Nov 12, 2024
Version 2
Whole-genome CRISPR screening of stably expressing Cas9 cancer organoid lines V.2
- Tessa Fowler1,
- Jade Smith1,
- Adam Jackson1,
- Agnieszka Andres1,
- Emily Souster1,
- Hazel Rogers1,
- Alexandra Beck1,
- Charlotte Beaver1,
- Mathew Garnett1
- 1Wellcome Sanger institute
External link: https://www.sanger.ac.uk/group/garnett-group/
Protocol Citation: Tessa Fowler, Jade Smith, Adam Jackson, Agnieszka Andres, Emily Souster, Hazel Rogers, Alexandra Beck, Charlotte Beaver, Mathew Garnett 2024. Whole-genome CRISPR screening of stably expressing Cas9 cancer organoid lines. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4q6zrvo5/v2Version created by Tessa Fowler
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 15, 2023
Last Modified: November 12, 2024
Protocol Integer ID: 100040
Keywords: Cancer Organoids, CRISPR/Cas9, gRNA Library titration, gRNA Library transduction, Antibiotic titration, Whole-genome CRISPR Screening, organoid, organoids
Abstract
This protocol is for whole-genome CRISPR screening of stably expressing Cas9 cancer organoid lines in triplicate using the commercially available minimal genome-wide human CRISPR Cas9 library. The protocol uses lentiviral transduction as a method for gRNA delivery. This method can be adapted for other gRNA libraries if the following is known;
- The number of days required for screening
- The size of the gRNA library
- The required coverage of the library
To allow for efficient scale up of the organoid culture for screening, a 5% suspension culture method is used. This is not essential to be able to perform whole-genome CRISPR screening of stably expressing Cas9 cancer organoid lines, but provides a more scalable, ergonomic and cost efficient culturing method.
Prior to commencing the screen, a puromycin antibiotic titration is used to identify the most suitable puromycin concentration for the selection of Cas9 positive cancer organoid lines transduced with gRNA library virus.
A gRNA library titration is then performed to determine the volume of library virus required to transduce Cas9 cancer organoids at 30% transduction efficiency which is calculated using FACS analysis. To avoid host organoids taking up more than one gRNA copy per cell, an MOI of 30% should be aimed for.
This protocol uses a 21-23 day screen process.
Process Diagram
Process diagram outlining key steps for CRISPR-Cas9 screening in organoids
Guidelines
It is important that all stages are carried out using the stably expressing Cas9 version of each cancer organoid line.
Avoid maintaining organoids in a single-cell state for extended periods, and refrain from freeze/thaw cycles of virus stocks. Always use freshly prepared virus stocks to ensure optimal viral integrity and confirm that the virus concentration is appropriate for the experimental setup.
Adequate dissociation is crucial for effective viral exposure and gene delivery. Be aware that some organoid lines may exhibit lower uptake of viral particles, which could render them unsuitable for this process.
Puromycin titration;
- Thoroughly mix the organoid suspension at each step to achieve an even single-cell distribution before plating, utilising reservoirs and multi-channel pipettes to reduce ergonomic strain and ensure homogenous solutions.
- If required, CellTiter-Glo 2.0 reagent is light-sensitive, so try to avoid exposure when using it.
gRNA library titration;
The volumes of library titrated may differ depending on the batch of lentivirus or library used. To achieve a 30% MOI, there may therefore be a requirement to dilute the virus prior to titrating. For this reason, an increased number of titration points may also be required. This may require some R&D to determine optimal volumes to titrate.
gRNA library transduction and screen;
The gRNA library transduction is carried out in triplicate with 12.5 x106 cells seeded per replicate. Expansion of the line to a minimum of 40 x106 cells is required for this process.
Following selection at Day 6, the organoid suspension should remain in puromycin selection media for the remainder of the screen.
Unless otherwise stated, all steps should be performed under sterile conditions in a microbiological safety cabinet.
