Harnessing the evolving CRISPR/Cas9 for precision oncology

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system, a groundbreaking innovation in genetic engineering, has revolutionized our approach to surmounting complex diseases, culminating in CASGEVY™ approved for sickle cell anemia. Derived from a microbial immune defense mechanism, CRISPR/Cas9, characterized as precision, maneuverability and universality in gene editing, has been harnessed as a versatile tool for precisely manipulating DNA in mammals. In the process of applying it to practice, the consecutive exploitation of novel orthologs and variants never ceases. It’s conducive to understanding the essentialities of diseases, particularly cancer, which is crucial for diagnosis, prevention, and treatment. CRISPR/Cas9 is used not only to investigate tumorous genes functioning but also to model disparate cancers, providing valuable insights into tumor biology, resistance, and immune evasion. Upon cancer therapy, CRISPR/Cas9 is instrumental in developing individual and precise cancer therapies that can selectively activate or deactivate genes within tumor cells, aiming to cripple tumor growth and invasion and sensitize cancer cells to treatments. Furthermore, it facilitates the development of innovative treatments, enhancing the targeting efficiency of reprogrammed immune cells, exemplified by advancements in CAR-T regimen. Beyond therapy, it is a potent tool for screening susceptible genes, offering the possibility of intervening before the tumor initiative or progresses. However, despite its vast potential, the application of CRISPR/Cas9 in cancer research and therapy is accompanied by significant efficacy, efficiency, technical, and safety considerations. Escalating technology innovations are warranted to address these issues. The CRISPR/Cas9 system is revolutionizing cancer research and treatment, opening up new avenues for advancements in our understanding and management of cancers. The integration of this evolving technology into clinical practice promises a new era of precision oncology, with targeted, personalized, and potentially curative therapies for cancer patients.

Background

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, a groundbreaking innovation in genetic engineering, has revolutionized our approach to surmounting complex diseases, including cancer [1, 2]. This microbial adaptive immune system, repurposed for precision genome editing, offers unparalleled accuracy and versatility, making it a pivotal tool in cancer research and therapy [2,3,4]. The ability to precisely manipulate the genome has opened new avenues for cancer screening, diagnosis, and treatment, heralding a new era in oncology. Cancer, a multifaceted and often lethal disease, is characterized by genetic mutations and alterations that drive tumor development, progression, and resistance to therapy [5,6,7,8,9,10]. Traditional methods for studying and treating cancer have been hampered by the complexity and heterogeneity of tumors [11, 12]. However, the advent of CRISPR/Cas9 has transformed our ability to dissect the genetic underpinnings of cancer. By enabling targeted modifications at specific genomic loci, CRISPR/Cas9 allows researchers to systematically investigate the roles of individual genes and genetic networks in cancer biology [13]. This precision not only facilitates the identification of novel cancer drivers and therapeutic targets but also enhances our understanding of tumorigenesis and metastasis. In cancer screening, CRISPR/Cas9 has demonstrated its potential to identify oncogenic mutations and variants associated with increased cancer risk. High-throughput CRISPR screens have been employed to uncover genetic vulnerabilities in cancer cells, offering insights into potential biomarkers for early detection. By targeting genes involved in DNA repair, cell cycle regulation, and apoptosis, CRISPR screens can pinpoint key players in cancer initiation and progression. This capability is particularly valuable in the development of personalized medicine, where genetic profiling can guide tailored screening strategies for individuals at high risk of developing cancer. The diagnostic applications of CRISPR/Cas9 are equally transformative. CRISPR-based diagnostics, such as CRISPR–Cas13a and CRISPR–Cas12a, have been developed to detect cancer-associated genetic alterations with high sensitivity and specificity. These systems leverage the unique properties of CRISPR enzymes to recognize and cleave target nucleic acids, generating detectable signals for the presence of cancer biomarkers. Such diagnostic tools can facilitate early detection and monitoring of cancer, improving patient outcomes through timely intervention. Perhaps the most profound impact of CRISPR/Cas9 lies in its therapeutic potential. The system’s ability to introduce precise genetic modifications enables the development of innovative cancer treatments. CRISPR/Cas9 has been employed to engineer T cells for adoptive cell therapy, enhancing their ability to recognize and destroy cancer cells. This approach, exemplified by chimeric antigen receptor (CAR) T-cell therapy, has shown remarkable success in treating hematologic malignancies. Additionally, CRISPR/Cas9-mediated gene editing has been explored to disrupt oncogenes, restore tumor suppressor functions, and modulate the tumor microenvironment, offering new therapeutic avenues for solid tumors.

