TITLE: CD34+ Cell RNP Nucleofection
AUTHORS: Daniel A Kuppers, Patrick Paddison

[DESCRIPTION]
Human CD34+ hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate “humanized mice”. To further extend the range of applications of these humanized mice, we developed a protocol to efficiently edit the genomes of human CD34+ HSPCs before transplantation. In the past, manipulating HSPCs has been complicated by the fact that they are inherently difficult to transduce with lentivectors, and rapidly lose their stemness and engraftment potential during in vitro culture. However, with optimized nucleofection of sgRNA:Cas9 ribonucleoprotein complexes, we are now able to edit a candidate gene in CD34+ HSPCs with almost 100% efficiency, and still be able to generate a humanized mouse with high engraftment and multilineage differentiation. 

[STEPS]
SECTION: Synthetic sgRNA reconstitution
5. Briefly centrifuge your tubes or plates containing synthetic modified single guide RNA (sgRNA) oligos to ensure that the dried RNA pellet is collected at the bottom.
SECTION: Synthetic sgRNA reconstitution
6. Carefully dissolve sgRNA in the provided nuclease-free 1X TE Buffer (Tris-EDTA, pH 8.0) by adding 15μl buffer to 1.5nmol of dried sgRNA. (This will result in a final concentration of 100μM (100pmol/μl)). Flick the bottom of the tube gently so the liquid covers the bottom, then quick spin reconstituted RNA down. Dissolved RNA should be stored at -20 °C when not in use. Under these conditions, RNA will be stable for at least one year.

NOTE: It is best to store RNA in 1X TE Buffer long-term and to dilute with water only the amount for use within a week. Freeze-thawing of RNA should also be kept to a minimum.
SECTION: CRISPR ribonucleoprotein (RNP) complex formation
7. Dilute Cas9 to an appropriate working concentration.
SECTION: CRISPR ribonucleoprotein (RNP) complex formation
7.1. Briefly centrifuge the stock Cas9 (10 mg/mL, 61μM) to ensure that all liquid is at the bottom.
SECTION: CRISPR ribonucleoprotein (RNP) complex formation
7.2. Create a working stock, by diluting Cas9:H2O 1:1.44 (e.g. 2.88μl H2O + 2μl Cas9, creating 4.88μl of 25pmol/μl Cas9). Keep diluted/undiluted Cas9 on ice.
SECTION: CRISPR ribonucleoprotein (RNP) complex formation
8. Dilute and pool sgRNAs to desired working concentration.
SECTION: CRISPR ribonucleoprotein (RNP) complex formation
8.1. Briefly centrifuge the stock sgRNAs (100 pmol/μl, 100μM) to ensure that all liquid is at the bottom.
SECTION: CRISPR ribonucleoprotein (RNP) complex formation
7.3. NOTE: If using more than 2 sgRNAs the Cas9 needs to be diluted to 50pmol/μl.
NOTE: It is best to dilute Cas9 only in H2O or PBS (pH 7.4), not directly in Nucleofector solution. We have found that concentrated Cas9 can precipitate when added directly to some buffers. Do not freeze the diluted Cas9.
SECTION: CRISPR ribonucleoprotein (RNP) complex formation
8.2. 1 sgRNA: dilute the stock sgRNA 1:1 with nuclease-free water to a concentration of 50 pmol/μl (e.g. 2μl H2O + 2μl sgRNA, creating 4μl of 50pmol/μl sgRNA)

2 sgRNA pool: mix the two stock sgRNAs (100pmol/μl 1:1) to get a final concentration of 50 pmol/μl of each sgRNA (100pmol/μl total)

3+ sgRNA pools: mix the sgRNA stock solutions for the desired number of sgRNAs to get a final concentration of 25 pmol of each sgRNA in a total volume of 2.5μl or less (e.g. 1μl each for 6 sgRNA , creating 6μl of 16.7pmol/μl of each sgRNA and 25pmol of each sgRNA in 1.5μl).