Materials
TrypLE™ Express Enzyme (1X), no phenol redThermo FisherCatalog #12604021 1X Dulbecco’s Phosphate Buffered Saline (DPBS) Thermo Fisher ScientificCatalog #14190094
10mg/ml PuromycinInvivoGenCatalog #ant-pr-1
CellTiter-Glo(R) 2.0 AssayPromegaCatalog #G9243 Black walled 96 well plateFisher ScientificCatalog #10419822
37% FormaldehydeAppliChemCatalog #A0823 eBioscience™ Fixable Viability Dye eFluor™ 780Thermo FisherCatalog #65-0865-18 10mg/ml PolybreneMerck Millipore (EMD Millipore)Catalog #TR-1003-G
Costar® 6-well Clear TC-treated Multiple Well Plates Individually Wrapped SterileCorningCatalog #3516 Falcon Round bottomed 5ml tube with cell strainer lidVWR InternationalCatalog #734-0001
Corning® 50ml mini bioreactorCorningCatalog #431720 Trypsin-EDTA (0.25%) phenol redThermo Fisher ScientificCatalog #25200072 Falcon™ 15mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-53A Y-27632 ROCK InhibitorSelleckchemCatalog #S1049 Corning® 125 mL Polycarbonate Erlenmeyer Flask with Vent CapCorningCatalog #431143 Corning® 250 mL Polycarbonate Erlenmeyer Flask with Vent CapCorningCatalog #431144 Microcentrifuge tube Safe-Lock write-on 1.5mL Eppendorf TubeEppendorfCatalog # 0030 120.086
Ultra-low attachment 6-well plateCorningCatalog #CLS3471 Corning® Ultra-Low Attachment 75cm² U-Flask Canted Neck Cell Culture Flask with Vent CapCorningCatalog #3814
Falcon 50mL Conical Polypropylene Centrifuge Tube with Flat Screw Cap, SterileScientific Laboratory Supplies LtdCatalog #352070
Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2Bio-TechneCatalog #3533-005-02
MinLibCas9 Library†addgeneCatalog #164896
Corning® Cell Recovery SolutionCorningCatalog #CLS354253
Organoid specific culture media- made to specification
Transduction media - organoid specific culture media + Y-27632 ROCK Inhibitor (final concentration 2.5 micromolar (µM))
Equipment
37 °C 5% CO2 Incubator
Light microscope
Microbiological safety cabinet (CLASS II)
Centrifuge
Cell counter
Chemical fume hood
Plate reader
Pipette boy
Stripette
Pipettes and tips
Multichannel pipette and tips
Safety warnings
- Lentiviral vectors can infect human cells. However, they are not able to replicate, so the pathogenicity is considered negligible and the risk is reduced by ensuring the correct use of PPE i.e. correct gloves, lab coat and eye protection.
- It is recommended to use centrifuge buckets safety caps and to ensure the caps are only be opened in a microbiological safety cabinet to prevent any biohazard aerosol exposure post-centrifugation.
- Many steps involve repetitive movements. Ensure the use of multichannel and electronic pipettes where possible to reduce ergonomic stress.
- All lentiviral waste should be inactivated and disposed of via recommended local waste routes.
Before start
- If required, ensure that all media is pre-warmed to room temperature before use.
- Where required, prepare an aliquot of 1 mg/ml puromycin (working concentration) by diluting a 10 mg/ml stock 1:10 with sterile water.
- On Day 4 of the puromycin titration process, bring CellTiter-Glo 2.0 reagent to room temperature. (It is light-sensitive and so it is advisable to keep the reagent covered at all times to avoid exposure to light when using).
- Prior to gRNA library transduction, thaw an aliquot of (10 mg/ml) polybrene
- Where required, thaw the required volume of lentivirus for each process stage.
- When fixing organoids, dilute 37% formaldehyde solution 1:10 with DPBS and store at 4 °C
Puromycin titration
Puromycin titration
3d 0h 22m
3d 0h 22m
Day 1: Titration plate set up
Note
This assay is set up using previously expanded organoids which are stably expressing Cas9.
Pre-warm organoid specific culture media to room temperature.
