Cell Transfection and RNA Extraction

(i) The cells were seeded in multiwell cell culture plates a day before transfection, for the cell density to be at 50–70% during transfection.

For experiments using short fragments of the CasRx sgRNA expression cassette (gBlocks^TM), prepare the Lipofectamine 2000/DNA mixture as follows:

For experiments using dual plasmids (CasRx and pre-sgRNA expression vectors), prepare the Lipofectamine 2000/DNA mixture as follows:

For experiments using the all-in-one pAAV–CasRx–pre-sgRNA plasmid, prepare the Lipofectamine 2000/DNA mixture as follows:

(ii) The transfection steps are as follows:

Step 1. Mix DNA in 150 μl (for the 6-well plate) or 50 μl (for the 12-well plate) OptiMEM.

Step 2. Mix Lipofectamine 2000 in 150 μl (for the 6-well plate) or 50 μl (for the 12-well plate) OptiMEM.

Step 3. Mix the DNA/OptiMEM mixture with the Lipofectamine 2000/OptiMEM mixture.

Step 4. Incubate at room temperature for 10 min.

Step 5. Remove the culture media from the plate wells and add 1.7 ml (for the 6-well plate) or 0.9 ml (for the 12-well plate) fresh culture media to each well.

Step 6. Add the DNA/Lipofectamine 2000 mixture to each well dropwise.

Step 7. Incubate at 37°C with 5% CO₂.

Step 8. Remove the culture media from the plate wells and add 2 ml (for the 6-well plate) or 1 ml (for the 12-well plate) fresh culture media to each well the next day.

Step 9. Incubate at 37°C with 5% CO₂ for another 48 h.

(i) Cells were seeded in multiwell cell culture plates a day before transfection, for the cell density to be at 50–70% during transfection. The ViaFect reagent/DNA mixture is prepared as follows:

(ii) The transfection steps are as follows:

Step 1. Mix DNA in 150 μl (for the 6-well plate) or 50 μl (for the 12-well plate) OptiMEM.

Step 2. Mix ViaFect in 150 μl (for the 6-well plate) or 50 μl (for the 12-well plate) OptiMEM.

Step 3. Mix the DNA/OptiMEM mixture with the ViaFect/OptiMEM mixture.

Step 4. Incubate at room temperature for 10 min.

Step 5. Remove the culture media from the plate wells and add 1.7 ml (for the 6-well plate) or 0.9 ml (for the 12-well plate) fresh culture media to each well.

Step 6. Add the DNA/ViaFect mixture to each well dropwise.

Step 7. Incubate at 37°C with 5% CO₂ for 48 h.

(iii) After transfection, place the samples into GENbag for 24 h.

(iv) For RNA isolation, HEK293FT or Müller cells were harvested using 300 μl RNA lysis buffer and total RNA was isolated using Quick-RNA^® Miniprep kit (Zymo Research) and eluted in 10 mM Tris–HCl, pH 8 (or the elution buffer from the kit) (Supplementary Method—Quick-RNA^® Miniprep kit). RNA was quantified using Nanodrop^TM.

It should be noted that (1) cells must not reach full confluency prior to transfection and (2) as RNA is unstable, the process must be performed as quickly as possible.