We thank Juliane Fohgrub, Ann-Christin Meinecke and Max Heiduk for excellent technical assistance. Funding was provided by Deutsche Krebshilfe (No 111350 and 70112925), Sander Stiftung (No 2014.104.1), Hector Stiftung (No M65.2) and the European Union (ERC No 639050).

Ethics approval was obtained from the ethical committee of the TU Dresden (#EK451122014).
1. Organoid Culture and Preparations Before Electroporation
<ol>
	<li>Establish organoids by tissue digestion as described previously and expand them with their corresponding entity specific culture medium in a basement matrix (overview see Table 1 and Table of Materials)11,12,13,14,15,16,17. 
	NOTE: For human tissue samples informed consent and approval of the study by an ethical committee is necessary.</li>
	<li>Prewarm 48-well plates at 37 &#176;C for post-electroporation seeding.</li>
	<li>Prepare basal medium w/o antibiotics as well as entity specific organoid culture medium w/o antibiotics (see Table 1) including 10 &#181;M Y-27632 and 3 &#181;M CHIR99021. 
	NOTE: The withdrawal of antibiotics is important to reduce toxic effects. Y-27632 and CHIR99021 ameliorate cell recovery10.</li>
	<li>Preparation of the organoids (see Figure 1)
	<ol>
		<li>Cultivate 5 wells of organoids in a 48-well plate per electroporation sample in culture medium. 
		NOTE: Proliferative organoids should be used (around 2-3 days after last splitting).</li>
		<li>Prepare 230 &#181;L of dissociation reagent (see Table of Materials) including 10 &#181;M Y-27632 per well.</li>
		<li>Remove the culture medium from each well and dissociate the organoids mechanically in 230 &#181;L of the prepared dissociation mixture. Pool 5 wells per electroporation sample into one 15 mL tube.</li>
		<li>Mix by vortexing and incubate for 5-15 min at 37 &#176;C until clusters of 10-15 cells occur. Therefore, check the dissociation microscopically. Stop the digestion by adding basal medium w/o antibiotics up to 10 mL. 
		NOTE: This step is very critical! The electroporation efficiency will be reduced, when incubation is too short, but long digestion will reduce survivability.</li>
		<li>Centrifuge at 450 x g for 5 min at room temperature, discard the supernatant and wash twice with 4 mL of electroporation buffer (see Table of Materials).</li>
	</ol>
	</li>
</ol>
2. Electroporation
NOTE: The following protocol is developed for electroporators capable of square waves and separated poring and transfer pulse sequences (see Figure 2). Optionally, impedance values as well as the voltages, currents and energies transferred into the sample can be measured as control for reproducible experiments.
<ol>
	<li>Resuspend the organoid pellet in 100 &#181;L of electroporation buffer (see Table of Materials) containing 30 &#181;g of plasmid DNA. 
	NOTE: The concentration of the used plasmid DNA should exceed 5 &#181;g/&#181;L for an optimal salt concentration during the electroporation process. Therefore, endofree plasmid maxi kits (see Table of Materials) for the preparation of vectors are recommended. A total amount up to 45 &#181;g of DNA can be used without cytotoxic effects.</li>
	<li>Dispense the complete DNA-organoid mixture into an electroporation cuvette with 2 mm gap width without producing air bubbles.</li>
	<li>Set the electroporation parameters according to Fujii et al.10 (see Table 2, Figure 2).</li>
	<li>Mix the cells slightly without foaming by tapping the cuvette with a finger. Place the cuvette into the cuvette chamber.</li>
	<li>Press the &#937; button of the electroporator and make a note of the impedance value. 
	NOTE: An impedance between 30-40 &#937; showed the best results. In general, it should be in the range between 30-55 &#937;. If this is not the case, please control the following aspects: gap width of the cuvette used, cable connections of the electroporator, possible air bubbles, correct volume and salt concentration of the electroporation mixture.</li>
	<li>Press the Start button to execute the electroporation program and control the values of currents, voltages and energies displayed. 
	NOTE: The values of measured voltages, currents and energies should correspond to the set electroporation parameters. For the comparison of repeated experiments, it can be helpful to note these data.</li>
	<li>After electroporation, immediately add 500 &#181;L of culture medium w/o antibiotics (with CHIR99021 and Y-27632; see step 1.3). Mix by pipetting up and down to dissociate the white foam. 
	NOTE: The white foam appears after the electroporation process and a significant number of cells is attached to it. So, dissociation of it is very important for not losing cells.</li>
	<li>Transfer the sample completely from the cuvette into a new 15 mL tube using the pipette belonging to the electroporation cuvettes (see Table of Materials). Rinsing the cuvette again with basal medium is recommended to obtain remaining cells.</li>
	<li>For regeneration of the cells, incubate them for 40 min at room temperature.</li>
</ol>
3. Seeding of Cells
<ol>
	<li>Centrifuge the cells at 450 x g for 5 min at room temperature and discard the supernatant.</li>
	<li>Resuspend the pellet in 100 &#181;L of basement matrix and seed 20 &#181;L drops in a prewarmed 48-well plate (see step 1.2). Incubate for 10 min at 37 &#176;C for polymerization and add 250 &#181;L of culture medium, which is supplemented with Y-27632 and CHIR99021 until the next splitting of the grown organoids (around 5-7 days).</li>
