TITLE: CRISPR-Cas9 Genome Editing in Human Primary T Cells
AUTHORS: STEMCELL Technologies

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">CRISPR-Cas9, an RNA-guided genome editing technology, is revolutionizing cell biology due to the ease and efficiency by which it enables genetic manipulation of mammalian cells. Through targeted modification of specific genes or regulatory regions, researchers can now rapidly generate precise genetic models to study normal and diseased cell physiology. Beyond genetic manipulation for research purposes, CRISPR-Cas9 genome editing also holds great potential for therapeutic applications, including immunotherapy and regenerative medicine. This protocol provides instructions for CRISPR-Cas9 genome editing of human primary T cells, including the isolation and activation of primary human T cells, preparation of a CRISPR-Cas9 RNP complex, delivery of an RNP complex into activated primary T cells using electroporation, and methods to evaluate genome editing efficiency.</div><div class = "text-block"><span>For a discussion of alternative strategies and expected outcomes, and protocol modifications, please see: </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Technical Bulletin: Genome Editing of Human Primary T Cells</span></a><span>. </span></div></div>

[STEPS]
?. [T Cell Isolation and Activation]
Isolate human T cells from peripheral blood using EasySep™ Human T Cell Isolation Kit. Refer to the Product Information Sheet for details.
?. [T Cell Isolation and Activation]
Count cells and adjust to 1 x 106 cells/mL in ImmunoCult™-XF T Cell Expansion Medium supplemented with:2 mM L-Glutamine50 μg/mL Gentamicin10 ng/mL Human Recombinant IL-2
?. [T Cell Isolation and Activation]
Activate human T cells by adding 25 μL/mL of ImmunoCult™ Human CD3/CD28 T Cell Activator. Incubate cell suspension at 37°C and 5% CO2 for 72 hours.
Optional: Assess T cell activation by binding the activation marker CD25 with Anti-Human CD25 Antibody, Clone BC96 and performing flow cytometry.
?. [Preparation of Stock Solutions]
Briefly centrifuge the vials of ArciTect™ crRNA and ArciTect™ tracrRNA before opening.
?. [Preparation of Stock Solutions]
Depending on the size of the ArciTect™ crRNA and ArciTect™ tracrRNA vials being used, add nuclease-free water to each vial to give a final concentration of 200 μM, as indicated in Table 1.  ABC1ArciTect™ crRNA OR ArciTect™ tracrRNA Vial SizeCatalog #Volume of Nuclease-Free Water (μL)22 nmol76010/7601610310 nmol76011/7601750420 nmol76012/76018100
ABC1ArciTect™ crRNA OR ArciTect™ tracrRNA Vial SizeCatalog #Volume of Nuclease-Free Water (μL)22 nmol76010/7601610310 nmol76011/7601750420 nmol76012/76018100
?. [Preparation of Stock Solutions]
Mix thoroughly. If not used immediately, aliquot and store at -80°C for up to 1 month. After thawing the aliquots, use immediately. Do not re-freeze.
?. [Preparation of ArciTect™ CRISPR-Cas9 RNP Complex]
Prepare a 60 μM guide RNA solution by combining components in an RNase-free microcentrifuge tube as indicated below in Table 2. Volumes are sufficient for a single electroporation reaction (10 μL/electroporation reaction); adjust as required. Mix thoroughly.  AB1ComponentVolume per Electroporation (μL)2Nuclease-free water23ArciTect™ Annealing Buffer (5X)24200 μM ArciTect™ crRNA35200 μM ArciTect™ tracrRNA36Total volume10
AB1ComponentVolume per Electroporation (μL)2Nuclease-free water23ArciTect™ Annealing Buffer (5X)24200 μM ArciTect™ crRNA35200 μM ArciTect™ tracrRNA36Total volume10
Note: ArciTect™ Annealing Buffer (5X) and ArciTect™ tracrRNA are both included in the ArciTect™ tracrRNA kit.
?. [Preparation of ArciTect™ CRISPR-Cas9 RNP Complex]
In a thermocycler or heating block, incubate guide RNA solution at 95°C for 5 minutes followed by 60°C for 1 minute. Cool to room temperature and place on ice.
Note: If not used immediately, store guide RNA solution at -80ºC for up to 1 month.