NOTE: The total combined volume of sgRNA and Cas9 can't exceed 20% of the total nucleofection volume. Therefore, to create pools containing greater than 8 sgRNAs, the stock concentrations of the individual sgRNAs will need to be increased accordingly.
SECTION: CRISPR ribonucleoprotein (RNP) complex formation
9. Assemble RNP complexes
SECTION: CRISPR ribonucleoprotein (RNP) complex formation
9.1. Create 20μl of complete Nucleofector Solution per nucleofection by mixing Nucleofector Solution P3 with the provided Supplement at a 4.5:1 ratio to (e.g. for one nucleofection, combine 16.4μl Nucleofector Solution + 3.6μl Supplement). Make a master mix for multiple nucleofections.
SECTION: CRISPR ribonucleoprotein (RNP) complex formation
9.2. Mix the nucleofection reaction components as listed below and incubate at room temperature for 15-20 minutes. Then proceed immediately to STEP 6 or store the RNPs on ice or at 4C for up to 24 hours.

Reaction for 1 or 2 sgRNA(s)
 

Reaction for 3+ sgRNAs
 
For 100μl reactions, scale reagent amounts based on the total number of cells to be nucleofected.
We have found that the optimal molar ratio of sgRNA:Cas9 is 2:1.
SECTION: Nucleofection of RNPs
10. Collect and prepare the CD34+ cells
SECTION: Nucleofection of RNPs
10.1. Label a 96-well or 24-well plate based on the number of cells to be nucleofected. 

NOTE: Cell survival is usually highest if the cells are kept at a high density for 24 hours post-nucleofection. For CD34+ HSPCs we typically plate each nucleofection at a cell concentration of 2.0-3.0 x 106/ml in CD34 expansion media.
SECTION: Nucleofection of RNPs
10.2. Harvest cells normally and count. For each 20μl nucleofection, aliquot 2-4 x 105cells into a separate 1.5 mL microcentrifuge tube and centrifuge 5 minutes at 350 x g, room temperature.

NOTE: For 100μl nucleofections use 1-2 x 106 cells.
SECTION: Nucleofection of RNPs
10.3. Aspirate the liquid as completely as possible without disrupting the cell pellet (We typically leave up to 2μL of media without issue). Resuspend the cell pellet in 20μL or 100 μL of RNP complexes. Work quickly, but carefully, and avoid leaving cells in Nucleofector Solution for longer than 30 minutes total (from the time you resuspend them to the time nucleofection is complete). Avoid bubble formation.
SECTION: Nucleofection of RNPs
11. Setup the 4D nucleofector and nucleofect the cells
SECTION: Nucleofection of RNPs
11.1. Following the manufactures instructions for using the Amaxa 4D Nucleofector X unit, turn on the core unit and then use the touch screen to select the appropriate options for nucleofecting a 16-well strip. Utilize program DS-150 for the CD34+ cells.

NOTE: For 100μl nucleofections select the option for the larger volume individual cuvettes.
SECTION: Nucleofection of RNPs
11.2. Transfer all of the cell-RNP solution to one well of a 16-well Nucleocuvette strip for each sample (or individual cuvettes for 100μl nucleofections), and cover with the provided lid. Make sure there are no bubbles in your Nucleocuvette.
SECTION: Nucleofection of RNPs
11.3. Insert the 16-well strip or individual cuvette into the open Nucleofector 4D X unit. Make sure the larger gap in the 16-well strip lid is at the top of the strip, so that the yellow indicator in the X unit fits through the large gap at the top of the lid. 
SECTION: Nucleofection of RNPs
11.4. Run the nucleofection program.  After run completion, the screen should display a “+” over the wells that were successfully electroporated. Remove the strip/cuvettes from the machine.
SECTION: Nucleofection of RNPs
11.5. Recover the cells in each well of the 16-well strip by adding 100μl of CD34 expansion media (250μl for individual cuvettes) and gently pipetting up and down 3-4 times.

