2, 3, and 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
Anti-GFP primary antibody (chicken, polyclonal)	Abcam	ab13970	RRID: AB_300798; dilution 1:2000
Anti-PCGF4 primary antibody (mouse, monoclonal)	Millipore	05-637	RRID: AB_309865; dilution 1:300
ApoTome fluorescent microscope	Zeiss
Ascorbic Acid	Sigma Aldrich	1043003	Forebrain medium 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
B27-supplement (+ vitamin A)	Gibco	17504044	Forebrain medium 3, 4   (https://doi.org/10.1016/j.stem.2020.02.002)15
Banana to Micrograbber Cable Kit	Harvard Apparatus BTX	45-0216
Biometra TRIO-Thermocycler (PCR machine)	Analytik Jena	846-2-070-723
Capillaries, borosilicate glass with filament, OD 1.2 mm; ID 0.69 mm; 10 cm length	Science Products	BF120-69-10	Need to be pulled to specific thickness
CHIR-99021	STEMCELL Technologies	72052	Forebrain medium 2  (https://doi.org/10.1016/j.stem.2020.02.002)15
Collagenase Type IV	Gibco	17104019
CRTDi004-A	Stem Cell Engineering Facility at CMCB DD	https://hpscreg.eu/cell-line/CRTDi004-A	Single-cell adapted hiPSC line from healthy donor
DAPI	Roche	10236276001	1 mg/mL stock, use 1:1000 diluted for IF
Dibutyryl-cAMP	STEMCELL Technologies	73884	Forebrain medium 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
Dimethyl sulfoxide (DMSO)	Sigma Aldrich	D2650
DMEM/F-12, HEPES	Fisher Scientific	31330095	Forebrain medium 1, 2, and 3  (https://doi.org/10.1016/j.stem.2020.02.002)15
Dorsomorphin	STEMCELL Technologies	72102	Forebrain medium 1  (https://doi.org/10.1016/j.stem.2020.02.002)15
Dumont #55 Forceps, straigth, 11 cm	Fine Science Tools	11295-51
ECM 830 Square Wave Electroporation System	Harvard Apparatus BTX	45-2052
Ethanol	Sigma Aldrich	32205-1L-M
Ethylenediaminetetraacetic acid (EDTA)	Sigma Aldrich	EDS-500G
Fast Green FCF	Sigma Aldrich	F7252
Fetal Bovine Serum (FBS)	GE Healthcare	SH30070.03
Flaming/Brown Micropipette Puller	Sutter Instrument Co.	P-97	For pulling of microcapillaries
Geneious Prime	GraphPad Software LLC d.b.a Geneious	Software version 2024.0.5
gLACZ	Kalebic et al., 2016; Platt et al., 2014		5'-TGCGAATACGCCCACGCGATCGG; underlined nucleotides = PAM
GlutaMAX	Gibco	35050038	Forebrain medium 1, 2, 3, and 4 (https://doi.org/10.1016/j.stem.2020.02.002)15
Glycine	Sigma Aldrich	G8898
gPCGF4 KO1	this paper		5'-TGAACTTGGACATCACAAATAGG (corresponds to the KO images in Figure 3I+J)
gPCGF4 KO2	this paper		5'-ACAAATAGGACAATACTTGCTGG (corresponds to the KO images in Figure 3I+J)
HEPES	made in house		1 M stock
Horse Serum, heat-inactivated	Gibco	26050088
Human GDNF Recombinant Protein	Thermo Fisher	450-10	Forebrain medium 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
Human/Mouse/Rat BDNF Recombinant Protein	Thermo Fisher	450-02	Forebrain medium 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
Hydrochloric acid (HCl)	Sigma Aldrich	258148	To make TrisHCl
ImmEdge (wax) Pen	Vector Laboraories	H-4000
Insulin solution human	Sigma Aldrich	I9278	Forebrain medium 3  (https://doi.org/10.1016/j.stem.2020.02.002)15
