Cancer remains an extremely complex disease despite tremendous advancements in the fields of diagnosis and treatment, such as radiation, immunotherapy, chemotherapy, and surgery. Unfortunately, there are currently no viable treatment options for malignant cancers that often metastasize and recur. This emphasizes the importance of exploring alternative therapeutic modalities as soon as feasible [1,2]. Since genome-editing technology has been proven to alter a cell's DNA sequence, it has become a cutting-edge approach for cancer treatment that uses antibodies and biological therapies [3]. The confirmation of the potential of gene editing to revolutionize cancer treatment comes from the alteration of immune cell adoptive therapy techniques and “tumor cell normalization” editing. The three primary methods for modifying DNA are zinc finger structure nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR/Cas9 system. ZFNs and TALENs are specialized biotechnological methodologies that assist in targeted genetic modifications by generating DNA double-strand breaks. However, their effectiveness in gene therapy and disease modeling is hindered by issues associated with their efficiency and targeting precision. Single guide RNA (sgRNAs) must have their first 20 nucleotides altered to target distinct loci and guide CRISPR/Cas9 editing, which simplifies target identification and reduces costs. Moreover, the CRISPR/Cas9 targeting method depends on ribonucleotide complexes, enhancing specificity compared to ZFN and TALEN, which rely on protein-DNA interactions [4].

The CRISPR/Cas system serves as a genetic defense mechanism adopted from which is found in bacteria and archaea. The gRNA adaptive immune system captures foreign genetic material, such as plasmids or phage DNA, and integrates it into the CRISPR repeat-spacer array in the bacterial chromosome. This creates a historical record of past infections, enabling defense against future attacks by the same invaders [5]. The CRISPR/Cas system is occasionally referred to as “genetic scissors” in the medical field of genome editing because of its effectiveness, ease of use, and adaptability. The key achievements of CRISPR/Cas since 1987 are shown in Fig. 1 A [6]. The CRISPR/Cas complex comprises several integral components: the Cas9 protein acts as a molecular scissor for precise DNA cleavage at designated sites, while the sgRNA provides a complementary sequence to guide Cas9. Additionally, tracrRNA (trans-activating CRISPR RNA) facilitates the correct binding of Cas9, and the Protospacer Adjacent Motif (PAM) sequence is vital for recognition and cleavage, with linker loops stabilizing the overall complex, as depicted in Fig. 1 B [7]. Fig. 1 C depicts the 3D crystal structure of Streptococcus pyogenes CRISPR-Cas9, which is catalytically active in association with sgRNA and dsDNA primed for target DNA cleavage.

Transcription of the CRISPR array produces mature crRNAs featuring a spacer sequence that matches the foreign genetic element at the 5 ‘end and a CRISPR repeat sequence at the 3’ end. The complementary pairing between the crRNA and the foreign target triggers sequence-specific damage by Cas nucleases during subsequent infections [[8], [9], [10]]. The CRISPR/Cas system employs a short-conserved sequence called PAM to identify target DNA for degradation. They were categorized into types I-VI, each utilizing distinct Cas proteins and crRNAs for interference (Fig. 2). Type II systems utilize Cas9 as a single protein DNA endonuclease, along with tracrRNA, to form a dual RNA hybrid with crRNA to guide Cas9 for DNA cleavage. The programmable nature of the CRISPR/Cas9 system enables precise targeting of DNA sequences within the genome by altering the gRNA sequence. Upon target DNA recognition, Cas9 induces DSBs that are repairable by either NHEJ or HDR, resulting in indels and precise genome modifications, respectively (Fig. 2 A). The system's simplicity, efficiency, and ease of use have made it a dominant tool for genome editing across various organisms, surpassing conventional DNA editing techniques such as TALENs and ZFNs [8]. Scientists have identified various CRISPR/Cas systems; however, only the class II system features a single effector Cas protein that is used in mammalian gene editing. As Fig. 2 B shows the class II systems primary effector proteins including Cas9 (type II), Cas12 (type V), and Cas13 (type VI), are indispensable tools in molecular biology and genetic engineering research [[11], [12], [13]]. The features of naturally occurring major CRISPR–Cas enzymes are listed in Table 1.

CRISPR/Cas9 has a wide range of applications in the regulation of gene transcription, extending beyond genome editing. When combined with transcriptional repressors or activators, catalytically inactive dead Cas9 (dCas9) variation promotes gene knockout (CRISPRko), gene suppression (CRISPRi), and activation (CRISPRa), as shown in Fig. 2 C. This finding supports the development of CRISPR-based gene therapy technologies, particularly those used to treat cancer. Targeting tumor stem cell-associated genes, editing metabolically important genes, manipulating chemo-tolerance genes, and improving cancer immunotherapy are only a few CRISPR/Cas9-mediated strategies that have demonstrated effectiveness in a variety of cancer types [14,15]. Despite the revolutionary potential of CRISPR/Cas9, issues regarding off-target consequences, in vivo cellular targeting, and nuclear transfer efficiency still exist. However, CRISPR/Cas9 editing methods and ongoing delivery system innovations present viable ways to overcome these challenges, opening the door for further breakthroughs in therapeutic interventions and scientific research [16].

Several studies have described the advantages, applications, and challenges of the CRISPR/Cas system in cancer treatment [14,[17], [18], [19]]. However, a comprehensive pipeline detailing the sequential strategies for CRISPR/Cas cancer therapy, including the processes for identifying and selecting target genes as well as optimizing gRNAs and sgRNAs, remains inadequately articulated. In this review, we systematically outline the processes of target gene identification and selection, gRNA optimization, and contemporary delivery methods, along with a compilation of patents and pertinent details regarding the ongoing clinical trials related to CRISPR/Cas-based cancer therapy.