?. [Preparation of ArciTect™ CRISPR-Cas9 RNP Complex]
To prepare the ribonucleoprotein (RNP) complex, combine 60 μM guide RNA (prepared in step 8) and ArciTect™ Cas9 Nuclease (or ArciTect™ Cas9-eGFP) in an RNase-free microcentrifuge tube as indicated below in Table 3. Volumes are sufficient for a single electroporation; adjust as required for the desired number of wells.  AB1ComponentVolume per Electroporation (μL)2Resuspension Buffer T6.834 μg/μL ArciTect™ Cas9 Nuclease*7.2460 μM guide RNA65Total volume10
AB1ComponentVolume per Electroporation (μL)2Resuspension Buffer T6.834 μg/μL ArciTect™ Cas9 Nuclease*7.2460 μM guide RNA65Total volume10
*Note: If using 3 μg/μL ArciTect™ Cas9-eGFP Nuclease, add 11.4 μL of Cas9-eGFP to 6 μL of 60 μM guide RNA and 2.6 μL of Resuspension Buffer T for a total volume of 20 μL. Note that 1 μg/μL ArciTect™ Cas9 should not be used for Neon® electroporation, as the total Resuspension buffer volume should represent ~90% of the total reaction volume.Note: Resuspension Buffer T is included in the Neon™ Transfection System 100 µL Kit.
?. [Preparation of ArciTect™ CRISPR-Cas9 RNP Complex]
Mix the RNP Complex Mixture by gently pipetting up and down 2 times; avoid creating air bubbles or foam in the tube.
?. [Preparation of ArciTect™ CRISPR-Cas9 RNP Complex]
Incubate the RNP Complex Mixture at room temperature (15 - 25°C) for 10 - 15 minutes.
?. [Electroporation of RNP Complex into T Cells]
For each electroporation condition (including positive and negative controls), prepare 2 mL of ImmunoCult™-XF T Cell Expansion Medium supplemented with:2 mM L-Glutamine50 μg/mL Gentamicin10 ng/mL Human Recombinant IL-2.
?. [Electroporation of RNP Complex into T Cells]
For each condition, add 2 ml of supplemented medium (prepared in step 12) to 1 well of a 6-well plate and place in a 37°C incubator. Store the remainder of the supplemented medium at 2 - 8°C.
?. [Electroporation of RNP Complex into T Cells]
Transfer 1.2 x 106 cells from the activated T cell suspension (prepared in section A) to a 15 mL conical tube. Centrifuge at  at room temperature.
?. [Electroporation of RNP Complex into T Cells]
Aspirate medium and resuspend cells in 100 μL of Resuspension Buffer T.
?. [Electroporation of RNP Complex into T Cells]
Using individual RNase-free microcentrifuge tubes for each condition, combine RNP Complex Mixture (prepared in steps 7 - 11) with activated T cells as indicated in Table 4.   AB1ComponentVolume Per Electroporation (μL)2RNP Complex203Activated T cell suspension1004Total Volume120
AB1ComponentVolume Per Electroporation (μL)2RNP Complex203Activated T cell suspension1004Total Volume120
?. [Electroporation of RNP Complex into T Cells]
Mix gently by pipetting up and down carefully 2 times; avoid creating air bubbles or foam in the tube.
Note: If air bubbles are present in the tip when the cells are electroporated, cell viability and transfection efficiency will be significantly reduced.Note: Refer to the manufacturer’s instructions for electroporation. Electroporation conditions may require optimization for different cell types.
?. [Electroporation of RNP Complex into T Cells]
Use a 100 μL Neon® pipette tip to draw up 100 μL of the mixture and place into the electroporation chamber containing 3 mL of Electrolytic Buffer E2. Electroporate mixture according to conditions indicated in Table 5.  AB1Electroporation Parameter2Electrical potential1400V3Pulse width30 milliseconds4Number of pulses1
AB1Electroporation Parameter2Electrical potential1400V3Pulse width30 milliseconds4Number of pulses1
?. [Electroporation of RNP Complex into T Cells]
Immediately add the electroporated cells to 1 well of the 6-well plate prepared in step 2. Incubate at 37°C and 5% CO2 for 2 - 3 hours.
?. [Electroporation of RNP Complex into T Cells]
Count cells and adjust cell density to 2.5 x 105 viable cells/ mL by adding IL-2-supplemented ImmunoCult™-XF T Cell Expansion Medium (prepared in step 12).
Optional: If using Cas9-eGFP nuclease, assess electroporation efficiency 12 - 24 hours after electroporation by flow cytometry.
?. [Electroporation of RNP Complex into T Cells]
Incubate at 37°C and 5% CO2 for 48 - 72 hours for genome editing to occur. Harvest cells for assessment of genome editing efficiency, genomic DNA can be amplified by PCR using primers flanking the target region and ArciTect™ High-Fidelity DNA Polymerase Kit, followed by sequencing of PCR products. Alternatively, ArciTect™ T7 Endonuclease Kit can be used to assess editing efficiency (% INDEL formation) following PCR amplification.