Aspirate media from each well of the organoid culture plate and add 2 mL TrypLE to each well.
Using a cell-scraper, detach BME2 drops containing the cancer organoids from the plate and transfer the organoid suspension to an appropriately sized tube.
Pipette the suspension up and down multiple times to dissociate organoids from the BME2.
Incubate at 37 °C 5% CO2.
Check the organoid suspension under the microscope every 00:15:00 , to assess and monitor the dissociation of the organoids.
Note
Mix the suspension thoroughly prior to each check to help dissociate the organoids. Stop the incubation once the organoids have broken down to single cells or a maximum time of 01:00:00 has passed.
15m
Centrifuge at 800 x g for 00:02:00 .
2m
Aspirate the supernatant and resuspend the pellet in 10 mL of organoid specific culture media.
Perform a cell count to calculate the total number of cells.
Note
A minimum of 12.5 x106 cells are required at this stage to ensure enough cells remain for transfer to 5% BME2 suspension culture (step 1.17).
Resuspend 2.4x106 cells in 2.7 ml of organoid specific culture media + 300 µL BME2 this will give a final seeding density of 8x104 cells per well once plated, Rows B-G of Fig 1.
Prepare a control stock solution containing organoid specific culture media with 5% BME2 (Row A of Fig 1).
Set up the titration plate as detailed in Fig 1 below;
Fig 1: Puromycin titration plate set up
Row A = 200 µl control stock solution per well (step 1.11)
Rows B - G = 100 µl cell suspension (step 1.10)
Note
- Always seed 3 wells per row as the titration is carried out in triplicate.
- A 96-well plate can be used to titrate up to 4 organoid lines at a time.
Incubate the plate at 37 °C 5% CO2 for 00:10:00 to allow the BME2 to polymerise.
10m
Using a 1 mg/ml stock, prepare puromycin antibiotic solutions at 2x concentrations in organoid specific culture media in tubes as outlined below (Table 1).
Safety information
Puromycin is toxic if swallowed and harmful in contact with skin.
Table 1: 2 x puromycin concentrations using 1 mg/ml puromycin stock.
Note
- Prepare a minimum of 2.5 ml of each 2x antibiotic so that the volume is adequate for loading a multi-channel pipette without bubbles.
- Antibiotic dilutions should be prepared fresh on the day and kept at 4 °C until required.
Remove the plate from the incubator and pipette 100 µL of the relevant 2x puromycin stock into each well (Rows B-G) to achieve the final required puromycin concentration according to the plate layout (Fig 1).
Incubate the plate for 72:00:00 at 37 °C 5% CO2.
3d
Plate any remaining cells from step 1.10 into a 5% BME2 suspension culture by seeding cells into a solution of 19 mL organoid specific culture media + 1 mL BME2 using an ultra-low attachment T75 flask.
Note
A minimum of 10x106 cells are required at this stage to ensure survival in 5% suspension culture.
Incubate the ultra-low attachment T75 flask at 37 °C 5% CO2.
Note
The seeded ultra-low attachment T75 flask is to be used for gRNA library titration set up (see section 3).
Day 4: Assessing viability using CellTiter-Glo 2.0 assay
Thaw CellTiter-Glo 2.0 reagent and equilibrate to room temperature prior to use.
Safety information
CellTiter-Glo is harmful to aquatic life with long lasting effects.
Note
- CellTiter-Glo reagent can be stored at -20 °C and is stable for up to 4 freeze-thaws; thawed reagent can be kept at 4 °C for up to 5 months.
- CellTiter-Glo is light-sensitive so should be kept covered, and used in a cell culture hood with the light off where possible.
Run a CellTiter-Glo 2.0 viability assay following the manufacturer’s instructions.
celltiterglo-2-0-assay-protocol.pdf Note
It has been found that adding the CellTiter reagent at 1:5 rather than the manufacturer recommended 1:2 dilution works efficiently.
It is recommended by the manufacturer to use white plates. However, the luciferase signal was found to be too strong so using black plates is recommended.
