Day 3: CRISPR-Cas9 editing and HDR-mediated gene addition

5. Count the cells and calculate the volume to collect the desired cell number for nucleofection. Consider keeping aside the same amount of non-edited cells as negative controls for the following assays.
Note: The required cell count depends on the downstream readouts.
CRITICAL: Use between 100,000 and 750,000 cells per cuvette to ensure optimal results. Collect the same number of cells across all experimental conditions.
6. HSPC nucleofection with Cas9 pre-assembled with the AAVS1 targeting sgRNA alone or in combination with the AAV6 GFP-expressing donor template is required for HDR-mediated longrange gene editing.
a. Assemble the Cas9 ribonucleoprotein (RNP) complex as per the table below and incubate it at 20°C–25°C for 20–30 min.
b. During the incubation time, mix the P3 solution with the Supplement 1 according to manufacturer’s instructions (P3 Primary Cell 4D-Nucleofector X kit):
c. Collect the calculated volume of cells. Top up with DPBS and centrifuge at 400 x g for 10 min. Discard the supernatant carefully.
CRITICAL: Dry out the pellet as much as possible without disturbing it. Use a 20 µL pipette for precision.
d. Add 3.16 µL of RNP complex + 17 µL of Nucleofection solution to the cell pellet. Mix gently and transfer to the cuvette. Proceed immediately to nucleofection.
e. Place the cuvette in the 4D-Nucleofector X Unit and run the program EO-100, as per the manufacturer’s instructions.
CRITICAL: Work quickly to minimize the time cells spend in the Nucleofection solution to preserve viability. Set the instrument before mixing cells with the solution.
f. After nucleofection, wait 2 min, then add 180 µL of warm HSPC Culture Medium to each cuvette. Handle the cuvette strips with care to prevent spillage.
g. Transfer all the content (approximately 200 µL) from the cuvette to a culture plate pre-filled with HSPC culture medium. Adjust the volume to achieve a final concentration of 500,000 cells/mL.

Note: When planning to transplant HSPCs 24 h after nucleofection in mice, aim for a cell concentration of 1,000,000 cells/mL.
h. In HDR-mediated gene addiction or correction strategy, immediately after the seeding and within 15 min of nucleofection, add the AAV6 donor template at a dose of MOI 10^4 to the culture (refer to the formula above).