Background

CRISPR-Cas9 technology enables gene knockouts (KO) and complete loss of protein translation in human cells, especially in primary cells (Seki and Rutz, 2018). In recently published research, we showed that combining an orthogonal human IL-2 and IL-2Rβ (ortho-hIL-2/ortho-hIL-2Rβ) system with CAR19 T cell therapy markedly enhanced the efficacy of the CAR T cell antitumor activity, in a well-established preclinical model of acute lymphoblastic leukemia (ALL) (Zhang et al., 2021). However, the unexpected deaths observed with high-dose ortho-hIL-2 led us to investigate mechanisms of toxicity. To explore whether T cell-mediated toxicity is related to acute xenogeneic graft-versus-host disease (GVHD) mediated through the T cell receptor (TCR), we knocked out the endogenous TCR of CAR T cells co-expressing ortho-hIL-2Rβ, using a CRISPR-Cas9 gene KO approach. We use guide RNAs (gRNA) targeting the TCR α constant (TRAC) and TCR β constant (TRBC) loci, followed by CD3 negative selection, resulting in TCR negative orthoCAR19 T cells.

Gene silencing is a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. Previous studies have utilized RNA interference (RNAi), small interfering RNA (siRNA), or short hairpin RNA (shRNA) technologies to effectively knock down genes of interest (Berns et al., 2004; O’Keefe, 2013). However, these knockdown approaches do not result in complete loss of gene/protein expression or KO and can often result in off-target effects (Jackson et al., 2006).

The CRISPR-Cas9 genome editing system consists of two components: a “guide” RNA (gRNA) and a Cas9. The Cas9 protein is an endonuclease that uses gRNA to form base pairs with DNA target sequences, enabling Cas9 to introduce a site-specific double-stranded break in the DNA. Through RNA-directed Cas9 nucleases, the CRISPR-Cas9 system can modify DNA with greater precision than existing technologies, such as transcription activator-like endonuclease (TALEN), and zinc-finger nuclease (ZFN) (Conant et al., 2022). While both TALEN and ZFN are artificial structures formed by joining restriction endonucleases with DNA-binding protein domains, CRISPR-Cas9 technology takes advantage of the antiviral defense mechanism of prokaryotes to perform genome editing at the targeted DNA sequence (Khan, 2019). Recent work done by Cui et al. revealed that both TALEN and ZFN inevitably generated massive off-target effects when targeting human papillomavirus 16 (HPV16), while the CRISPR-Cas9 system was shown to be more efficient and specific (Cui et al., 2021). Therefore, CRISPR-Cas9 technology shows better efficiency, feasibility, and practicality in multi-role clinical applications, as compared to TALEN and ZFN. Inference of CRISPR Edits (ICE) was used to analyze CRISPR editing data in this study. ICE is a free and easy-to-use software tool that offers fast and reliable analysis of CRISPR experiments, highly comparable to that of next-generation sequencing (NGS) data. CRISPR editing can be assessed and analyzed by uploading the Sanger sequencing data and specifying a guide sequence(s). A knockout score will be generated, which represents the proportion of cells that have either a frameshift or insertion-deletion mutation (indels). This score is a useful means to quantify the number of contributing indels that are likely to result in a functional KO of the targeted gene.

Our results showed that the CRISPR-Cas9 system is easy to operate, has a short cycle time, low cost, higher accuracy, and is more controllable in targeting TCR α and TCR β genes together than existing technologies such as TALEN and ZFN. The combination of CRISPR/Cas9 with CD3 negative selection leads to a complete TCR depletion in orthoCAR19 T cells.