+Electroporation of primary human CD34 hematopoietic stem and progenitor cells.
²

Experimental preparation
1. Guide RNA
1) Form the crRNA:tracrRNA duplexi.
Resuspend the Alt-R CRISPR-Cas9 crRNA and Alt-R CRISPR-Cas9 tracrRNA in TE buffer to final
concentrations of 200 μM. (For assistance, use the Resuspension Calculator at
www.idtdna.com/SciTools.
ii.
Mix the two RNA oligos in equimolar concentrations in a sterile PCR tube to a final duplex
concentration of 100 μM. The following table shows an example for a final volume of 5 μL.
Component

iii.
iv.
v.

Amount (μL)

200 μM Alt-R CRISPR-Cas9 crRNA

2.5

200 μM Alt-R CRISPR-Cas9 tracrRNA

2.5Total volume

5Heat at 95℃ for 5 min.
Remove from heat, and allow to cool to RT on the bench.
Keep at RT for about 10 min, the place on ice. (Store crRNA:tracrRNA duplex at -20℃ after use).
Note: Alt-R CRISPR-Cas9 crRNA need predesign and custom in www.idtdna.com/CRISPR-Cas92)²²

Resuspend sgRNAi.
Order sgRNA from www.idtdna.com/CRISPR-Cas9, the product is a 2 μM powder.
ii.
Add 20 μL TE into sgRNA powder (100 pmol/μL).
iii.
Place on ice (Store crRNA:tracrRNA duplex at -20℃ after use).
2. Cas9 Nuclease1) Alt-R™ S.p. Cas9 Nuclease V3 dilution.
Stock is 500 µg or 100 µg, add 50/10 μL PBS make the concentration into 10 µg/μL2) Guide-it™ Recombinant Cas9
Stock is 3 µg/μL.
3. Cell culture preparation
TMMedium: StemSpan SFEMII (STEMCELL Technologies) supplemented with 100 ng/ml rhSCF (#255-SC, R&DSystems), 10 ng/ml rhIL-6 (#206-IL, R&D Systems), 1 ng/ml rhIL-3 (#203-IL, R&D Systems) and 1% penicillin–
streptomycin (PS, #09367-34, Nacalai).
5CB Cells thawing amount: 2.5x10 cells/sample (before 48 hours thawing and culture)Day 1
1. Thaw CB cells and start pre-culture 48 hours.
2. Adjust sgRNA and Cas9 protein concentration.
Day 3
1. Add each well 500 μL culture medium into 24 well plate and put it into the incubator.
2. Pick up P3 Primary Cell 4D-Nucleofector™ X Kit S | Lonza kit box.
3. Add the entire supplement to Nucleofector Solution P3 as below (each sample):
Component

4.
5.

Amount (μL)

P3 Primary Cell Nucleofector® Solution

16.4

Supplement 1

3.6

Total volume

20

Let the supplemented Solution P3 reach room temperature.
Prepare RNP complexes as below:
Component

Amount (μL)

100 pmol/μL Alt-R CRISPR-Cas9 sgRNA

2.6

10 µg/μL Alt-R™ S.p. Cas9 Nuclease V3

1.7

Total volume

4.3

Or for Takara Cas9 nuclease
Component²Amount (μL)

100 pmol/μL Alt-R CRISPR-Cas9 sgRNA

2.63 µg/μL Guide-it™ Recombinant Cas95.6

Total volume

8.2Gently swirling the pipet tip while pipetting.
6. Incubate the mixture in RT 15 min.
7. Collect CB cells into 50 mL falcon tube and centrifuge ( 1500 rpm, 5 min, 4℃).
8. Aspirate supernatant, use cold PBS wash 2 times.
9. Aspirate PBS and add supplemented Solution P3 20 μL into CB cells. Add supplemented Solution P3 withCB cells into RNP complexes. Use tip gently mix and lightly pipet 3 times (Be careful with the bubbles).
10. Transfer 25 μL to the relevant well of Amaxa plate, gently tap after adding and make sure no air bubbles
are present.
11. Open 4D-Nucleofector System, insert plate into the machine. Enter the Protocol and chose DZ-100. SelectOK to load the strip, and select Start.
12. After electroporation, remove the plate from the instrument, quickly add 100 μL prewarmed (beforeexperiment incubate in incubator) CB cells culture medium, resuspend cells by gently pipetting up and down,and transfer cells into 24 well plate.
Day 5
Collect cells and check KO efficiency.