Gene knock-outs in human CD34+ hematopoietic stem and progenitor cells and in the human immune system of mice

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 transplant these modified cells in immunodeficient mice with high engraftment levels and multilineage hematopoietic differentiation. The result is a humanized mouse from which we knocked out a gene of interest from their human immune system.

1 NOTE: 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.

2
Prepare an amount of CD34 expansion media appropriate for the number of cells to be thawed.

3
Equilibrate the CD34+ gradually to ensure maximum viable recovery.

3.1
Prepare 35ml of room temperature thawing media, per tube of CD34+ cells, by 0.2um filtering 1x PBS with 0.5% BSA.

3.2
Thaw the cells rapidly in a 37C water bath.

3.3
Transfer the cells to a 50 ml conical tube.

3.4
Serially dilute the thawed cells with thawing media while gently mixing the cells.

3.6
Repeat this process with 2 ml of thawing media for a total volume of 4 ml.

3.7
Follow the same process of doubling the volume until you have a total volume of 32 ml.

3.8
Collect the cells by centrifugation for 10 minutes at 400 x g, room temperature.

3.9
Aspirate the media and resuspend the cells in 1ml of thawing media.

3.10
Take a small aliquot of cells to count and centrifuge the remainder at 400 x g for 5 mins

4
If expanding the cells, count the cells daily to ensure they are maintained at concentration of 2-3 x 10 6 /ml.

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.

NOTE: 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. We have found that the optimal molar ratio of sgRNA:Cas9 is 2:1. We have found that the optimal molar ratio of sgRNA:Cas9 is 2:1.

10.2
Harvest cells normally and count. For each 20μl nucleofection, aliquot 2-4 x 10 5 cells into a separate 1.5 mL microcentrifuge tube and centrifuge 5 minutes at 350 x g, room temperature.

10.3
Aspirate the liquid as completely as possible without disrupting the cell pellet 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.

11
Setup the 4D nucleofector and nucleofect the cells Setup the 4D nucleofector and nucleofect the cells

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 DS-150 for the CD34+ cells.

NOTE:
NOTE: For 100μl nucleofections select the option for the larger volume individual cuvettes.

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.

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.

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.

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: NOTE: Excessive pipetting can greatly increase cell death.

11.6
Transfer the cells to the 96-well or 24-well plate prepared above.
in vivo in vivo use use

13.2
Pool cells from nucleofections, with a minimum 5 x 10 5 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.

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 10 6 cells/vial and 1ml of CryoStor CS10 if freezing up to 10 x 10 6 cells/vial.

13.4
Aliquot into cryovials and freeze using your desired method.
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.

15
Extract gDNA using the E.Z.N.A. MicroElute Genomic DNA Kit (Omega Bio-Tek), according to the manufacturer's protocol.

16
Perform PCR amplification of the region around the sgRNA cut site(s):

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.

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.

16.3
Perform PCR amplification (50μl reaction) on the edited and control samples using a highfidelity polymerase such as Phusion (ThermoFisher Scientific), following manufacturer's suggestions for PCR conditions.

22
Perform Sanger sequencing on the PCR amplicons for your edited and control samples.

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.