</ol>
4. Determination of Transfection Efficiency
NOTE: In general, it is recommended to electroporate a vector carrying a fluorescence marker as additional transfection control. Dependent on the chosen marker and its chromophore maturation the fluorescence will be visible within around 24-48 h post transfection18.
<ol>
	<li>Check the fluorescence microscopically after 24-48 h in the transfection control (see Figure 4B).</li>
	<li>Fluorescent activated cell scanning (FACS)
	<ol>
		<li>Harvest the cells analogously to step 1.4.2-1.4.4 and digest around 10-20 min until there are single cells. Add up to 10 mL of phosphate-buffered saline (PBS).</li>
		<li>Centrifuge at 450 x g for 5 min at room temperature and discard the supernatant.</li>
		<li>Optionally for discrimination of living cells: Resuspend the pellet in 1 mL of phosphate-buffered saline (PBS) and add a suitable antibody (see Table of Materials) or propidium iodide (PI). Mix very carefully only by tapping and incubate for 30 min at room temperature in the dark. Wash with 10 mL of PBS, centrifuge and discard the supernatant.</li>
		<li>Resuspend the cell pellet in 200 &#181;L of PBS and optionally filter the suspension through a 100 &#181;m cell strainer into a FACS tube.</li>
		<li>Analyze the cells by a FACS machine using an appropriate gating strategy (see Figure 3; Figure 4A) and determine the transfection efficiency.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This protocol gives detailed instructions for an efficient, quick and easy to perform electroporation of different organoid entities. Additional to the presented tumor organoids from PDAC, CRC, CCC and GC, it works successfully for organoids derived from healthy tissue as well. The protocol can be performed within one day. In published organoid transfection protocols the whole procedure lasted four days including two days of preparations with different types of cultivation media10,21. In our protocol no special pretreatment is required. By washing with electroporation buffer before electroporation the antibiotic components of the media were washed out and an adjustment of saline concentrations for optimal impedance values was achieved. Nevertheless, some critical aspects should be considered for a successful electroporation:
Cells 
In the electroporation protocol by Fujii et al.10 it is recommended to dissociate organoids to single cells and to filter them through a 20 &#181;m cell-strainer. In our hands digestion to single cells strongly decreases the survivability of cells. As suggested in Merenda et al.21, we also dissociated organoids to clusters of 10-15 cells and could not determine a decreased efficiency compared to single cell dissociation. After electroporation, it is a very important step to dissociate the white foam, so that no attached cells are getting lost.
For 2D cell culture, it has been shown that a regeneration time after electroporation of more than 10 min up to 40 min increases survivability and transfection efficiency especially of large plasmids22. In test experiments, the same could be documented for organoids, leading to an incubation step of 40 min after electroporation in this protocol. In order to increase recovery from the electroporation, we cultured them with Rho-associated protein kinase (ROCK) inhibitor Y-27632 for five to seven days23. Similarly, the additional supplementation of glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021 is meant to help single cells to recover10.
Settings 
One of the advantages of the used electroporator is that the impedance can be measured before electroporation for optimal conditions. According to the manufacturer, the impedance values should be 30-55 &#937;. In our hands, impedance values of 30-40 &#937; have shown optimal efficiencies. In a preliminary experiment, different voltages and pulse length values of the poring pulse were varied to find the optimal proportion of efficiency to survivability. In summary, we could confirm the described values of Fujii et al.10 in the different entities described here.
DNA 
The effect of different DNA amounts was tested in preliminary experiments up to 45 &#181;g of DNA per sample. No cytotoxic effects could be detected. Transfection efficiency was increased in a dose dependent way with saturation &#62; 30 &#181;g. So, we used 30 &#181;g per sample in the final protocol, but of course it can be increased (e.g., for the electroporation of more plasmids in parallel). Additionally, the purity and concentration of the DNA seems to be very important. A concentration exceeding 5 &#181;g/&#181;L has shown optimal transfection efficiencies.
As expected, the 9.3 kB plasmid could be transfected with a lower efficiency than the smaller 4.2 kB plasmid (see Figure 4). The use of even larger plasmids than 10 kB is anticipated to further decrease the efficiency. For future applications, it might be interesting to test minicircle DNA as a vector, since these gene carriers lack the bacterial backbone of a plasmid which makes them smaller24. This should result in an enhanced transfection efficiency. Furthermore, for CRISPR-based manipulations of organoids a direct electroporation of sgRNAs bound to Cas9 as a ribonucleoprotein (RNP) complex could be an alternative or addition25.

