Recent advances and applications of CRISPR-Cas9 in cancer immunotherapy

Abstract

The incidence and mortality of cancer are the major health issue worldwide. Apart from the treatments developed to date, the unsatisfactory therapeutic effects of cancers have not been addressed by broadening the toolbox. The advent of immunotherapy has ushered in a new era in the treatments of solid tumors, but remains limited and requires breaking adverse effects. Meanwhile, the development of advanced technologies can be further boosted by gene analysis and manipulation at the molecular level. The advent of cutting-edge genome editing technology, especially clustered regularly interspaced short palindromic repeats (CRISPR-Cas9), has demonstrated its potential to break the limits of immunotherapy in cancers. In this review, the mechanism of CRISPR-Cas9-mediated genome editing and a powerful CRISPR toolbox are introduced. Furthermore, we focus on reviewing the impact of CRISPR-induced double-strand breaks (DSBs) on cancer immunotherapy (knockout or knockin). Finally, we discuss the CRISPR-Cas9-based genome-wide screening for target identification, emphasis the potential of spatial CRISPR genomics, and present the comprehensive application and challenges in basic research, translational medicine and clinics of CRISPR-Cas9.

Keywords: CRISPR-Cas9; Cancer immunotherapy; Cellular therapy; Mechanism; NK/macrophage.

Fig. 1

Naturally occurring Cas proteins and the engineered Cas proteins. These systems are divided into two categories: Class I utilize multiple Cas proteins to form effector complexes while Class II perform targeting and nuclease activity with a single Cas protein. a Type I-E, also known as Cascade, is a DNA nuclease. b Type III, is a DNA/RNA nuclease. The activation of both the HD and the Palm domains of the Cas10 subunit is crucial to confer immunity. c Type II (Cas9), has high GC protospacer adjacent motifs (PAM). TracrRNA:crRNA is usually designed as a single RNA complex (sgRNA). Cas9 is the most widely characterised protein. d Type V (Cas12), has high AT PAM, can process its own crRNA and possess an RNA processing site. e Type VI (Cas13), has no PAM requirement, targets RNA specifically. The Cas9 protein loses cleavage activity by introducing D10A and H840A mutations into the RuvC and HNH domains respectively. f, g CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi), which activated or repressed target genes by recruiting various effector domains, resulting in transient transcriptional and epigenetic modulation

Fig. 2

Role of PD-1/PD-L1 axis in tumor progression and utilization of CRISPR-Cas9 to block the PD-1 and CTLA-4 in combination. a When PD1−/PD-L1 binding, downstream signaling brings about tumor gene expression as angiogenesis (offer tumor nutrition and promote metastasis), EMT phenomenon (decrease adhesion between epithelial cells and develop tumor invasion and metastasis), and accelerating cancer stem cell generation [cancer stem cells possess (1) self-renewal; (2) proliferation; (3) differentiation traits]. b Tumor cells up-regulate the PD-L1 to activate PD-1 on the surface of T cells, then diverse downstream events such as CD28 signal transmission are terminated and multifarious signaling pathways are activated, such as RAS, NF-κB, PI3K-PKB, WNT, Hh, ultimately giving rise to gene expression that tumor proliferation and survival are developed. Another pivotal immune checkpoint is CTLA-4, binding to the B7 receptor on the antigen-presenting cells, which has a inhibitory role in T-cell function. Hence, utilizing CRISPR-Cas9 to block the PD-1 and CTLA-4 in combination may generate higher antitumor responses in CAR-T cells than knocking out PD-1 alone

Fig. 3

Generate “off-the-shelf” allogeneic CAR-T cells via CRISPRs. Given the presence of endogenous TCR and HLA on donor T lymphocytes, the most significant challenge with universal products is the potential risk of Graft-Versus-Host-Disease (GVHD) and alloreactivity (host versus graft response). GVHD is caused by targeting patient somatic cells mediated by donor T cell TCR-αβ receptors, resulting in an allogeneic T cell attack. Conversely, alloreactivity occurs when patient T cell TCR-αβ receptors recognize exogenous HLA molecules on donor T cells, giving rise to rapid rejection. CAR-T capacity and safety can be enhanced by CRISPR-Cas9 which efficiently knocks out multiple genetic loci with a single pass

Fig. 4

A multitude of factors and regulations may influence gene editing. a Higher CG content stabilizes the hybrid and facilitates the efficiency of the Cas9. b gRNA and Cas protein are PAMPs for the innate immune cells, and the immune response may affect the results of gene editing. c Nucleosome breathing and remodeling have already been investigated that may enhance Cas9 activity. d Modification of sgRNAs responsible for target DNA recognition can affect the specificity of Cas9 cleavage. e The CRISPR system can be tightly spatially or temporally controlled. Light irradiation induces heterodimerization between pMag and nMag