NOTE: Excessive pipetting can greatly increase cell death.
SECTION: Nucleofection of RNPs
11.6. Transfer the cells to the 96-well or 24-well plate prepared above.
SECTION: CD34+ HSPCs culture
1. NOTE: Cells should be thawed out approximately 4 hours before nucleofection for use in vivo. Cells for in vitro experiments can be expanded for up to 4 days in CD34 expansion media prior to nucleofection, with no effect on editing efficiency or post-nucleofection cell viability.
SECTION: CD34+ HSPCs culture
2. Prepare an amount of CD34 expansion media appropriate for the number of cells to be thawed. 
SECTION: CD34+ HSPCs culture
3. Equilibrate the CD34+ gradually to ensure maximum viable recovery.
SECTION: CD34+ HSPCs culture
3.1. Prepare 35ml of room temperature thawing media, per tube of CD34+ cells, by 0.2um filtering 1x PBS with 0.5% BSA.
SECTION: CD34+ HSPCs culture
3.2. Thaw the cells rapidly in a 37C water bath.
SECTION: CD34+ HSPCs culture
3.3. Transfer the cells to a 50 ml conical tube.
SECTION: CD34+ HSPCs culture
3.4. Serially dilute the thawed cells with thawing media while gently mixing the cells. 
SECTION: CD34+ HSPCs culture
3.5. Slowly add 1 ml media to the 1 ml of cells and allow 3 minutes for cells to equilibrate.
SECTION: CD34+ HSPCs culture
3.6. Repeat this process with 2 ml of thawing media for a total volume of 4 ml.
SECTION: CD34+ HSPCs culture
3.7. Follow the same process of doubling the volume until you have a total volume of 32 ml.
SECTION: CD34+ HSPCs culture
3.8. Collect the cells by centrifugation for 10 minutes at 400 x g, room temperature.
SECTION: CD34+ HSPCs culture
3.9. Aspirate the media and resuspend the cells in 1ml of thawing media.
SECTION: CD34+ HSPCs culture
4. If expanding the cells, count the cells daily to ensure they are maintained at concentration of 2-3 x 106/ml.
SECTION: CD34+ HSPCs culture
3.10. Take a small aliquot of cells to count and centrifuge the remainder at 400 x g for 5 mins
SECTION: CD34+ HSPCs culture
3.11. Resuspend the cells in CD34 expansion media at 2-3x106/ml.
SECTION: Cell manipulation post-nucleofection
12. in vivo use
SECTION: Cell manipulation post-nucleofection
12.1. The nucleofected CD34+ HSPCs should be cultured in the CD34 expansion media for 4-hours and then have recovered sufficiently for transplantation.
SECTION: Cell manipulation post-nucleofection
13. Freezing cells post nucleofection
SECTION: Cell manipulation post-nucleofection
13.1. NOTE: Starting at 4-hours post-nucleofection, we have found that CD34+ HSPCs can be frozen from any time point without loss of editing efficiency as measured by ICE analysis.
NOTE: Freezing fewer than 5 x 105 cells per vial typically results in dramatically lower cell recovery post-thaw.
SECTION: Cell manipulation post-nucleofection
13.2. Pool cells from nucleofections, with a minimum 5 x 105 cells for each cryo vial, into an appropriately sized tube and add 2.5x the volume of 1x PBS to wash. Centrifuge for 5 minutes at 350 x g, room temperature.
SECTION: Cell manipulation post-nucleofection
13.3. Aspirate the liquid as completely as possible without disrupting the cell pellet. Resuspend the cell pellet in 500ul of CryoStor CS10 (Sigma) if freezing less than 1 x 106 cells/vial and 1ml of CryoStor CS10 if freezing up to 10 x 106 cells/vial. 
SECTION: Cell manipulation post-nucleofection
13.4. Aliquot into cryovials and freeze using your desired method.
SECTION: Editing efficiency analysis
14. Harvest nucleofected cells and control cells (or other suitable sources of unedited template as discussed above) collecting 50,000-100,000 cells per sample in a 1.5 mL microcentrifuge tubes. Add 500μl 1x PBS to wash. Centrifuge for 5 minutes at 350xg, to pellet the cells and resuspend in 100μl 1x PBS. The cells can either be used immediately or stored at -20°C or -80°C for several months before gDNA extraction.
SECTION: Editing efficiency analysis
15. Extract gDNA using the E.Z.N.A. MicroElute Genomic DNA Kit (Omega Bio-Tek), according to the manufacturer’s protocol. 
SECTION: Editing efficiency analysis
16. Perform PCR amplification of the region around the sgRNA cut site(s):
SECTION: Editing efficiency analysis
16.1. Design PCR primers to be a minimum of 250-350bp outside of the sgRNA cut site. If multiple sgRNAs are being used, design primers to be approximately 250bp outside of the outermost cut sites. We usually target a 700-900bp (wild-type size) amplicon.
SECTION: Editing efficiency analysis
16.2. Design at least one sequencing primer that is approximately 20-80bp inside either end of the PCR amplicon. Ensure the sequencing primer is a minimum of 100bp away from any cut site. If no suitable sequencing primer can be designed, one of the PCR primers can be substitute.
SECTION: Editing efficiency analysis
16.3. Perform PCR amplification (50μl reaction) on the edited and control samples using a high-fidelity polymerase such as Phusion (ThermoFisher Scientific), following manufacturer’s suggestions for PCR conditions. 
SECTION: Editing efficiency analysis
17. PCR set-up for Phusion High-Fidelity DNA Polymerase