Using the luminescence data plot a kill curve to ascertain the lowest concentration of puromycin which results in approximately 100% cell death after 72 hours. (Fig 2).
Note
To create the kill curve;
- Average the triplicate luminescence values to get a single value for each condition.
- Subtract the average background luminescence (Row A, media only) from the other averaged conditions (Rows B-G).
- Divide the average luminescence minus background for each concentration 1, 2, 3, 4 and 5 µg/ml by the 0 µg/ml average to obtain a relative percentage viability.
- Plot these percentage viabilities on a graph against the puromycin concentration.
Expected result
Fig 2: Example Kill curve showing a 'kill concentration' of 3 µg/ml
Guide RNA library titration of Cas9 expressing organoid lines
Guide RNA library titration of Cas9 expressing organoid lines
4m
4m
Day 1: gRNA library titration set up
Pre-warm organoid specific culture media to room temperature and thaw a 10 mg/ml aliquot of polybrene.
Prepare transduction media by adding 12.5 µL of 10 millimolar (mM) Y-27632 (Rock inhibitor) to 50 mL organoid specific culture media (2.5 micromolar (µM) final concentration).
Note
Organoids need to remain in transduction media during the setting up and plating of the guide RNA library titration stages within this protocol (steps 3.9 to 4.4).
Collect the organoid suspension culture (from step 1.18) in a 50 ml tube and centrifuge at 800 x g for 00:02:00 .
2m
Aspirate the supernatant and resuspend the pellet in 15 mL to 20 mL of TrypLE.
Pipette the suspension up and down multiple times to dissociate organoids from the BME2.
Incubate at 37 °C 5% CO2.
Check the suspension under a microscope every 15 minutes to assess and monitor the dissociation of the organoids. Mix the suspension thoroughly prior to each check to help dissociate the organoids.
Note
Generally the suspension becomes cloudy once the majority of organoids are dissociated to small clumps of cells. Stop the incubation once the organoids have broken down to single cells or a maximum time of 01:00:00 has passed.
Centrifuge at 800 x g for 00:02:00 .
2m
Aspirate the supernatant and resuspend the pellet in an appropriate volume of transduction media.
Note
15-20 ml is advised.
Perform a cell count to calculate the total number of cells.
Prepare a preparation mix using the organoid suspension and transduction media to achieve a final concentration of 5.6x106 cells in a total volume of 11.2 mL plus 14 µL 10 mg/ml polybrene.
Note
This protocol provides the volumes required to set up a 6 point titration, plus 1 well of dead volume, seeding 8x105 cells/well with a final polybrene concentration of 10 µg/ml. If more or less points are required, adjust volumes accordingly.
Prepare 6x bioreactor tubes as outlined below in table 2 to achieve 8x105 cells per tube, adding appropriate transduction media and library virus volumes.
Table 2: Library titration volumes.
Thaw an aliquot of the gRNA library to room temperature. If the gRNA library is not being used
neat, dilute the required volume of the gRNA library in organoid complete culture media.
Note
- Any dilution made to the library at this stage will also need to be made when carrying out the gRNA library transduction at screen. go to step #6.10
- Once defrosted, the gRNA library should be used within 1 hour.
- Avoid freeze/thaw cycles of the gRNA library.
Safety information
Lentiviral vectors can infect human cells. Ensure correct use of PPE and utilise recommended waste routes to reduce the risk.
Add the appropriate volume of gRNA library virus (Table 2) to each bioreactor tube.
Mix well by pipetting and transfer the bioreactor tubes to the incubator at 37 °C 5% CO2 for overnight incubation.
Passage all remaining organoids in 5% BME2 organoid specific suspension culture media.
Note
The passaged organoids are to be used for gRNA library transduction set up (section 6).
Day 2: Plating
Transfer the 6x bioreactor tubes to the centrifuge
Safety information
Lentiviral vectors can infect human cells. Ensure correct use of PPE and utilise recommended waste routes to reduce the risk.