Moreover, CRISPR/Cas9 is instrumental in overcoming therapeutic resistance, a major challenge in cancer treatment. By targeting genes that confer resistance to chemotherapy, radiotherapy, and immunotherapy, CRISPR/Cas9 can sensitize cancer cells to these treatments, enhancing their efficacy. The system also facilitates the study of resistance mechanisms, providing insights that can inform the development of combination therapies to prevent or overcome resistance. Despite its vast potential, the clinical application of CRISPR/Cas9 in cancer research and therapy is accompanied by significant technical and safety considerations. Off-target effects, delivery challenges, and immune responses to CRISPR components are critical issues that need to be addressed. Ongoing advancements in CRISPR technology, including the development of high-fidelity Cas9 variants and improved delivery methods, are aimed at mitigating these challenges and enhancing the system’s clinical applicability. In the subsequent sections, we will comprehensively examine the evolutionary trajectory of CRISPR/Cas9 and elucidate its applications in oncology with meticulous detail.

The origin and development of the CRISPR/Cas9

Advent and rise of CRISPR/Cas9

Gene editing, a transformative technology, allows for precise genomic alterations through insertions, deletions, or substitutions [1]. The evolution of gene editing has progressed through three distinct generations. Initially, engineered endonucleases, such as meganucleases and zinc-finger nucleases (ZFNs), were employed for specific DNA targeting [14,15,16]. This was followed by the second generation, which leveraged TALE repeat units to enhance precision [17]. The current era is marked by the advent of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology, which has revolutionized gene editing with its unmatched efficiency and broader applicability [1]. CRISPR/Cas9, renowned for its precision in gene editing, is widely utilized in biology and medicine. Originally functioning as a bacterial and archaeal defense mechanism against phages and plasmids, CRISPR/Cas9 was first identified in 1987 by a Japanese research team that discovered unique repetitive sequences in Escherichia coli [18]. Although the significance of these sequences was not fully understood at the time, subsequent research in 2002 by Ruud Jansen coined the term “CRISPR” (clustered regularly interspaced short palindromic repeats) and identified the associated (cas) genes [19]. In 2005, Mojica et al. uncovered that the intervening sequences were mosaics comprising repeated units interspersed with unique sequences derived from external sources [20]. This understanding was further consolidated by studies on Streptococcus thermophilus, revealing its susceptibility to phages linked to CRISPR sequences and identifying specific protospacer adjacent motifs (PAMs) [21,22,23,24]. This led to the classification of CRISPR–Cas systems into three primary types, each further divided into subtypes, with CRISPR/Cas9 categorized under the second type [25]. The pioneering work by Church et al. in adapting the type II CRISPR system for use in human cells through custom guide RNA set the stage for rapid advancements in gene editing across various species [26,27,28] (Fig. 1).

Fig. 1

The timeline of the CRISPR/Cas9 system. After over four decades of development since the discovery of CRISPR/Cas9 in prokaryotes, CASGEVY™, the first gene therapy protocol approved by the FDA, has evolved into an indispensable tool for disease research and treatment. In 1987, the initial discovery of CRISPR sequences in Escherichia coli by Yoshizumi Ishino and his team laid the groundwork. Between 2005 and 2012, multitudes of researchers, including Emmanuelle Charpentier and Jennifer Doudna, unveiled the mechanisms behind CRISPR/Cas9, showing how it could be harnessed as a tool for precise DNA editing. Their work culminated in the development of a simplified system for gene editing, for which they were later awarded the Nobel Prize in Chemistry in 2020. Afterward, Feng Zhang and his colleagues have successfully adapted CRISPR/Cas9 for gene editing in eukaryotic cells, demonstrating its potential for precise genetic manipulation in complex organisms, including humans since 2013. This breakthrough paved the way for CRISPR/Cas9’s use in therapeutic applications. In 2016, CRISPR/Cas9 entered the clinical trial phase for cancer treatment, marking a significant milestone in its application. Trials include efforts to modify patient immune cells to target specific cancer cells more effectively. Regulatory approval of CRISPR/Cas9-based therapies has begun, with treatments for conditions like sickle cell disease and β-thalassemia leading the way, represented by the approval of CASGEVY™. These approvals are a testament to CRISPR’s potential as a therapeutic tool and mark a new era of gene-edited medicines