   Component   50 ml reaction     5X Phusion Buffer (HF or GC)   10 ml     dNTPs (10 mM each)   1 ml     Forward primer (10 mM)   2.5 ml     Reverse primer (10 mM)   2.5 ml     DMSO   1.5 ml     Phusion polymerase   0.5 ml     100 ng gDNA template   x ml     H2O   To 50 ml total   
NOTE: We typically include a final concentration of 3% DMSO (which aids with template denaturation) in our PCR reactions with gDNA templates. 
SECTION: Editing efficiency analysis
18. Cycling conditions for Phusion High-Fidelity DNA Polymerase

   Temperature   Time   Cycles     98˚C   30 sec   1     98˚C   5-10 sec     25-35     Annealing Temp.   10-30 sec     72˚C   15-30 sec/kb     72˚C   5-10 min   1     4 ˚C   Hold   --------   

SECTION: Editing efficiency analysis
19. Run 10 μl of the PCR reactions on an agarose gel to ensure that there is only one product of the correct size in the control sample. When using multiple sgRNAs, the edited sample may have multiple bands if your CRISPR-Cas9 RNPs created multiple deletions. If you have multiple products in the control sample, design new PCR primers. 
SECTION: Editing efficiency analysis
20. NOTE: If you were able to design nested sequencing primers, we’ve had success submitting products with multiple bands, so long as the desired product constitutes the majority of the total. Alternatively, you may be able to gel purify the product if the combination of sgRNAs used produce fragments clearly distinguishable from the background in the control samples.
SECTION: Editing efficiency analysis
21. Use a column PCR purification kit (Monarch PCR & DNA Clean-up Kit ) to purify the remainder of the edited and control sample PCR products, according to the manufacturer’s protocol. 
SECTION: Editing efficiency analysis
22. Perform Sanger sequencing on the PCR amplicons for your edited and control samples.
SECTION: Editing efficiency analysis
23. Perform editing analysis using the Sanger trace files and the freely-available webtool “Inference of CRISPR edits” (ICE), https://ice.synthego.com/#/. Enter the trace files (.ab1 files) for your targeted and control samples, as well as the sequence(s) of the sgRNA(s) that you used. These tools generate a computational prediction of which overlapping DNA sequences could explain the observed trace in the targeted sample. It provides both predictions of % wild-type versus % edited sequences in the cell pool and taking into account the sequence context of the edits a predicted % knockout.