Introduction

Through targeted modification of specific genes or regulatory regions with CRISPR genome-editing technology, 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. Early attempts to apply CRISPR-Cas9 for genome editing in human primary T cells used either viral vectors^1,2 or plasmids^3,4 for Cas9 and gRNA expression, resulting in low targeting efficiency and high toxicity. More recently, ribonucleoprotein (RNP)-based CRISPR-Cas9 expression systems have achieved high efficacy across a number of targets.^5-7

The following document provides instructions for genome editing of human primary T cells using an RNP-based CRISPR-Cas9 system, including optimization of pre- and post-editing culture conditions and methods to evaluate genome editing efficiency. The protocol details instructions for isolation and activation of primary human T cells, preparation of a CRISPR-Cas9 RNP complex using sgRNA or crRNA:tracrRNA duplexes, and delivery of RNP complex into activated primary T cells using electroporation (Figure 1). We describe one strategy to generate knockout primary T cells with high efficiency and present a case study and data on CRISPR-Cas9 genome editing of the TCRαβ locus across a number of activation conditions; see the discussion section of this document for alternative strategies and expected outcomes, as well as protocol modifications.

Background

The mechanism of CRISPR-Cas9 genome editing is shown in Figure 1. The CRISPR-Cas9 system is composed of two key components: a guide RNA (gRNA) and a CRISPR-associated endonuclease (Cas) protein. Briefly, Cas9 endonuclease is targeted to the desired genomic locus through complementary base pairing of the associated single guide RNA (sgRNA) or CRISPR RNA (crRNA) at a site 5’ to a protospacer adjacent motif (PAM) sequence (NGG for Streptococcus pyogenes Cas9). The crRNA partners with a trans-acting CRISPR RNA (tracrRNA), which acts as a scaffold for the interaction with Cas9, to form the guide RNA (gRNA). Once complexed with the gRNA, Cas9 is targeted to the desired genomic locus and will generate a DNA break 3 to 4 bases upstream of the PAM sequence. Endogenous DNA repair systems then function to repair the DNA break. Non-homologous end joining (NHEJ) mediates direct ligation of the broken DNA molecule, and due to the error-prone nature of NHEJ, this can result in the formation of insertion or deletion (INDEL) mutations. If a donor DNA sequence template is provided, knock-in of precise DNA sequences can be accomplished through the homology-directed repair (HDR) pathway (Figure 2).

While this technology has been successfully applied in numerous cell lines, its application in primary human immune cells has been hampered by challenges in efficient delivery and expression of CRISPR-Cas9 components. As mentioned before, early attempts to apply CRISPR-Cas9 for genome editing in primary human T cells used either viral vectors^1,2 or plasmids^3,4 for Cas9 and gRNA expression, which resulted in low targeting efficiency and high toxicity. These expression systems also pose concerns about safety in clinical translation due to the risk of unwanted genetic mutations and immunogenicity.⁵ More recently, electroporation of activated T cells with ribonucleoprotein (RNP) complexes made from recombinant Cas9 protein and in vitro-transcribed (IVT) or synthetic gRNAs has achieved high efficacy across a number of targets.^6-10 In many cell types, including human T cells, IVT gRNAs cause high cytotoxicity due to the presence of a 5’ triphosphate single-stranded RNA (ssRNA) that activates a type I interferon-mediated immune response.¹¹ Synthetic gRNAs are therefore a preferred format for the RNP-based method. ArciTect™ is an RNP-based CRISPR-Cas9 expression system that includes custom synthetic gRNA (sgRNA and crRNA) and is designed to fully support genome editing of human primary T cells.

Beyond CRISPR-Cas9 expression methods, the culture systems for expansion and activation of primary human T cells also represent critical elements for successful genome editing, with cell activation being required in most experimental contexts.¹² While T cells can be isolated from a number of sources using a variety of isolation techniques, to date most genome editing studies involving T cells have used primary human T cells isolated from peripheral blood mononuclear cells (PBMCs) by immunomagnetic selection.^{7, 12-14}