To reduce the risk of aerosols, it is advised where possible that centrifuge buckets are sealed using safety caps and only opened in a microbiological safety cabinet.
Centrifuge at 800 x g for 00:02:00 .
2m
Aspirate the supernatant from the tubes and resuspend each organoid pellet in1.9 mL of transduction media and add 100 µL of 100% BME2 to make a 5% BME2 solution. Mix well and transfer all 2 ml into each well of a ultra-low attachment 6 well plate (6wp). (Fig 3).
Fig 3: Example plate layout showing virus volumes per well. Virus volumes can be adjusted.
Incubate at 37 °C 5% CO2 until Day 6.
Formaldehyde fixation of organoids
Formaldehyde fixation of organoids
4m
4m
Day 6: Fixing and staining organoids for flow cytometry analysis.
Prepare Live/Dead stain solution or antibodies.
Note
Other antibodies can be used but this protocol uses an e780 viability dye. For this reagent prepare a 1:10,000 dilution of e780 dye in PBS. Mix well and store at 4 °C (The solution can be used for 1 week from the time it was prepared).
Collect the suspension culture from each well of the ultra-low attachment 6wp into individual 2 ml tubes.
Centrifuge at 800 x g for 00:02:00 .
2m
Aspirate the supernatant from each tube and resuspend in 1 mL of Trypsin-EDTA (0.25%).
Safety information
Trypsin-EDTA may cause allergic reactions, asthma symptoms, or breathing difficulties if inhaled.
Incubate for 00:15:00 , until organoids have broken down to single cells.
Note
Mix the solution every few minutes during the incubation. Some lines take longer to dissociate. Do not leave any longer than 00:30:00 .
15m
Once organoids have broken down to single cells stop the reaction by adding 1 mL (diluting 1:1) of media containing serum.
Centrifuge at 800 x g for 00:02:00 .
2m
Aspirate supernatant and resuspend pellets in 200 µL e780 dye solution.
Incubate at room temperature for 00:05:00 .
5m
Add 1 mL PBS to each tube
Centrifuge at 800 x g for 00:02:00 .
2m
Aspirate the supernatant and resuspend in 500 µL of 3.7% formaldehyde. Mix well by pipetting to ensure cells are fixed as single cells.
Safety information
3.7% formaldehyde must be prepared and used only in a chemical fume hood, using chemical resistant gloves. Waste must be kept in the fume hood and disposed of via the recommended route.
Incubate at 4 °C for 00:10:00 .
10m
Centrifuge at 800 x g for 00:02:00 .
2m
Carefully aspirate the supernatant (in chemical fume hood).
Note
Organoid pellets may become transparent and therefore difficult to see. It may also be sticky so can easily stick to pipette tips.
Resuspend the pellet in 500 µL (dependent on pellet size) PBS or alternative FACs buffer, and store at 4 °C until ready for analysis by flow cytometry.
Using collected flow cytometry data create a plot showing side scatter area vs BFP+ expression, gating the positively expressed BFP+ organoids.
For each titration point create an overlay plot (Fig 4) using your un-transduced control (well 1 of Fig 3).
Expected result
Fig 4: Example flow cytometry analysis of percentage of BFP (%BFP) expression. Blue = tested virus concentration. Red = e780 un-transduced control.
Using the %BFP expression for each titration point, plot a titration curve to calculate the volume of virus required to obtain 30% BFP expression. (Fig 5).
Note
A 30% transduction efficiency is aimed for to try and ensure that each host cell has only taken up one gRNA.
Expected result
Fig 5: Example gRNA library titration curve. For 30% BFP expression a volume of 41 µl of virus per 2 ml would be required.
Guide RNA library transduction and screen of Cas9 expressing organoid lines
Guide RNA library transduction and screen of Cas9 expressing organoid lines
2m
2m
Day 1: gRNA library transduction
Pre-warm organoid specific culture media to room temperature. Thaw a 10 mg/ml aliquot of polybrene and a 10 millimolar (mM) aliquot of Y-27632 (Rock inhibitor).