In recent years, a novel 3D cell culture system, termed organoids, was developed for various normal and cancerous tissues. Organoids are functionally and morphologically very close to their tissue of origin. They can be generated from different species, are easily expandable, genomically stable and genetically modifiable, which makes them an ideal model system for genetic investigations1,2,3. Genetic engineering techniques like the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system enable diverse manipulations. The selection of clones can be realized by defined media conditions, for example, by WNT ligand withdrawal for APC (Adenomatosis Polyposis Coli) knockout clones4,5. Alternatively, selection markers have to be introduced by homologous directed repair of a targeting vector6,7. Due to the fact that often more than one plasmid needs to be introduced, an efficient transfection becomes a crucial parameter. Additionally, to reduce unspecific off-target effects, a transient expression of the Cas9 endonuclease is desirable8.
Electroporation is a comparatively simple method to transfect cells with DNA, RNA, proteins or other macromolecules. By means of electric pulses, the cell membrane becomes more permeable and causes an increased uptake9. In a previously published electroporation protocol of colon organoids a 30 % efficiency with a piggy-bac GFP (green fluorescent protein) expressing vector (7.4 kB) was reached in a four days procedure10. The following protocol was developed to facilitate an efficient transfection of cancerous or healthy organoids with large plasmids encoding for single guide RNA (sgRNA) and the Cas9 endonuclease sequence (e.g. px458 as vector with 9.3 kb). The whole electroporation process can be performed within one day, without special electroporation buffers, and with at least comparable efficiencies between different gastrointestinal organoids, namely pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), cholangiocarcinoma (CCC) and gastric cancer (GC) organoids.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>For establishment and culture medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>[Leu15] Gastrin</td>
			<td>Sigma-Aldrich</td>
			<td>G9145</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Tocris Bioscience</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12</td>
			<td>Invitrogen</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement, minus vitamin A</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Life Technologies</td>
			<td>17101-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase XI</td>
			<td>Sigma-Aldrich</td>
			<td>C9407-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma-Aldrich</td>
			<td>D5319</td>
			<td></td>
		</tr>
		<tr>
			<td>D-sorbitol</td>
			<td>Roth</td>
			<td>6213.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Thermo Scientific</td>
			<td>1859330</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Roth</td>
			<td>8040</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Tocris Bioscience</td>
			<td>1099</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>hFGF-10</td>
			<td>Preprotech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Roth</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>basement matrix</td>
		</tr>
		<tr>
			<td>mEGF</td>
			<td>Invitrogen</td>
			<td>PMG8043</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Invitrogen</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Roth</td>
			<td>3957.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Roth</td>
