TITLE: Cas9 transduction of cancer cell lines
AUTHORS: Emily Souster, Verity Goodwin, Adam Jackson, Charlotte Beaver, Rizwan Ansari, Fiona Behan, Mathew Garnett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div><div class = "text-block">Process diagram:</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/b94e6458af3e4aa17dc6c162a5d28b87f529d83e9d05c0764f6eb3fffa72f21d/ccgpbdsax.png" /></div></div>

[STEPS]
?. [Day 1: Transduction]
Detach, collect and count cells by following Steps 1-8 of protocol: Passaging adherent cancer cell lines.
?. [Day 1: Transduction]
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%.
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.
?. [Day 1: Transduction]
Freeze 2 vials of parental cells for storage in the event that the Cas9 transduction fails and needs to be repeated. See protocol: Freezing cancer cell lines.
?. [Day 1: Transduction]
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.   ABCDEF1  <b style="mso-bidi-font-weight:
  normal">Flask    <b style="mso-bidi-font-weight:
  normal">Number of Cells    Volume
  of Cells + Media    <b style="mso-bidi-font-weight:
  normal">Volume of 10mg/ml Polybrene    <b style="mso-bidi-font-weight:
  normal">Volume of Cas9 virus    <b style="mso-bidi-font-weight:
  normal">Total Volume  2  T25   2.5x10^6  3.5ml    4µl    1.5ml    5ml  3  T75  2.5x10^6  13.5ml    12 µl    1.5ml    15ml
Be aware that the total volume should not exceed 5ml for a T25 or 15ml for a T75.
ABCDEF1  <b style="mso-bidi-font-weight:
  normal">Flask    <b style="mso-bidi-font-weight:
  normal">Number of Cells    Volume
  of Cells + Media    <b style="mso-bidi-font-weight:
  normal">Volume of 10mg/ml Polybrene    <b style="mso-bidi-font-weight:
  normal">Volume of Cas9 virus    <b style="mso-bidi-font-weight:
  normal">Total Volume  2  T25   2.5x10^6  3.5ml    4µl    1.5ml    5ml  3  T75  2.5x10^6  13.5ml    12 µl    1.5ml    15ml
Lentiviral vectors can infect human cells. Ensure correct use of PPE to reduce the risk.
?. [Day 1: Transduction]
Place the flask in a   , 5% CO2 incubator over night and discard any remaining cells.
37 °C
?. [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. For passage, see protocol: Passaging adherent cancer cell lines.
?. [Day 3: Inspect]
Inspect the cells. If >80% confluent, passage at an appropriate split ratio, keeping all cells. For passage, see protocol: Passaging adherent cancer cell lines.
?. [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.-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).  ABCDEF1Size of flaskVolume of media (ml)  10ug/ml
  Blasticidin (µl)    25ug/ml
  Blasticidin (µl)    50ug/ml
  Blasticidin (µl)    75ug/ml
  Blasticidin (µl)  2T257  7    17.5    35    52.5  3T7512  12    30    60    90  4T15024  24    60    120    180
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.
ABCDEF1Size of flaskVolume of media (ml)  10ug/ml
  Blasticidin (µl)    25ug/ml
  Blasticidin (µl)    50ug/ml
  Blasticidin (µl)    75ug/ml
  Blasticidin (µl)  2T257  7    17.5    35    52.5  3T7512  12    30    60    90  4T15024  24    60    120    180
Blasticidin is toxic if swallowed and harmful in contact with skin.
From this point onwarwds, Cas9 cells are maintained in media containing blasticidin- until the point at which they are screened.
?. [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]
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.
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]
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 LinesFreeze 5 vials of Cas9 positive cells according to the protocol: Freezing cancer cell lines.