Reaching the pinnacle of its developmental trajectory

The advancement of the CRISPR/Cas9 system has established several principles for effective genome manipulation [29]. The balance between cleavage efficiency and target specificity varies across different CRISPR/Cas systems. Larger Cas enzymes, such as SpCas9, might exhibit higher editing efficiency but lower specificity compared to smaller nucleases. Additionally, the efficacy of CRISPR/Cas9 nucleases is influenced by the expression levels of target genes, with higher efficiency observed in more actively transcribed genes, likely due to differences in chromatin accessibility [29]. Diverse DNA repair pathways caused by cleavage influence the outcomes of gene editing. Homology-directed repair (HDR), characterized by higher fidelity, is preferred for precise gene insertions, deletions, or modifications [30]. Manipulating the repair pathway can favor HDR over non-homologous end joining (NHEJ), leading to more accurate gene insertions, deletions, or modifications without the random integrations associated with NHEJ [31]. Using cell cycle synchronization, employing DNA repair inhibitors, or modifying the CRISPR/Cas9 system to improve template DNA’s access to the break site. Besides, the chosen HRD template could potentially influence the specificity of editing. Single-stranded DNA (ssDNA) decreases erroneous integration regardless of target homology in comparison to double-stranded DNA (dsDNA) [32]. The design of single-stranded oligodeoxynucleotide (ssODN) donor templates also affects HDR efficiency following CRISPR cleavage [29]. These strategies aim to increase the precision of gene edits while minimizing off-target effects, crucial for therapeutic applications where unintended genomic alterations could jeopardize genetic integrity [29].

With the maturation of CRISPR/Cas9 theory and technology, this revolutionary technology secured the Nobel Prize and has been proven instrumental in the precise detection of nucleic acids for molecular diagnosis and treatment of various intractable diseases, especially cancers [13, 33,34,35,36]. The first successful attempt to employ CRISPR/Cas9 in humans involved the reinfusion of CCR5 hematopoietic stem and progenitor cells (HSPCs) into HIV patients in 2019, yielding modestly promising outcomes [37]. In the same year, Métais et al. initiated the editing of HSPCs using a Cas9 variant with three nuclear localization sequences and infused them into humanized mice to suppress γ-globin gene expression for treating sickle cell disease and β-thalassemia [38]. Subsequent advancements have led to clinical trials evaluating CRISPR/Cas9-edited CD34+ HSPCs products, demonstrating significant improvements in treating sickle cell disease and β-thalassemia [39]. In 2023, CASGEVY™, a CRISPR genetically modified autologous CD34+ HSPCs for sickle cell disease and transfusion-dependent β-thalassemia, was authorized by the FDA and MHPR, heralding a new era for the clinical application of CRISPR therapy [40].

Advancements in SpCas9: pioneering precision in CRISPR genome editing

As the foremostly employed Cas9 homolog, Streptococcus pyogenes Cas9 (SpCas9) originates from the nucleic acid sensing and recognition system inherent in Streptococcus pyogenes [4, 41, 42]. Its functionality involves the recognition of a 20nt length specific sequence, as determined by the CRISPR RNA (crRNA) which ends up with the protospacer adjacent motif (PAM) sequence of NGG, and subsequent cleavage through identification of the corresponding sequence within the host genomic sequence [43]. Building upon the seminal research conducted by Zhang et al., a series of revolutionary refinements and manipulations of the sequence and structure of SpCas9 and its auxiliary guiding RNA has emerged to enhance the precision of genome editing. The research into CRISPR/Cas9 surged, marking a pivotal era in gene editing. Despite its potential in rectifying genetic mutations, challenges like limited targeting due to fixed PAM sequences, delivery difficulties due to the size of the effectors, and off-target effects were noted. Requisite modification is warranted.

