Cas13d wants to compete with Cas9 to benefit tumor patients by enhancing precision CAR T therapy

A recent article published in Cell¹ reported that the multiplexed effector guide arrays (MEGA) based on the CRISPR-Cas13d system can contribute to improving chimeric antigen receptor (CAR) T cell exhaustion by massively multiplexed, quantitative, and reversible perturbation of the transcriptome in primary human T cells. This study reminds us that Cas9 may be no longer the dominating force or the only choice in the gene-editing and precision therapy field, and other contenders, including Cas13d, Cas12a as well as numerous unknown systems will come into the fray in not long future.

The successful application of CAR T therapy, as everyone knows, could tremendously benefit tumor-targeted therapy but is plagued by the following challenges, such as T cell exhaustion, cytotoxicity, and off-target effects. To address these issues, Tieu and colleagues developed a MEGA platform by harnessing the Cas13d system that is characterized by the RNA-guide RNA endonuclease activity without PAM sequence recognition, the ability to process poly-crRNA guide arrays to facilitate efficient simultaneous targeting of multiple RNA transcripts in single cells, and the smaller molecular weight compare with Cas9 (Figure 1). First, the authors have succeeded in optimizing MEGA HA-28ζ CAR T cells that robustly suppress the exhaustion marker (LAG3, PD-1, and TIM3) upregulation on transcriptional and surface protein levels and have positively affected the tumor-killing activity of chimeric T cells. Moreover, the MEGA expression and effective processing did not induce interferon (IFN) pathway activation, which is a critical signal for tumor surface recognition of CAR T cells and may be one reason of tumor-killing activity enhancement in chimeric T cells.² More importantly, single-vector bicistronic configurations show that this system has low viral titers, which may benefit from the crRNA-guided cleavage of lentiviral RNA of Cas13d, whereas non-induction of IFN signaling is also extremely important to CAR T cell-mediated cytotoxicity elimination.²

Indeed, previous studies have also provided evidence for CRISPR-Cas9 on pathogenic RNA-targeted elimination and IFN signal inhibition via its powerful gene silencing ability.^{2, 3} Nevertheless, one typical advantage of the MEGA platform is that it can process a long array of nearly 10 targeted genes simultaneously dispensing with independent gRNA guiding, although the knockdown efficiency is uneven when without prior optimization of spacer sequence or position. This has phased significance for data validation of CRISPR-based whole-genome screening or conventional RNA-seq analysis in biological research.^1-3 MEGA provides a powerful example in experimental co-validation of multiple candidate genes in the purinergic signaling and the PI3K/Akt pathway, and its multiplexing capability allows for expending to investigate the comprehensive regulation of metabolic pathways, immune pathways, and other cellular functions. Compared with Cas9-based systems that configure one or two gene targeting capabilities,^{2, 3} the Cas13d-based MEGA indubitably showcases its strong competitiveness in understanding multi-gene functions of one signaling pathway or bioprocess.

MEGA exhibited excellent performance in specificity and efficiency of targeting knockdown, which is likely due to Cas13d own structural and functional ascendancy in mammalian cells (Figure 1). As the Cas13′s effector is the only known Cas endonuclease that absolutely binds and cleaves targeted ssRNA, the Cas9- and Cas12-based systems are not considered to be a potential RNA-targeting candidates because of their off-target effects, less efficient, along with high nuclease activity to cleave DNA.⁴ The cleavage of double-stranded DNA is a key constraint on the development of the Cas9 system in CAR T therapy, as it increases the risk of off-target and toxicity and causes unnecessary DNA damage.^{1, 4} Significantly, MEGA did not exhibit “collateral activity” in primary human T cells, which is consistent with the previous research findings in mammalian cells or plants.⁴ Despite the disappearance of the collateral activity may merely contribute to reducing the toxicity of MEGA to T cells or imposing targeting and cutting efficiency, these works provide full confidence for further testing application of Cas13d (or the Cas13 family) in CAR T therapy.

The other commendable achievement in Tieu and colleagues' work is that MEGA is fused with a dihydrofolate reductase domain to Cas13d C-terminus to enable tunable and reversible control of gene knockdown (Figure 1). Under normal cultivation conditions, the fusion system is recognized and degraded by proteasomes, and the addition of trimethoprim (an FDA-approved small-molecule antibiotic) disrupted this balance and enabled the Cas13d to stably perform RNA cleavage. This configuration design is not pioneering; even so, the RNA-targeting cleavage activity broadens Cas13d-based system application potential compared to permanent DNA cleavage of Cas9-based system in bioengineering. On the one hand, the reversibility of RNA editing and the drug-depended control make MEGA as a safer alternative to gene therapy; on another hand, the successful application of regulable fusion elements renders a strategy for improving the stability of the CRISPR-Cas13d system applicable to biosensing, in vitro diagnostics, in vivo imaging and the other fields.⁵ The unique advantage of multitargeted RNA will also make Cas13d a reliable RNA viral biomarker.

In summary, the CRISPR-Cas system boasts great potential in precision therapy. Researchers are trying to break through the engineering barriers by engineering/re-engineering the effector molecule, searching homologous small molecules, fusing other active proteins, and so on.^{1, 3, 4} However, the special molecular configurations and molecular mechanisms of Cas13d boast critical characteristics that serve it well as a strong rival to Cas9 in bioengineering (Figure 1). These may include: (i) Containing two HEPN domains that is enough to catalyze HEPN-independent processing of a pre-crRNA into a mature crRNA and produce HEPN-dependent RNA cleavage activity; (ii) Guiding the ribonucleoprotein to RNA transcripts for sequence-specific degradation without PAM restriction or even PFS preference; and (iii) Targeting cleavage in various disease models with higher efficiency.^{1, 4} The initial success of MEGA is a decisive occasion for the Cas13d-based targeting platform in precision therapy, which may not challenge the status of conventional Cas9 system in genetic engineering, but it will offer more biotechnology tool selection for biomedical scientists.

Hongbiao Ran: Conceptualization (lead); formal analysis (lead); visualization (lead); writing—original draft (lead). Jianxin Jiang: Writing—review and editing (supporting). Ping Lin: Funding acquisition (lead); supervision (lead); writing—review and editing (lead). All authors have read and approved the final manuscript.

The authors declare no conflict of interest.

The authors have nothing to report.