TITLE: Generating stably-expressing Cas9 cancer organoid lines
AUTHORS: Jade Smith, Tessa Fowler, Agnieszka Andres, Adam Jackson, Emily Souster, Hazel Rogers, Alexandra Beck, Charlotte Beaver, Mathew Garnett

[DESCRIPTION]
This protocol aims to establish a robust Cas9 expression system in cancer organoids through a three-stage process. The first stage involves titrating blasticidin to determine the optimal concentration for eliminating wild-type cells while supporting the survival and growth of Cas9-expressing cells. The second step introduces the Cas9 gene, and the specified blasticidin concentration selects and maintains Cas9-positive cells. The final stage assesses Cas9 activity, ensuring functionality is above 75%. This established system facilitates precise and genome-wide gene modification using the 'lentiCas9-Blast' CRISPR-Cas9 guide RNA system. 


 



Process Diagram


 



[BEFORE_START]
If required, ensure that all media is pre-warmed before use.

If required, Cell Titer-Glo 2.0 reagent is light-sensitive, so try to avoid exposure when using it.

If needed, thaw an aliquot of polybrene.

When needed, thaw an appropriate amount of Cas9 lentivirus for the number of transductions you will carry out.


[GUIDELINES]
Ensure the cell suspension is mixed thoroughly at each step to create an even single-cell suspension before plating, using reservoirs and multi-channel pipettes where possible to minimise ergonomic strain and to maintain homogenous solutions throughout.

Avoid prolonged maintenance of organoids in a single-cell state. 

Refrain from the freeze/thaw cycle of virus stocks.

Prioritise using freshly prepared virus stocks to maintain optimal viral integrity.

Ensure the correct concentration of virus is used in the experimental setup.

Adequate dissociation is critical for efficient viral exposure and subsequent gene delivery.

Be aware that some cell lines may exhibit lower uptake of viral particles; which may make them unsuitable for this process.

Black-walled 96-well plates are used in this protocol, as we have found luminescence can carry over into neighbouring wells in clear plates. 

[STEPS]
2. Day 4: Assess cell viability using CellTiter-Glo 2.0 assay


1.16. Incubate the plate for 4320 min at 37 °C, 5% CO2.  


1.15. Add 100 µL of the blasticidin antibiotic stock into the corresponding wells in Fig.1.

1.14. Make up blasticidin antibiotic solutions at 2x concentration in organoid specific culture media, containing no BME2 in 5 mL tubes.

 
 

 

1.13. Plate control wells (in triplicate) containing 200 µL organoid specific culture media containing 5% BME2 and no cells.

1.12. Incubate plate at 37 °C, 5% CO2 for 10 min to allow the BME2 to polymerise.

1.11. Add 100 µL of the cell suspension containing BME2 to Rows B-F (according to Fig.1).  
1.10. Make up enough volume to plate all required wells for running each required antibiotic concentration in triplicate (plus extra 10% for dead volume). 

 

 

SECTION: Blasticidin titration
1.9. Resuspend 2.4x106 cells in 2.7ml of organoid specific culture media +  300 µL   BME2 (This will give a final seeding density of 8x104 cells per well once plated, Rows B-F of Fig.1).

Prepare a control stock solution containing organoid specific culture media with 5% BME2 (Row A of Fig.1).

 


SECTION: Blasticidin titration
1.7. Centrifuge at 800 x g for 2 min.

SECTION: Blasticidin titration
1.6. Check organoid suspension under the microscope every 15 min, to assess and monitor the dissociation of the organoids. 


 

SECTION: Blasticidin titration
1.5. Incubate at 37 °C 5% CO2.

SECTION: Blasticidin titration
1.4. Pipette suspension up and down multiple times to dissociate organoids from the BME2.
SECTION: Blasticidin titration
1.3. Using a cell-scraper, detach BME2 drops containing the cancer organoids from the plate and transfer organoid suspension to an appropriately sized tube.

SECTION: Blasticidin titration
1. Day 1: Titration plate set up

 

SECTION: Blasticidin titration
1.2. Aspirate media from well plates and add 2 mL TrypLE to each well. 

SECTION: Blasticidin titration
1.1. Pre-warm organoid specific culture media to room temperature or place in a water bath at 37 °C  for 10 min

2.1. Run a CellTiter-Glo 2.0 viability assay following the manufacturer’s instructions. 

   

 

2.2. Generate a kill curve graph using the data collected. The ‘kill concentration’ is the concentration of an antibiotic at which organoid growth is completely inhibited.

