·       CRISPR design and preparation

  1. Click here for a Table of crRNA sequences validated with CRIMSON OR design crRNA using idtDNA webtool: https://www.idtdna.com/site/order/designtool/index/CRISPR_PREDESIGN. See note a. below.
  2. order 2-3 crRNA (2 nmol) with on-target score > 60%. Be sure to order negative control crRNA.
  3. prepare crRNA/tracRNA complexes (not needed for sgRNA):
  1. Resuspend crRNA to 200 uM (2 nmol in 10 uL) RNAse free 10 mM Tris (PH 7.5)*
  2. Resuspend tracRNA to 200 uM (5 nmol in 25 uL) RNAse free 10 mM Tris (PH 7.5)
  3. Mix crRNA 1:1 with tracRNA in RNAse free PCR tube. Mix at least 2.5 uL of each.
  4. Heat at 95c for 5 min in a thermal cycler with heated lid
  5. during step d, resuspend enhancer to 100 uM (2 nmol in 20 uL) in RNAse free 10 mM Tris (PH 7.5), place on ice
  6. remove crRNA/tracRNA from heat and cool to room temperature on bench top
  7. Keep at room temperature for 10 min., then place on ice
  8. proceed to CRISPR/CAS9 transfection or store crRNA/tracRNA duplex at -20c

a. There are many options for CRISPR design software. We prefer using idtDNA for custom or pre-designed crRNA. We usually check the top 10 designs and aim for crRNA that have >60% on-target score. You will want to test 2-3 non-overlapping crRNA, since not all will work the first time (in our experience at least one of three will, see figure below). Combining two different crRNA often results in even better KO efficiency. We usually choose the crRNA 2 nmol initially. The XT and sgRNA (which doesn’t require tracRNA) versions may work better for some targets (we have tried both), but aren’t usually necessary since we can usually identify a crRNA with sufficiently high efficiency. Depending on the experiment, crRNA with higher off-target scores are preferred. Off targets can be examined through their website to make sure no predicted targets are platelet function genes.

·       CD34+ cell culture

CD34+ cells are cultured in SFEM/TPO/SCF for 3 days and then in SFEM/TPO until day 13. Alternatively, for a larger cell yield, CD34+ cells can be pre-expanded for 5-6 days ~50 fold and frozen in aliquots for future use using the cocktial IL6, SRI, UM171,Flt3L, TPO, SCF published in https://pubmed.ncbi.nlm.nih.gov/29370156/. Thawed, pre-expanded cells are treated as day 0 cells and cultured using the same protocol below.

  1. thaw C34+ cord blood cells or isolate fresh using magnetic selection
  2. Add 0.5-1×10^6 cells/mL cells to prewarmed SFEM-ST media (cytokines added fresh) in tissue culture plate:
    1. SFEM + gentamycin*
    2. 25 ng/mL SCF
    3. 20 ng/mL TPO
  3. Incubate at 37c 5% co2.
  4. passage on day 3 by centrifugation (300xg), replate at 0.5-1×10^6 cells/mL in SFEM-ST media.
  5. Transfect cells (see next section) between day 1 and day 6 (we prefer day 5) and replate in SFEM-ST media.
  6. passage on day 6 and 9 or 10 as in step 4, but use SFEM-T media. Starting on day 6, keep cells in larger wells (i.e. 1 mL (0.5-1×10^6/mL) in a 12 well plate, 2-3 mL in a 6 well plate) to maximize the surface area exposed to air.
    1. SFEM + gentamycin*
    2. 50 ng/mL TPO**

*Gentamycin is optional but recommended to prevent bacterial growth. Gentamycin is known to affect mitochondria in other mammalian cells. 10/21 update: switched to using Pen/Strep as the antibiotic of choice with no noticeable differences.**use 100 ng/mL TPO day 6 on, and keep cells at 0.5×10^6/mL for enhanced proplatelet formation.

·       CRISPR/CAS9 transfection

On the day of electroporation:

Pre-warm SFEM-ST media 

Add 425 uL (for 1×10^6 cells) of media per transfection  to each well of a 24  well plate. To another well, add extra 100 uL per transfection. place in incubator to warm.

Place Amaxa solution P3 (supplemented) at room temperature

Prepare cells:

  1. Resuspend cells in the wells by gently pipetting.
  2. Transfer cells to a 15 mL sterile tube.
  3. Rinse the culture dish surface with an additional 1 mL of prewarmed culture medium and add this volume to the tube.
  4. Mix cells gently, count and assess viability (>90%) by hemacytometer or automated counter
  5. Transfer the number of cells needed into a new 15 mL sterile tube. Between 2.5e5 and 4e6 cells per transfection. We prefer 1e6 cells/transfection.
  6. Place the cells in incubator with cap loose while preparing rest of experiment.