Innovations in sgRNA: enhancing efficiency and specificity

The components of the primordial CRISPR system are tanglesome. The first step is to simplify the composition, with the initial effort leading to an innovative fusion of guide RNA and scaffold RNA, yielding sgRNA. The former is complementary to the target DNA sequence and determines the specificity of cleavage, while the latter acts as the framework connected with Cas9. This RNA fusion cleverly mimics the intricacies of its natural counterpart, crRNA, while introducing an additional element called trans-activating crRNA (tracerRNA), the incorporation of which is closely resembled natural genetic manipulation components [41]. The manufacturing optimizations for tracerRNA involve designing synthetic sgRNA scaffolds based on structural studies and mutational analyses, aiming to maintain functional integrity while introducing variations for improved targeting efficiency or additional functionalities [44]. Besides, through precise modulation of the scale of sgRNA and meticulous adjustment of its length, the researchers primarily ensured molecular-level activity and precision in genome editing [45, 46]. However, the practical application of CRISPR/Cas9 is significantly hindered by its off-target effects, despite extensive utilization of computational approaches to optimize suitable sgRNA [47,48,49].

Structural insights and modifications: towards optimized SpCas9 functionality

Cryoelectronic microscopy unveiled the crystal structure of spCas9 and its functional complex, showcasing a bilobed architecture with recognition and nuclease lobes. The lobes form a positively charged groove for the sgRNA: DNA heteroduplex. The recognition lobe, REC domain binds sgRNA and DNA. The nuclease lobe (NUC) domain houses a PAM-interacting domain (PI), as well as two cleavage domains known as the HNH domain and RuvC domain. These domains exert endonuclease activity on the complementary and noncomplementary strands respectively. SpCas9 initiates its action by recognizing the PAM-proximal guide region and the repeat: anti-repeat duplex of sgRNA, forming the SpCas9–sgRNA binary complex. This binary complex then identifies the DNA sequence complementary to the 20nt guide region of the bound sgRNA, resulting in the formation of the final SpCas9–sgRNA–target DNA ternary complex. Prior to ternary complex formation, the PI domain recognizes the PAM sequence on the noncomplementary strand, leading to R-loop formation. Once the ternary complex is assembled, the mobile HNH domain cleaves the complementary strand, and the RuvC domain cleaves the single-stranded, noncomplementary strand [50, 51]. Biochemical studies emphasize the significance of PAM recognition for both target DNA binding and cleavage, suggesting a potential inactive-to-active conformational transition in the SpCas9–sgRNA complex upon PAM recognition [52]. Besides, the discovery has revealed that the occurrence of off-target events is attributed to the stronger binding strength of SpCas9 to the nontarget DNA strand compared to DNA rehybridization forces. Modulating these interactions can enhance specificity, as evidenced by mutations. Similar strategies can be employed to improve specificity in other Cas9 family proteins, providing valuable insights for engineering nucleases with enhanced precision [53]. Additionally, other methods portray the detailed and sophisticated dynamic and kinetic profiles of CRISPR/Cas9 in gene manipulation as well. Through employing single-molecule Förster resonance energy transfer (smFRET) to observe Cas9–DNA interactions in real-time, researchers discover that Cas9 probes DNA for PAM sequences through a combination of one-dimensional (1D) lateral diffusion along the DNA strand and three-dimensional (3D) diffusion. This lateral diffusion allows Cas9 to translocate across the DNA to adjacent PAM sites, increasing the likelihood of finding and binding to its target sequence effectively [54]. These structural insights clarify Cas9’s RNA-guided DNA targeting mechanism, enabling and invigorating the rational engineering of advanced genome-editing technologies. The dCas9 variant, in light of structure trait and more profoundly deep mutational scanning parsing, engineered with mutations in Cas9’s RuvC and HNH regions, is rendered enzymatically inactive as an endonuclease. However, it retains the ability to bind DNA through guide RNA and can be utilized as a precise tool for transcription regulation [55, 56]. By virtue of their defined microstructure, the separate mutations in RuvC or HNH domains enable constitutive focus on specific DNA chain cleavage. Consequently, the RuvC mutant Cas9 (D10A) exclusively induces incisions on the target chain, while the HNH mutant Cas9 (H840A) selectively generates incisions on the non-targeting chain. The nickaseCas9 (nCas9) refers to either Cas9 (D10A) or Cas9 (H840A) [57, 58]. The compromised or deficient catalytic function of both these Cas9 proteins forms the basis for the epigenetic editor, base editor, and primer editor, tailoring themselves for a more sophisticated and comprehensive range.