With improved expression and delivery methods, CRISPR-Cas9 genome editing is now rapidly being incorporated into the development of next-generation immunotherapies. One example is chimeric antigen receptor (CAR) T cell therapy, wherein CRISPR-Cas9 enables generation of allogeneic immune effector cells that are compatible for patient infusion.¹⁵ For this purpose, T cells can be engineered to escape immune rejection through deletion of endogenous T cell receptors (TCRs) and human leukocyte antigen (HLA) class I molecules, which shows high efficacy when combined with deletion of inhibitory receptors such as programmed death-1 (PD-1) or cytotoxic T-lymphocyte antigen protein 4 (CTLA-1).^9-10,16-17 This strategy can then be coupled with targeting CAR constructs to the endogenous TCR alpha constant (TRAC) locus18 to generate universal allogeneic CAR-T cells.¹⁸

CRISPR-Cas9 Genome Editing in Primary Human T Cells

Materials Required

A. T Cell Isolation and Activation

1. Isolate human T cells from peripheral blood (e.g. Catalog #70500) using EasySep™ Human T Cell Isolation Kit. Refer to the Product Information Sheet (Document #DX20004) for details.
   
   Optional: Source frozen primary T cells (e.g. Catalog #70024). Refer to the Product Information Sheet (Document #DX27569) for details.
2. Count cells and adjust to 1 x 10⁶ cells/mL in ImmunoCult™-XF T Cell Expansion Medium supplemented with 2mM L-Glutamine, 50µg/mL gentamicin, and 10 ng/mL (130 IU/mL for the specific lot*) Human Recombinant IL-2.
   
   *International Units (IU) need to be calculated per lot. For more information visit www.stemcell.com/international-units-conversion-data-for-cytokines.
3. 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.
4. Optional: Assess T cell activation by binding the activation marker CD25 with Anti-Human CD25 Antibody, Clone BC96 (Catalog #60158) and performing flow cytometry.

B. Preparation of ArciTect™ sgRNA working solution and ArciTectTM crRNA and ArciTectTM tracrRNA stock Solutions

1. Briefly centrifuge the vials before opening.
2. Add nuclease-free water to each vial to give a final concentration of 200 μM (crRNA and tracrRNA) or 100μM (sgRNA), as indicated in Table 1 and Table 2, respectively.



Table 1. Preparation of 200 µM* ArciTect™ crRNA or ArciTect™ tracrRNA





Table 2. Resuspension Volume for 100 µM* ArciTect™ sgRNA


3. Mix thoroughly. If not used immediately, aliquot and store at -20°C for up to 6 months or at -80°C for longer than 6 months. After thawing the aliquots, use immediately. Do not refreeze.

C. Annealing of crRNA:tracrRNA Duplexes

RNP complexes can be prepared using either the two-part crRNA:tracrRNA gRNA, which requires pre-annealing (see below), or sgRNA. If working with sgRNA, proceed directly to section D.

1. Prepare a 60 μM crRNA:tracrRNA solution by combining components in a DNase- and RNase-free microcentrifuge tube as indicated in Table 3. Volumes are sufficient for a single electroporation reaction; adjust as required. Mix thoroughly.



Table 3. Preparation of 60 μM (60 pmol/μL) crRNA:tracrRNA Duplex




2. In a thermocycler or heating block, incubate crRNA:tracrRNA mixture at 95°C for 5 minutes followed by 60°C for 1 minute. Cool to room temperature (15 - 25°C) and place on ice. If not used immediately, store at -80ºC for up to 1 month.

D. Preparation of ArciTect™ CRISPR-Cas9 RNP Complex Mix

1. To prepare the RNP Complex Mix, combine the components in the order listed in Table 4 in a microcentrifuge tube. Adjust component volumes according to the desired number of transfections.
2. Mix thoroughly.



Table 4. Preparation of RNP Complex Mixture for Electroporation




3. Mix the RNP Complex Mixture by gently pipetting up and down 2 times; avoid creating air bubbles or foam in the tube.
4. Incubate the RNP Complex Mix at room temperature (15 - 25°C) for 10 - 15 minutes.