Isopropanol	Fisher Scientific	BP2618-1
Knockout Serum Replacement	Gibco	10828-010	Forebrain medium 1  (https://doi.org/10.1016/j.stem.2020.02.002)15
Laser Scanning Confocal 980 Microscope	Zeiss
Low Melting Point Agarose, ultra pure	Thermo Fisher	16520100	for embedding of hCOs during vibratome sectioning
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free	Corning	354230	For organoid culture
Matrigel hESC-Qualified Matrix, LDEV-free	Corning	354277	For hiPSC culture
MgCl2, 1 M	Thermo Fisher	AM9530G
Microinjector PicoPump + Foot Switch	World Precision Instruments	SYS-PV820
Microloader Pipette Tips 0.5 to 20 µL	Eppendorf	5242956003	For loading of glass capillaries
Mowiol 4-88	Sigma Aldrich	81381
mTeSR 1	STEMCELL Technologies	85850	Stem cell medium
N-2 supplement	Gibco	17502048	Forebrain medium 2, and 3  (https://doi.org/10.1016/j.stem.2020.02.002)15
NaCl	Sigma Aldrich	S5886
NaHCO3	Sigma Aldrich	S5761
NEB Next High Fidelity 2x PCR Mastermix	New England Biolabs	M0541S
Neubauer counting chamber	Brand	718605
Neurobasal medium	Gibco	21103049	Forebrain medium 4  (https://doi.org/10.1016/j.stem.2020.02.002)15
Non-Essential Amino Acids	Gibco	11140050	Forebrain medium 1, 2, 3, and 4 (https://doi.org/10.1016/j.stem.2020.02.002)15
Nuclease-Free Duplex Buffer	Integrated DNA technologies	1072570
O. C. T. Compound	Tissue-Tek	4583	Embedding medium for frozen tissue specimen
P3 Primary Cell 4D-Nucleofector X Kit S	Lonza	V4XP-3032
Paraformaldehyde	Fisher Chemical	P/0840/53
pCAG-GFP	Addgene	11150
Peel-A-Way Embedding Mold TruncatedT12	Polysciences Inc.	18986-1	For embedding of hCOs for vibratome and cryo-sectioning
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140122	Forebrain 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, Sterile	Corning	BP50-02
Petri Dish Platinum Electrode Chamber, 5 mm gap	Harvard Apparatus BTX	45-0504
Phosphate buffered saline (PBS)	made in house
Proteinase K	Sigma Aldrich	P2308	From Titrachium album; 10 mg/mL stock
QIAquick Gel Extraction Kit	Qiagen	28706
RHO/ROCK pathway inhibitor (Y-27632)	STEMCELL Technologies	72308
SB-431542	STEMCELL Technologies	72232	Forebrain medium 2  (https://doi.org/10.1016/j.stem.2020.02.002)15
Sodium Citrate Dihydrate	Sigma Aldrich	W302600-1KG-K
Sodium Dodecyl Sulfate (SDS)	Sigma Aldrich	L3771
Sucrose	Sigma Aldrich	S7903
SuperFrost Plus adhesion slides	Fisher Scientific	10149870
SZX10 with a KL 300 LED	Olympus	SZX10	Or alternative stereoscope
Thermo Shaker	Grant bio	PSC24N
Triton-X	Sigma Aldrich	T9284	a.k.a. Octoxinol 9
Trizma base	Sigma Aldrich	T1503	To make TrisHCl
Trypan Blue Solution, 0.4%	Thermo Fisher	15250061
TrypLE Express Enzyme (1x)	Thermo Fisher	12604021	Dissociation enzmye to make single-cell suspensions
Tween 20	Sigma Aldrich	P1379	a.k.a. Polysorbate 20
Tyrode's salt	Sigma Aldrich	T2145-10x1l
Vibrating Microtome (vibratome)	Leica	VT1200 S
Wide-Bore 1000 µL Universal Fit Filter Tips	Corning	TF-1005-WB-R-S
β-Mercaptoethanol	Gibco	21985-023	Forebrain medium 1, 3, and 4  (https://doi.org/10.1016/j.stem.2020.02.002)15