The evolution of SpCas9 variants: engineering for precision and broad spectrum targeting

The first generation of refined SpCas9 for precise gene editing avoiding off-target effect

In the relentless pursuit of precision within genome editing, challenges persist due to constraints imposed by the recognition of specific sequences guided by a particular PAM and the occurrence of unintended off-target effects [59, 60]. The off-target effect arises from the evolutionary flexibility inherent in bacteria. This flexibility, crucial for bacterial survival, enables hosts to discern exogenous DNA-bearing sequence alterations resulting from evolutionary pressures. This attribute is advantageous for bacteria, but it becomes an adverse hindrance when applying RGNs to human genome editing endeavors [61]. In order to tackle this issue, the first step is to figure out and ascertain the off-target events accurately. Various artificial enhancement of computational algorithms facilitates the progress [48, 62,63,64]. Recently, Zhu and colleagues came up with a cutting-edge approach, known as Tracking-seq, for in situ detection of off-target effects that can be widely applied to commonly used genome-editing tools, including Cas9, base editors, and prime editors [65]. Besides, an alternative approach lies in molecular structural refinement [66]. Kleinstiver et al. have made significant advancements in enhancing the feasibility of SpCas9 based on crystal structural analysis. Precise modifications were made to SpCas9 variants, specifically VQR (D1135V/R1335Q/T1337R) and VRER (D1135V/G1218R/R1335E/T1337R), to alter PAM specificity and broaden the versatility of this endonuclease for genome editing. The VQR mutations increased specificity towards NGAN PAM, while the VRER variant exhibited higher recognition activity on NGCC PAM [67, 68]. Notably, the T1337R substitution imparts an additional nucleotide recognition preference, as evidenced by both the EGFP disruption assay and crystal structure analysis, which are in agreement. Furthermore, compared to the wild type, the D1135E substitution enhances specificity by facilitating steric accommodation of the bound DNA and eliminating a potentially repulsive electrostatic interaction [67, 69]. Additionally, the discovery of potential orthologs SaCas9 and St1Cas9, particularly SaCas9, holds great promise for ushering in a new era in the CRISPR system [67]. Furthermore, Slaymaker et al. advanced the refinement trajectory, marked by the introduction of “enhanced specificity” SpCas9 (eSpCas9). This variant encompasses K855A mutations, K810A/K1003A/R1060A mutations, or K848A/K1003A/R1060A mutations. The eSpCas9s exhibit diminished tolerance for mismatched target sites, whether involving single-base or double-base mismatches. In the context of genome-wide editing, eSpCas9 significantly diminishes off-target cleavages when contrasted with the conventional SpCas9 [53]. Afterward, in their quest for molecular mastery, Kleinstiver et al. deftly harnessed the capabilities of Cas9-HF1, a variant endowed with heightened fidelity. They discerningly probed the efficacy of this variant when directed towards both standard non-repetitive sequences and the more intricate terrain of repetitive sequences. The results of their empirical inquiry stood as a testament to the efficacy of Cas9-HF1, demonstrating a conspicuous reduction in off-target events, whether navigating the well-charted territories of non-repetitive sequences or braving the intricacies of repetitive ones [70, 71]. The amelioration of off-target events relies on the putative theory that through attenuating non-specific interactions between the Cas9–RNA complex and its substrate DNA, these evolved SpCas9 variants exhibit enhanced specificity compared to the wild-type while maintaining high on-target activity [72]. However, instances where these engineered SpCas9 enzymes are unable to efficiently cleave the target sequence may arise. To tackle this issue, researchers have developed Sniper-Cas9, which possesses the same functionality as the wild-type SpCas9 while effectively subduing off-target effects [73].