E. Preparation of T Cell Suspension for Electroporation

1. For each electroporation condition (including positive and negative controls), prepare 2 mL of ImmunoCult™-XF T Cell Expansion Medium supplemented with 2 mM L-Glutamine, 50 μg/mL gentamicin, and 10 ng/mL Human Recombinant IL-2.
2. For each condition, add 2 mL of supplemented medium (prepared in step 1) to 1 well of a 6-well plate (for 100 μL tip reactions) or 1 mL of supplemented medium to 1 well of a 12-well plate (for 10 μL tip reactions) and place in a 37°C incubator. Store the remainder of the supplemented medium at 2 - 8°C.
3. Transfer 1.2 x 10⁶ cells (for 100 μL tip reactions) or 1.5 x 10⁵ cells (for 10 μL tip reactions) from the activated T cell suspension (prepared in section A) to a 15 mL conical tube. Centrifuge at 300 x g for 5 minutes at room temperature.

F. Electroporation of T cells with RNP Complex Using Neon® Transfection System

1. Aspirate supernatant from cell pellet (prepared in section E). Resuspend cells in 100 μL of Resuspension Buffer T, if using 100 μL tips, or 7.5 μL of Resuspension Buffer T, if using 10 μL tips, and pipette up and down to mix.
2. Using individual RNase-free microcentrifuge tubes for each condition, combine RNP Complex Mixture (prepared in section D) with activated T cells as indicated in Table 5.



Table 5. Mixture of RNP Complex and T Cells for Electroporation




3. 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.
4. 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.
5. Electroporate mixture according to conditions indicated in Table 6.
   Note: Refer to the manufacturer’s instructions for electroporation. Electroporation conditions may require optimization for different cell types.



Table 6. Recommended Electroporation Conditions for Human T Cells Using a Neon® Transfection System

G. Post-Editing Culture

1. Immediately add the electroporated cells to 1 well of the 6- or 12-well plate prepared in Section E step 2. Incubate at 37°C and 5% CO₂ for 2 - 3 hours.
2. 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 1).
3. 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 (Catalog #76026), followed by sequencing of PCR products. Alternatively, ArciTect™ T7 Endonuclease Kit (Catalog #76021) can be used to assess editing efficiency (% INDEL formation) following PCR amplification. For further information, refer to the Technical Bulletin: Evaluation of Genome Editing.

Case Study: Evaluation of Optimal Culture Methods for High Efficiency TRAC Knockout in Human Primary T Cells

To demonstrate genetic knockout with the ArciTect™ CRISPR-Cas9 system, we targeted the TRAC locus using the protocol described in this document. In addition, we tested multiple T cell activation reagents and dynamics to identify a condition with the highest knockout efficiency. Loss of function due to INDELs at this locus is readily identifiable by the lack of TCR expression on the cell surface. The practical importance of targeting this locus lies in the generation of universal allogeneic T cells for CAR T cell therapies.¹⁸

We first designed multiple crRNAs (Figure 3A, D), as different crRNA sequences can exhibit varying efficiency at the target site (see Technical Bulletin: Genome Editing with Direct Cas9 RNP Delivery Design Considerations). We tested the efficacy of the four crRNAs in activated human T cells, with TRAC knockout efficiency detected by flow cytometry, and INDEL generation detected by the ArciTect™ T7 Endonuclease I Kit. All four crRNAs significantly reduced the frequency of TCRαβ+ cells (Figure 3B) and resulted in cut bands in the T7 endonuclease I assay (Figure 3C), confirming the presence of genomic mismatches that are indicative of INDELs. Based on these results, we proceeded with crRNA2, as it showed the highest knockdown efficiency by flow cytometry.

We then used the validated crRNA2 to compare knockout efficiency in a variety of T cell activation conditions. First, T cells were isolated from the human peripheral blood of four independent donors. Cells were cultured in Immunocult™-XF T Cell Expansion Medium supplemented as described with either ImmunoCult™ Human CD3/CD28 T Cell Activator or CD3/CD28/ CD2 T Cell Activator for 2 or 3 days, for a total of 4 test conditions. On day 0, approximately 10 - 20% of cells expressed the activation marker CD25 (data not shown), which increased to over 80% after 2 or 3 days in the presence of the activators (Figure 4). T cell activation was greater when cells were incubated with the CD3/CD28/CD2 T Cell Activator compared to the CD3/CD28 T Cell Activator (Figure 4). The percentage of activated cells was also greater when cells were incubated with either activator for 3 days compared to 2 days (Figure 4). For more information about optimization of T cell activation and expansion conditions, refer to the Technical Bulletin: Optimization of Human T Cell Expansion Protocol: Effects of Early Cell Dilution.