Collect the organoid suspension culture from step 3.16 go to step #3.16 in a 50 ml tube and centrifuge at 800 x g for 00:02:00 .
2m
Aspirate the supernatant and resuspend the pellet in 30 mL of TrypLE.
Pipette the suspension up and down multiple times to dissociate organoids from the BME2.
Incubate at 37 °C 5% CO2.
Check the organoid suspension under the microscope every 15 minutes to assess and monitor the dissociation of the organoids. Mix the suspension thoroughly prior to each check to help dissociate the organoids.
Note
Generally the suspension becomes cloudy once the majority of organoids are dissociated to small clumps of cells. Stop the incubation once the organoids have broken down to single cells or a maximum time of 01:00:00 has passed.
Centrifuge at 800 x g for 00:02:00
2m
Aspirate the supernatant and resuspend the pellet in an appropriate volume of organoid specific culture media.
Note
25 -35 ml is advised
Perform a cell count to calculate the total number of cells.
Note
- For this protocol, using the minimal genome-wide human CRISPR Cas9 library requires a minimum of 40x106 cells at this stage.
- The number of cells required may vary for transduction using alternative gRNA libraries as the number of cells required at transduction is dependent on library size and required coverage.
Thaw an aliquot of the gRNA library to room temperature. If the gRNA library is not being used neat, dilute the required volume of the gRNA library in organoid complete culture media.
Note
- Any dilution made to the library at this stage must match the dilution carried out when setting up the gRNA library titration. go to step #3.13
- Once defrosted, the gRNA library should be used within 1 hour.
- Avoid freeze/thaw cycles of the gRNA library.
Safety information
Lentiviral vectors can infect human cells. Ensure correct use of PPE and utilise recommended waste routes to reduce the risk.
Carry out each library transduction in triplicate. Prepare each replicate in an appropiate size erlenmeyer flask as per Table 3. Mix gently by pipetting.
This protocol uses the minimal genome-wide human CRISPR Cas9 library requires 12.5x106 cells per replicate at transduction.
Table 3: Replicate transduction mixture set up for minimal genome-wide human CRISPR Cas9 library. The volume of library virus required to obtain 30% transduction efficiency will be previously established following library titration and scaled to account for transduction labware size at screen.
Prepare an un-transduced control using the appropriate volumes of reagents listed in Table 4. Approximately 1.6 x106 cells should be seeded into a 50 ml bioreactor tube.
Table 4: Un-transduced control transduction mixture set up.
Incubate the control bioreactor and replicate flasks prepared in step 6.11 and 6.12 overnight at 37 °C 5% CO2.
Day 2: Seeding
Collect and transfer the replicate solutions into labelled 50 ml tubes and centrifuge alongside the control bioreactor tube at 800 x g for 00:02:00 .
Safety information
Lentiviral vectors can infect human cells. Ensure correct use of PPE and utilise recommended waste routes to reduce the risk.
To reduce the risk of aerosols, it is advised where possible that centrifuge buckets are sealed using safety caps and only opened in a microbiological safety cabinet.
2m
For the replicates, aspirate the supernatant and resuspend each replicate pellet in 19 mL organoid specific culture media with 5 µL of 10 millimolar (mM) Y-27632 (Rock inhibitor) and 1 mL of BME2 to make a 5% BME2 solution.
Mix the suspension well by gentle pipetting and transfer each replicate into 1x ultra low-attachment T75 flask and incubate at 37 °C 5% CO2.
For the un-transduced control sample, aspirate the supernatant from the tube and resuspend the pellet in 1.9 ml of organoid specific culture media containing 0.5 µL 10 millimolar (mM) Y-27632 (Rock inhibitor) and add 100 µL of BME2 to make a 5% BME2 solution.
Mix well by pipetting and transfer into 1 well of an ultra-low attachment 6wp and incubate at 37 °C 5% CO2.
Day 6: Puromycin selection and flow cytometry analysis.
Using a pipette, collect 0.5 - 1 ml of organoid suspension from each replicate and transfer to individual 2 ml tubes. Collect all of the control and transfer to another 2 ml tube, this is done to test transduction efficiency using flow cytometry.