Row A is used as negative control to show background luminescence
Row B is the positive control to compare against wells without antibiotics
 

SECTION: Cas9 transduction of cancer organoids
3. Day 1: Transduction setup 
SECTION: Cas9 transduction of cancer organoids
3.1. Prepare transduction media, add 5 µL ROCKi Y-27632 (10 mM) to 20 mL of organoid specific culture media (2.5 µM final concentration; dilution 1:4000).  

SECTION: Cas9 transduction of cancer organoids
3.2. Aspirate media from well plates and add 2 mL TrypLE to each well of a 6 well plate. 
SECTION: Cas9 transduction of cancer organoids
3.3. Using a cell-scraper, detach BME2 drops containing the cancer organoids from the plate and transfer organoid suspension to an appropriately sized tube.
SECTION: Cas9 transduction of cancer organoids
3.4. Pipette suspension up and down multiple times to dissociate organoids from the BME2.
SECTION: Cas9 transduction of cancer organoids
3.5. Incubate at 37 °C 5% CO2.
SECTION: Cas9 transduction of cancer organoids
3.6. Check organoid suspension under the microscope every 15 min, to assess and monitor the dissociation of the organoids. 

 

SECTION: Cas9 transduction of cancer organoids
3.7. Centrifuge at 800 x g for 2 min.
SECTION: Cas9 transduction of cancer organoids
3.8. Aspirate supernatant and resuspend in 10 mL of transduction media (more or less can be added depending on the size of the cell pellet).

SECTION: Blasticidin titration
1.8. Aspirate supernatant and resuspend in 5 mL organoid specific culture media.

SECTION: Cas9 transduction of cancer organoids
3.9. Perform a cell count to calculate the total number of cells.
SECTION: Cas9 transduction of cancer organoids
3.10. Prepare a preparation mix using the cell suspension and transduction media to achieve a final concentration of between 2x106 and 4x106 cells (with the minimum cells needed to perform a Cas9 transduction being 2x106). 


 

SECTION: Cas9 transduction of cancer organoids
3.11. Please consult Fig.4 for the overall volumes, where the transduction media constitutes the remaining volume. Please consult Fig.4 for the overall volumes, where the transduction media constitutes the remaining volume. Add preparation mix, Cas9 transduction virus and polybrene into a 50 mL bioreactor tube using the table below.

 


 

 
 
SECTION: Cas9 transduction of cancer organoids
3.12. Incubate the 50 mL bioreactor tube prepared in step 3.11   at 37 °C, 5% CO2.

SECTION: Cas9 transduction of cancer organoids
4. Day 2: Plating cells
SECTION: Cas9 transduction of cancer organoids
4.1. Centrifuge at 800 x g for 2 min.

SECTION: Cas9 transduction of cancer organoids
4.2. Aspirate the supernatant.
SECTION: Cas9 transduction of cancer organoids
4.3. Seed 2x106 cells in 230 µL of 80% BME2.  Seed as 15 µLdrops in a one well of 6 well plate (230 μl per well).

SECTION: Cas9 transduction of cancer organoids
4.4. Incubate at 37 °C for 15 min then add 2 mLs of transduction media.

SECTION: Cas9 transduction of cancer organoids
4.5. Incubate cells at 37 °C, 5% CO2.

SECTION: Cas9 transduction of cancer organoids
5. Day 6: Blasticidin selection
SECTION: Cas9 transduction of cancer organoids
5.1. Prepare relevant organoid specific culture media with blasticidin (using the concentration based on the results obtained from the kill curve refer to Fig.3). 
 

SECTION: Cas9 transduction of cancer organoids
5.2. Replace media on plates or flasks with media containing blasticidin at 25 mg/mL(obtained at Fig.3). For example: if the concentration is 25 µg/mL add 12.5 µl to 5 mL media ((volume x 2.5) / 1000 (to convert mL to µL)).

 

SECTION: Cas9 transduction of cancer organoids
5.3. Expand until required number of cells for endpoint experiments has been reached (e.g. assessment of Cas9 activity).

 


SECTION: Assessment of Cas9 activity assay
6. Day 1: Assay set up
SECTION: Assessment of Cas9 activity assay
6.1. Prepare transduction media, by adding 7.5 µL of  ROCKi Y-27632 10 millimolar (mM) to 30 mL of organoid specific culture media. 

 


SECTION: Assessment of Cas9 activity assay
6.2. Aspirate media from well plates and add 2 mL TrypLE to each well. 
SECTION: Assessment of Cas9 activity assay
6.3. Using a cell-scraper, detach BME2 drops containing the cancer organoids from the plate and transfer organoid suspension to an appropriately sized tube.
SECTION: Assessment of Cas9 activity assay
6.4. Pipette suspension up and down multiple times to dissociate organoids from the BME2.