Prepare CRISPR/CAS9 RNP complexes

If not already done, complex crRNA with tracRNA as described in step 3 of “CRISPR design and preparation above“. 

RNP formation:

RNP formation should be done immediately before transfection, while cells are simultaneously being prepared below, and the time between preparation and transfection should be standardized. 

  1. For each transfection, mix the following in a 0.2 mL PCR tube (we prefer 8 tube strips), gently swirling the pipet tip while pipetting. Sufficient for 2 transfections, scale up as necessary.
    • PBS (sterile/RNAse free) – 2.1 uL
    • crRNA/tracRNA duplex – 1.2 uL (120 pmol)
    • Alt-R spCas9 nuclease V3 (provided as 62 uM stock) – 1.7 uL (105 pmol)
  2. Heat RNP complexes at 37c in thermal cycler for 4 min
  3. While RNP is forming, immediately centrifuge cells at 300xg, 5 min, room temp
  4. Remove RNP complexes to room temperature for 10 min prior to nucleofection. Prolonged incubation may reduce efficiency.
  5. During step 4, immediately resuspend cells in 1 mL PBS, transfer to a sterile microtube, rinse original tube with 500 uL PBS and add rinse to rest of sample
  6. Centrifuge cells again at 300xg, 5 min, room temp
  7. Completely remove supernatant
  8. Resuspend cells in 20 uL per transfection of room temp, supplemented, solution P3 (i.e. for 4 transfections at 1e6 cells/transfection, add 80 uL to 4e6 cells).
  9. Transfer 20 uL of the cell suspension into individual sterile PCR tubes
  10. proceed immediately to nucleofection

Amaxa nucleofection

  1. working quickly (very important – efficiency will improve with practice), add RNA complexes and enhancer to cells in PCR tubes as follows:
    1. Electroporation Enhancer – 1 uL (3.85 uM) (note, we now add this to the bottom of the PCR tube in advance while cells are centrifuging.)
    2. Cells – 20 uL
    3. RNP complex from “RNP formation” step – 2.5 uL (final is 4.6 uM crRNA/tracRNA, 4 uM cas9, may need to titrate for different loci (0.5 – 4 uM))*
  2. Avoiding bubbles, pipet mixture up and down 2 times (consider using multichannel for multiple transfections) and transfer 25 uL to the 16 well electroporation strip. Note: the cuvette can only be loaded onto the nucleofector in 1 direction, so check you are loading samples accordingly.
  3. On the Amaxa device select the volume/wells to be transfected, program DZ100 
  4. Select ok to load strip
  5. Place nucleofector strip into the 4D device
  6. Select start
  7. Immediately retrieve media from the incubator and add 75 uL prewarmed media (from the extra well on the plate) to each well. Let sit for 3-5 min at room temp.
  8. gently transfer the entire volume to the 425 uL SFEM-ST media in the 24 well plate
  9. Incubate 24 hours. Usually we transfect on day 5 and passage on day 6 to fresh SFEM-T media and continue culture as described in the section CD34+ Cell Culture. Visually inspect cells on day 8 by light microscope – see example below.

·       Assessing cutting efficiency

Picture1-3 CRISPR/CAS9 in CD34+ cells

Light microscope image (20x objective) of a well of CRISPR/CAS9 transfected CD34+ cells 3 days after transfection (day 8 of culture)

*For transfecting multiple crRNA at the same time, add 2.5 uL of each RNP complex.

ASSESSING CUTTING EFFICIENCY

Cutting efficiency can be assessed as soon as 3 days after transfection. Since MKs become multinucleate, we usually assess cutting on day 13 cells.

  • Pellet 2×10^5 cells from KO and control
  • Isolate DNA (i.e. Qiagen genomic DNA extraction kit)
  • Design primers to amplify that flank ~200 basepairs on one side and 400 base pairs on the other side of the target site
  • Amplify cut region via PCR
  • Clean up PCR and perform Sanger sequencing
  • Plug KO and control sanger sequencing files into TIDE analysis for total efficiency score: https://tide.nki.nl/ or Synthego ICE for KO score: https://www.synthego.com/products/bioinformatics/crispr-analysis

Note that these software seem to sometimes underestimate by 10-20% the actual % of knock out we observe by protein analysis.