Jul 23, 2020
Cas9 transduction of cancer cell lines
- Emily Souster1,
- Verity Goodwin1,
- Adam Jackson1,
- Charlotte Beaver1,
- Rizwan Ansari1,
- Fiona Behan1,
- Mathew Garnett1
- 1Wellcome Sanger Institute
- Cellular Generation and Phenotyping
Protocol Citation: Emily Souster, Verity Goodwin, Adam Jackson, Charlotte Beaver, Rizwan Ansari, Fiona Behan, Mathew Garnett 2020. Cas9 transduction of cancer cell lines. protocols.io https://dx.doi.org/10.17504/protocols.io.bg4ijyue
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: June 03, 2020
Last Modified: July 23, 2020
Protocol Integer ID: 37738
Abstract
The creation of stably Cas9 expressing cancer cell lines allows targeted and genome-wide gene modification using the CRISPR-Cas9 guide RNA system. The outcome of this process is the production of a cell line with greater than 75% Cas9 activity.
Process diagram:
Guidelines
- We transduce cancer lines in only T25s or T75s because we use a fixed volume of Cas9 lentivirus; the transduction efficiency would be affected if this volume of virus was used in larger labware. If you think you need to use larger labware for the Cas9 transduction, be sure to adjust the volume of Cas9 lentivirus accordingly.
Materials
MATERIALS
Falcon™ 15mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-53A
TrypLE™ Express Enzyme (1X), no phenol redThermo FisherCatalog #12604021
Cell culture treated T75 flasksCorningCatalog #430641
DPBSInvitrogen - Thermo FisherCatalog #14190
10mg/ml BlasticidinInvivoGenCatalog #ant-bl-1
10mg/ml PolybreneMilliporeCatalog #TR-1003-G
Cas9 Lentivrus
Cell culture treated T25 flasksVwrCatalog #734-1532
Select an appropriate culture media for your cell line. Common culture medias used for cancer cell lines are serum supplemented Advanced DMEM F-12 or RPMI, in the presence of pen-strep.
Equipment
Light Microscope
Microbiology safety cabinet (MSC)
Pipette Boy
Stripettes
Centrifuge
Pipettes and tips
Nucleocounter/Cell counter
37 °C , 5% CO2 incubator
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 managed by ensuring correct use of PPE throughout the protocol (including correct gloves, lab coat, eye protection).
All lentiviral waste should be deactivated with 1% Virkon solution for a minimum of 1 hour, preferably overnight.
Before start
- Pre-warm culture media to room-temperature
- Thaw an aliquot of polybrene
- Thaw an appropriate amount of Cas9 lentivirus for the number of transductions you will carry out.
Note
Once thawed, the Cas9 virus can be freeze-thawed only once more if required.
Day 1: Transduction
Day 1: Transduction
Detach, collect and count cells by following Steps 1-8 of protocol: Passaging adherent cancer cell lines.
Using the cell count from the source labware, determine the appropriate flask size for transduction.
This will be the flask size that is best able to accomodate 2.5x106 cells at a confluency of 70%.
Note
This will vary between cell lines but will generally be either a T25 or T75 flask. We do not tend to transduce in a T150 even for large cell lines.
For large cells lines that have up to 1x105 cells/cm2 at 70% confluency, a T75 should be used.
For smaller cell lines with greater than or equal to 1x105 cells/cm2 at 70%, a T25 should be used.
See Guidelines for more information.
Freeze 2 vials of parental cells for storage in the event that the Cas9 transduction fails and needs to be repeated.
According to the volumes in Table 1, prepare a sterile 15ml falcon tube to contain 2.5x106 cells and top up to either 3.5ml or 13.5ml with media depending on transduction labware.
Add the remaining transduction reagents as in Table 1. Mix well and transfer to the appropriate flask.
Note
Be aware that the total volume should not exceed 5ml for a T25 or 15ml for a T75.
| Flask | Number of Cells | Volume of Cells + Media | Volume of 10mg/ml Polybrene | Volume of Cas9 virus | Total Volume | |
| T25 | 2.5x10^6 | 3.5ml | 4µl | 1.5ml | 5ml | |
| T75 | 2.5x10^6 | 13.5ml | 12 µl | 1.5ml | 15ml |
Table 1: Reagent volumes for Cas9 transduction.
Safety information
Lentiviral vectors can infect human cells. Ensure correct use of PPE to reduce the risk.
Place the flask in a 37 °C , 5% CO2 incubator over night and discard any remaining cells.
Day 2: Media Change
Day 2: Media Change
Aspirate media and replace with fresh media: 5ml to each T25 or 12ml to each T75.
If cells are >90% confluent, passage the cells into a T75 (1:3 from T25) or T150 (1:2 from T75), keeping all cells.
Day 3: Inspect
Day 3: Inspect
Inspect the cells. If >80% confluent, passage at an appropriate split ratio, keeping all cells.
Day 4: Antibiotic Selection
Day 4: Antibiotic Selection
Harvest the cells by following steps 1-7 of the protocol: Passaging adherent cancer cell lines and treat with blasticidin at the concentration determined by protocol: Blasticidin titration of cancer cell lines. See Table 2 for appropriate blasticidin volume.
Note
For optimum selection efficiency, the cells need to be in suspension when first exposed to blasticidin. Therefore it is important that the cells are harvested and re-seeded with blasticidin at the point of selection.
-If the cells are in a T25, transfer all to a T75 (1:3 split)
-If the cells are in a T75, transfer all to a T150 (1:2 split)
-If the cells are in a T150, transfer all to one or two T150s (1:1 or 1:2 split depending on confluency and growth of the cell line).
| Size of flask | Volume of media (ml) | 10ug/ml Blasticidin (µl) | 25ug/ml Blasticidin (µl) | 50ug/ml Blasticidin (µl) | 75ug/ml Blasticidin (µl) | |
| T25 | 7 | 7 | 17.5 | 35 | 52.5 | |
| T75 | 12 | 12 | 30 | 60 | 90 | |
| T150 | 24 | 24 | 60 | 120 | 180 |
Table 2: Volumes of blasticidin and media for Cas9 antibiotic selection.
Safety information
Blasticidin is toxic if swallowed and harmful in contact with skin.
Note
From this point onwarwds, Cas9 cells are maintained in media containing blasticidin- until the point at which they are screened.
Day 7: Media change
Day 7: Media change
Inspect cells. Aspirate media and replace with fresh culture media to remove the majority of dead cells.
Day 8-13: Expansion
Day 8-13: Expansion
Passage cells when required using appropriate split ratios if necesary. See protocol: Passaging adherent cancer cell lines.
The aim is to expand cells to 2x confluent T150 flasks by day 14, so do not discard cells from slow growing cell lines.
Note
Some lines may struggle and require passages with low split ratios such as 1:1 or even temporarily downgrading by 2:1.
If cells are not >50% confluent in 2x T150 flasks by Day 14, allow to expand for another week before proceeding to the Cas9 activity assay and banking.
If cells fail to expand to this point within 4 weeks of Cas9 transduction, the cell line has failed the Cas9 transduction process due to lack of growth.
Day 14: Cas9 activity & bank
Day 14: Cas9 activity & bank
Detach cells from both T150 flasks according to Passaging adherent cancer cell lines protocol linked above.
Proceed directly to protocol: Assessment of Cas9 activity in Cas9 Transduced Cancer Cell Lines
Freeze 5 vials of Cas9 positive cells according to the protocol: Freezing cancer cell lines.