			<td>K300.2</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetyl-L-Cystein</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamid</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>Conditioned medium produced from HEK293 cells (Hek293-mNoggin-Fc)</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen</td>
			<td>ant-pm-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Prostaglandin E2</td>
			<td>Tocris Bioscience</td>
			<td>2296</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human HGF</td>
			<td>Preprotech</td>
			<td>100-39H</td>
			<td></td>
		</tr>
		<tr>
			<td>Rspondin</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>Conditioned medium produced from HEK293 cells (HA-Rspo1-Fc-293T)</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S7067</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>VWR</td>
			<td>27,480,294</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12604021</td>
			<td>Dissociation reagent</td>
		</tr>
		<tr>
			<td>Wnt3A</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>Conditioned medium produced from L-Wnt3a cells (from Sylvia Boj)</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 100 &#181;m</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well plate</td>
			<td>Corning</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Nepa Electroporation Cuvettes 2mm gap w/pipettes</td>
			<td>Nepa Gene Co., Ltd.</td>
			<td>EC-002S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes 15 ml</td>
			<td>Greiner</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes 15 ml low binding</td>
			<td>Eppendorf</td>
			<td>30122208</td>
			<td>Tubes for FACS preparing</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator Nepa21</td>
			<td>Nepa Gene Co., Ltd.</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS FL Auto</td>
			<td>Invitrogen</td>
			<td>AMAFD1000</td>
			<td>Fluorescence microscope</td>
		</tr>
		<tr>
			<td>LSRFortessa</td>
			<td>BD Bioscience</td>
			<td>647800E6</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Reagents and plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Live/dead fixable blue dead cell stain kit</td>
			<td>Invitrogen</td>
			<td>L34962</td>
			<td>Includes antibody for live/dead staining for FACS analysis</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutlin-3</td>
			<td>Selleckchem</td>
			<td>S1061/07</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985047</td>
			<td>Electroporation buffer</td>
		</tr>
		<tr>
			<td>2 gRNA concatemer vector</td>
			<td>AddGene</td>
			<td>84879</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-EGFP</td>
			<td>Nepa Gene Co., Ltd.</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>px458 plasmid</td>
			<td>AddGene</td>
			<td>48138</td>
			<td>coding for sgRNA and Cas9</td>
		</tr>
		<tr>
			<td>px458_Conc2 plasmid</td>
			<td>AddGene</td>
			<td>134449</td>
			<td>px458 plasmid containing 2x U6 promotors for two different sgRNAs</td>
		</tr>
		<tr>
			<td>sgRNA_hTP53_1a</td>
			<td>Eurofins Genomics</td>
			<td>n.a.</td>
			<td><img src="/files/ftp_upload/60704/60704tomeq1.jpg" /></td>
		</tr>
		<tr>
			<td>sgRNA_hTP53_1b</td>
			<td>Eurofins Genomics</td>
			<td>n.a.</td>
			<td><img src="/files/ftp_upload/60704/60704tomeq2.jpg" /></td>
		</tr>
		<tr>
			<td>sgRNA_hTP53_2a</td>
			<td>Eurofins Genomics</td>
			<td>n.a.</td>
			<td><img src="/files/ftp_upload/60704/60704tomeq3.jpg" /></td>
		</tr>
		<tr>
			<td>sgRNA_hTP53_2b</td>
			<td>Eurofins Genomics</td>
			<td>n.a.</td>
			<td><img src="/files/ftp_upload/60704/60704tomeq4.jpg" /></td>
		</tr>
	</tbody>
</table>