SECTION: Assessment of Cas9 activity assay
6.5. Incubate at 37 °C 5% CO2.
SECTION: Assessment of Cas9 activity assay
6.6. Check organoid suspension under the microscope every 15 min, to assess and monitor the dissociation of the organoids. 


 

SECTION: Assessment of Cas9 activity assay
6.7. Centrifuge at 800 x g for 2 min.
SECTION: Assessment of Cas9 activity assay
6.8. Aspirate supernatant and resuspend in 10 mL of transduction media (more or less can be added depending on the size of the cell pellet).

SECTION: Assessment of Cas9 activity assay
6.10. Prepare a preparation mix using the cell suspension and transduction media to achieve a final concentration of 2.8x106 cells in 5.95 mL(equivalent to 8x105 cells in 1.7 mL accounting for dead volume) and polybrene.

SECTION: Assessment of Cas9 activity assay
6.9. Perform a cell count to calculate the total number of cells.


SECTION: Assessment of Cas9 activity assay
6.11. Transfer 1.7 mL of the preparation mix into 3 x 50 mL bioreactor tubes to include; Mock, Control and Reporter transductions with the final cell numbers, add 300 µL of each respective reagent to the tubes as shown in the table below. 

 



SECTION: Assessment of Cas9 activity assay
6.12. Place bioreactor tubes in the incubator at 37 °C, 5% CO2 for15 min incubation.

SECTION: Assessment of Cas9 activity assay
7. Day 2: Plating cells
SECTION: Assessment of Cas9 activity assay
7.1. Transfer the 3 x 50 mL bioreactor tubes to the centrifuge.

SECTION: Assessment of Cas9 activity assay
7.2. Centrifuge at 800 x g for 2 min.
SECTION: Assessment of Cas9 activity assay
7.3. Aspirate the supernatant for each bioreactor tube.
SECTION: Assessment of Cas9 activity assay
7.4. Resuspend the cells in each 230 µL of organoid specific culture media containing 80% BME2 (to account for pipetting loss) into each Mock / Reporter / Control tube. 

SECTION: Assessment of Cas9 activity assay
7.5. Plate into 1 well of a 6 well plate for each Mock / Reporter / Control, dispensing small 15 µL droplets using a pipette.

SECTION: Assessment of Cas9 activity assay
7.6. Place plate in incubator at 37 °C, 5% CO2 for 10 min until the BME droplets solidify.

SECTION: Assessment of Cas9 activity assay
7.7. Add 2 mLof transduction media to each well.

 


SECTION: Formaldehyde fixation of organoids
8. Day 6: Fixing and staining organoids for flow cytometry analysis
SECTION: Formaldehyde fixation of organoids
8.1. Prepare Live/Dead stain solution or antibodies.
 
 

SECTION: Formaldehyde fixation of organoids
8.2. Aspirate media from wells and resuspend in 1 mL of Trypsin-EDTA (0.25%) in a 2 mL tube. 

SECTION: Formaldehyde fixation of organoids
8.3. Incubate for 15 min, until organoids have broken down to single cells. 

 

SECTION: Formaldehyde fixation of organoids
8.4. Once organoids have broken down to single-cells stop the reaction by adding 1 mL (diluting 1:1) in media containing serum.

SECTION: Formaldehyde fixation of organoids
8.5. Centrifuge at 800 x g for 2 min.
SECTION: Formaldehyde fixation of organoids
8.6. Aspirate supernatant and resuspend pellets in 200 µL Live/Dead dye solution (or specific antibody of choice).

SECTION: Formaldehyde fixation of organoids
8.7. For the Live/Dead solution, incubate at room temperature for 5 min. (Follow specific guidelines for your antibodies).

SECTION: Formaldehyde fixation of organoids
8.8. Add 1.8 mL  of PBS (1:10 dilution). 

SECTION: Formaldehyde fixation of organoids
8.9. Centrifuge at 800 x g for 2 min.
SECTION: Formaldehyde fixation of organoids
8.10. Aspirate supernatant and resuspend in 500 µL of 3.7% formaldehyde. Mix well by pipetting to ensure cells are fixed as single cells.

 

SECTION: Formaldehyde fixation of organoids
8.11. Incubate at 4 °C for 10 min.

SECTION: Formaldehyde fixation of organoids
8.12. Centrifuge at 800 x g for 2 min.
SECTION: Formaldehyde fixation of organoids
8.13. Carefully aspirate supernatant (in chemical fume hood). 

 


SECTION: Formaldehyde fixation of organoids
8.14. Resuspend the pellet in 500 µL (dependant on pellet size) PBS or alternative FACs buffer, and store it at 4 °C until ready for analysis by flow cytometry.