Fix each collected organoid sample and analyse by flow cytometry following steps 5.1 to 5.17. 5.1
Note
The un-transduced control once fixed should be kept at 4 °C for re-analysis at Day 21-23 (Assessment of final selection efficiency).
The pass rate for transduction efficiency is 30% (previously calculated from step 5.19) go to step #5.19 . However, a value between 15-50% is acceptable. This is to try and ensure only one gRNA has been transduced per cell. If a value outside of this range is obtained at this stage the screen should be failed (Fig 6). If the pass rate has been reached, continue to step 8.4.
Expected result
Fig 6: Example flow cytometry analysis of transduction efficiency for screen replicates showing %BFP expression. Red = transduced replicate. Blue = e780 un-transduced control.
For each replicate; collect the suspension culture in a 50 ml tube.
Mix the suspension well by pipetting to break down any larger clumps of BME2/aggregates
before centrifugation.
Centrifuge at 800 x g for 00:02:00 .
Safety information
Lentiviral vectors can infect human cells. Ensure correct use of PPE and utilise recommended waste routes to reduce the risk.
To reduce the risk of aerosols, it is advised where possible that centrifuge buckets are sealed using safety caps and only opened in a microbiological safety cabinet.
2m
Aspirate the supernatant and resuspend the pellet in 10 mL organoid specific culture media.
Prepare an ultra-low attachment T75 flask with the appropriate remaining volume of media, BME2 and puromycin (Table 5 and 6) (Puromycin should be added at a concentration previously calculated ingo to step #2.3 Fig 2).
Note
Ultra-low attachment T75 flasks have a working volume of between 20 - 40 ml. This is dependent on the confluency of the organoid suspension culture.
Safety information
Puromycin is toxic if swallowed, harmful in contact with skin
Table 5: Volumes for preparing 5% BME media solution
Table 6: Puromycin selection volumes to add per specific total flask volumes.
Mix the organoid suspension well by pipetting, then transfer the organoid suspension to the individual ultra-low attachment T75 flasks.
Incubate at 37 °C 5% CO2.
Day 9-20: Maintenance of screens.
Note
Media change, or passage 1:2 during week 3 as appropriate.
Do not discard any organoids at this point and re-seed all at passage. Screens are maintained in puromycin until the point of pellet.
Collect suspension for each replicate in 50 ml tubes.
Centrifuge at 800 x g for 00:02:00 .
Safety information
To reduce the risk of aerosols, it is advised where possible that centrifuge buckets are sealed using safety caps and only opened in a microbiological safety cabinet.
2m
Aspirate the supernatant and resuspend the pellet in an appropriate volume of TrypLE.
Note
Small pellets (less than 2 ml in size) can be resuspended in up to 10 ml, whilst larger pellets may need to be resuspended in up to a maximum of 40 ml per tube.
Mix well by pipetting and incubate at 37 °C 5% CO2 for 00:10:00 .
Note
Check organoid suspension under the microscope after 5 minutes to assess and monitor the dissociation of the organoids. Pipette the organoid suspension up and down to help dissociate the organoids. Generally the suspension becomes cloudy once the majority of organoids are dissociated to smaller clumps of cells. Do not leave any longer than 00:30:00 .
10m
Centrifuge at 800 x g for 00:02:00 .
Safety information
To reduce the risk of aerosols, it is advised where possible that centrifuge buckets are sealed using safety caps and only opened in a microbiological safety cabinet.
2m
Aspirate the supernatant and resuspend each pellet in 20 mL organoid specific culture media.
For a 1:2 split, prepare 2x ultra-low attachment T75 flasks with the appropriate remaining volume of media, BME2 and puromycin per replicate (Table 5 and 6). 8.8
Mix the organoid suspension well by pipetting, then transfer half the organoid suspension to each new ultra-low attachment T75 flask.
Incubate at 37 °C 5% CO2.