universal and efficient electroporation protocol for genetic engineering of gastrointestinal organoids

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.

Organoids of four different cancer entities (CRC, CCC, PDAC, GC) were electroporated at least 3 times using 30 &#181;g of a small plasmid (pCMV-EGFP, 4.2 kb) or a large plasmid (px458, 9.3 kb). Both vectors carry a GFP cassette allowing the determination of transfection efficiency 48 h after electroporation by flow cytometry. To analyze only living cells, staining with a life-death antibody before scanning was performed. The gating strategy is shown in Figure 3.
In all four organoid entities the 4.2 kB sized plasmid was transfected with higher efficiency compared to the larger one (see Figure 4). The most efficient transfection of the small plasmid was reached in PDAC organoids with 92.1 &#177; 5.2 % GFP positive cells, whereas the large plasmid was transfected with an efficiency of 46.7 &#177; 3.7 % (mean &#177; standard deviation, n = 3). Compared to pancreatic cancer organoids, the larger plasmid was more efficiently transfected into CRC organoids with a mean efficiency of 53.4 &#177; 11.7 %, while the small plasmid was transfected with a mean efficiency of 84.3 &#177; 5.8 %. The most difficult entity to transfect were gastric cancer organoids: for both the large and the small plasmid, the lowest transfection efficiency was reached in this entity (32.3 &#177; 12.7 % and 74.1 &#177; 5.5 %, respectively). CCC organoids showed a mean transfection efficiency of 83.0 &#177; 13.1 % for the small plasmid and for the large plasmid 39.5 &#177; 10.4 % were obtained.
As proof of concept, human normal stomach organoids were electroporated with a px458_Conc2 plasmid encoding for Cas9, GFP and two sgRNAs targeting TP53. The Cas9-induced double strand breaks on exon 8 were repaired by non-homologous end-joining (NHEJ), resulting in frameshifts and consequently a knockout of the gene (see Supplementary Figure 1).
Table 1: Composition of basal media, digestion mixtures and cultivation media. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60704/60704_table1.xlsx" target="_blank">Please click here to view this file (Right click to download).</a>
<img alt="Table 1" src="/files/ftp_upload/60704/60704table2.jpg" /> 
Table 2: Electroporation settings according to Fujii et al.10.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60704/60704fig1.jpg" /> 
Figure 1: Electroporation preparation workflow. First, organoids ought to be dissociated to clusters of 10-15 cells and antibiotics should get washed out. After electroporation the white foam needs to be dissociated. Cells can be seeded after regenerating for 40 min at room temperature. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60704/60704fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60704/60704fig2b.jpg" /> 
Figure 2: Two-step electroporation. Two poring pulses with higher voltage und short duration (175 V and 157.5 V, each for 5 ms, pause for 50 ms, voltage decay 10%) lead to the formation of pores in cell membranes. The following transfer pulses deliver the DNA into the cells: five positive transfer pulses (with 20 V, 12 V, 7.2 V, 4.32 V and 2,592 V, each for 50 ms, pause for 50 ms, voltage decay 40%), followed by five polarity exchanged transfer pulses (with 20 V, 12 V, 7.2 V, 4.32 V and 2,592 V, each for 50 ms, pause for 50 ms, voltage decay 40%). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60704/60704fig2blarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60704/60704fig3b.jpg" /> 
Figure 3: Representative gating strategy shown by CCC organoids. All electroporated organoids were analyzed by flow cytometry 48 h after electroporation. Cells electroporated without plasmid DNA were used as negative controls. The gates were set as following: (A) gating for cell shape, (B,C) gating for single cells (doublet discrimination), (D) gating for living cells (stained with an antibody for apoptotic cells) and (E,F) finally gating for eGFP expressing cells (FITC channel). FSC = forward scatter; SSC = side scatter. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60704/60704fig3blarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60704/60704fig4.jpg" /> 
Figure 4: Electroporation efficiency of four organoid entities. (A) FACS analysis (n = 34, mean standard deviation and each single value are shown) and (B) visual comparison by fluorescence microscope. Scale bar = 1,000 &#181;m. BF = bright field; CCC = cholangiocarcinoma; CRC = colorectal cancer; GC = gastric cancer; PDAC = pancreatic ductal adenocarcinoma. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60704/60704fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/60704/60704suppfig1a.jpg" /> 
Supplementary Figure 1: Exemplary CRISPR/Cas9-based knockout of TP53 in normal human stomach organoids.The px458_Conc2 vector (see Table of Materials) was cloned by combining the 2 gRNA concatemer vector, a generous gift from Bon-Kyoung Koo19, with px45820, resulting in a plasmid encoding for 2 sgRNAs, Cas9 and GFP. Two sgRNAs targeting TP53 were introduced in px458_Conc2 vector by golden gate cloning (analogously to Andersson-Rolf et al.19). 10 &#181;g of plasmid DNA were electroporated in human normal gastric organoids (A). Clones were selected by Nutlin3 administration (B) and the TP53 knockout was confirmed by TOPO TA cloning and sequencing of the alleles, here exemplary shown for one clone (C). The sgRNAs are underlined in the reference. Scale bar = 200 &#181;m. BF = bright field. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60704/60704suppfig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids at JoVE.com

Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

This protocol describes an efficient electroporation method for the transfection of four different gastrointestinal organoid entities with larger plasmids (to the extent of 10 kB). It can be performed within one day and does not need extensive preparation or special, cost-intensive electroporation buffers.

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the ...

universal-efficient-electroporation-protocol-for-genetic-engineering

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

16.9K Views.  Technische Universität Dresden. This protocol describes an efficient electroporation method for the transfection of four different gastrointestinal organoid entities with larger plasmids (to the extent of 10 kB). It can be performed within one day and does not need extensive preparation or special, cost-intensive electroporation buffers.

Video: Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis.

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

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