Day 21-23: Assessment of final selection efficiency
Note
All steps in section 10 should be completed prior to, but can be completed on the same day as section 11.
Collect 0.5-1 ml of organoid suspension from each replicate and transfer to separate 2 ml tubes.
Fix each collected organoid sample and analyse by flow cytometry following steps 5.1 to 5.17.go to step #5.1
Note
The un-transduced control, fixed at Day 6 go to step #8.2 and kept at 4 °C should be re-analysed alongside the fixed replicates at Day 21-23.
The pass rate for selection efficiency is between 60-100%. This ensures efficient selection of organoids that have been transduced with the viral library. If a value below this range is obtained at this stage the screen should be failed. (Fig 7). If the pass rate has been reached continue to step 11.1.
Expected result
Fig 7: Example flow cytometry analysis of screen replicates at assessment of final selection efficiency showing %BFP expression. Blue = transduced replicate. Red = e780 un-transduced control.
Day 21-23: Pelleting using cell recovery solution
Collect the organoid suspension for each replicate in 50 ml tubes.
Centrifuge at 800 x g for 00:02:00 .
Safety information
To reduce the risk of aerosols, it is advised where possible that centrifuge buckets are sealed using safety caps and only opened in a microbiological safety cabinet.
2m
Aspirate the supernatant and resuspend each pellet in an appropriate amount of cell recovery solution (up to 30 mL per replicate) to remove BME. Mix well by pipetting.
Incubate on ice for01:00:00 .
1h
Once the sample tubes have been on ice for 00:30:00 ; mix the suspensions well by pipetting and take aliquots from each replicate to perform a cell count.
30m
Following 01:00:00 on ice, based on the required number of cells per pellet, transfer the required volume of suspension per pellet into 15 ml tubes.
Note
For the minimal library used in this example 25x106 cells are recommended per pellet.
If more than 15 ml of suspension is required, multiple rounds of centrifugation are advised.
1h
Centrifuge at 800 x g for 00:02:00 .
2m
Aspirate the supernatant and resuspend in 10 ml of 4 °C PBS to wash and remove the cell recovery solution from the pellets.
Note
It is advised to dispense the PBS quickly onto the pellet and to avoid mixing the suspension as the pellet will be prone to sticking to the stripette resulting in a loss of organoids.
Centrifuge at 800 x g for 00:02:00 .
2m
Aspirate the PBS and store the pellets at -80 °C ready for downstream processing.
Protocol references
Verity Goodwin, Emily Souster, Charlotte Beaver, Adam Jackson, Rizwan Ansari, Mathew Garnett , Fiona Behan 2020. Guide RNA Library Transduction of Cas9 Cancer Cell Lines. protocols.io dx.doi.org/10.17504/protocols.io.bg2njyde
Gonçalves, E., Thomas, M., Behan, F. M., Picco, G., Pacini, C., Allen, F., Vinceti, A., Sharma, M., Jackson, D. A., Price, S., Beaver, C. M., Dovey, O., Parry-Smith, D., Iorio, F., Parts, L., Yusa, K., & Garnett, M. J. (2021). Minimal genome-wide human CRISPR-Cas9 library. Genome biology, 22(1), 40. https://doi.org/10.1186/s13059-021-02268-4
Price, S., Bhosle, S., Gonçalves, E. et al. A suspension technique for efficient large-scale cancer organoid culturing and perturbation screens. Sci Rep 12, 5571 (2022). https://doi.org/10.1038/s41598-022-09508-y
Emily Souster, Verity Goodwin, Adam Jackson, Charlotte Beaver, Rizwan Ansari, Fiona Behan, Mathew Garnett 2020. Puromycin titration of cancer cell lines. protocols.io dx.doi.org/10.17504/protocols.io.bg2gjybw
Verity Goodwin, Emily Souster, Charlotte Beaver, Adam Jackson, Rizwan Ansari, Mathew Garnett , Fiona Behan 2020. Guide RNA Library Titration of Cas9 Cell Lines. protocols.io dx.doi.org/10.17504/protocols.io.bgxujxnw