In the light of the study on the target discrimination of recognition, Chen et al. furtherly exploited the hyper-accurate Cas9 variant (HypaCas9) to achieve a balanced equilibrium between targeted recognition and nuclease activation for precision of genome edition [74]. Moreover, by combining mutations from previously eSpCas9 and Cas9-HF1, Péter István Kulcsár et al. introduced a new “Highly enhanced Fidelity” nuclease variants (HeFSpCas9) that showed improved specificity for certain targets that were problematic for earlier versions, which emphasized the importance of matching specific high-fidelity nucleases with particular genomic targets to achieve the most effective and precise gene editing, minimizing the risk of unintended modifications [75]. Subsequent investigations have underscored the criticality of maintaining an exact 20-nucleotide guide RNA sequence for enhancing the precision and efficacy of genome editing [76]. Soon after, evoCas9 was developed through a yeast-based assay designed to identify optimized SpCas9 variants with both high on-target activity and reduced off-target activity. This process involved screening a library of SpCas9 variants with random mutations in the REC3 domain. The key advantages of evoCas9 over previous Cas9 variants include a significantly improved fidelity (79-fold over wild-type and a fourfold average improvement over other engineered variants) while maintaining near wild-type on-target editing efficiency (90% median residual activity). Moreover, evoCas9 demonstrated substantially improved specificity, with no detectable off-target sites for several tested guide RNAs, and maintained this specificity even after long-term expression, limiting unspecific cleavage of difficult-to-discriminate off-target sites [77].

The secondary generation of refinement for enhancing the scope of genome editing accessibility

In the year 2018, advancements in the exploitation and development of evolutionary SpCas9 variations reached an unprecedented pinnacle. In addition to mitigating off-target issues, the development of novel SpCas9 variants has the potential to expand recognition sites within the genome, thereby significantly broadening the targeting scope of CRISPR–Cas9 genome editing. This achievement was accomplished through a combination of phage-assisted continuous evolution (PACE) and an innovative selection strategy that enhances DNA binding to specific non-G PAM sequences. The evolved SpCas9 variants were successfully tested in human cells, demonstrating efficient indel formation, base editing capabilities, and the ability to edit previously inaccessible genomic loci [78]. Additionally, the development of xCas9, a SpCas9 variant with expanded PAM compatibility, allows it to recognize a broader range of DNA sequences for genome editing. This variant was created using PACE and demonstrates improved specificity and efficiency in various applications, including targeted transcriptional activation, nuclease-mediated gene disruption, and base editing in human cells. xCas9’s broad PAM compatibility significantly extends the range of genomic targets accessible for editing, without sacrificing DNA specificity. This innovation enhances the versatility and applicability of CRISPR technology in gene editing, with potential implications for research and therapeutic applications [79]. Another engineered SpCas9 variant, SpCas9-NGv1 is able to recognize a broader scope of PAM sequences as well, specifically NG PAMs, enabling more flexible target site selection. Furthermore, when fused with cytidine deaminase, SpCas9-NGv1 supports targeted C-to-T substitutions, demonstrating its utility in precise base editing applications [80].

Practicable Cas9 orthologs

The utilization of SpCas9 would be challenging due to its relatively large size and refined genome editable scope. To overcome these limitations, researchers have explored various Cas9 orthologs garnered from diverse bacterial species, with respective disparate attributes expanding the gene editing toolkit repertoire. The development of these Cas9 variations and orthologs also soundly expedite more precise and efficient genetic modifications. This expanding repertoire advances therapeutic strategies for genetic diseases, offering the potential for more effective and customizable treatments.

In spite of the initial deployment of SpCas9 delivered via adeno-associated virus (AAV) vectors in mammalian systems, Zhang et al. encountered impediments attributed to the considerable size of SpCas9, thereby impeding its further technological applicability in genome editing [81]. Consequently, smaller Cas9 orthologs have surfaced as pragmatic alternatives. Notably, Staphylococcus aureus (SaCas9) has garnered considerable acclaim owing to its efficiency comparable to SpCas9 and a reduced size of one kilobase. Subsequently, SaCas9 was elucidated as possessing the most fitting guide sequence spanning 21 to 23 nucleotides and a highly specific PAM of NNGRRT [82]. Meanwhile, the accuracy and off-target rate were directly measured through the direct in situ breaks labeling, enrichment on streptavidin, and next-generation sequencing (BLESS) system, and the conclusive findings unequivocally demonstrate the eligibility of SaCas9 as a robust and reliable programmable tool. Ulteriorly, the investigators delved into an in-depth examination at 2.6 and 2.7 Å resolutions of the SaCas9 crystal structure. The recognition of Cas9 nuclease relies significantly on the 8 bp PAM proximal RNA–DNA base pairing within the heteroduplex. Additionally, the recognition of sgRNA scaffolds, which exhibit variations in different orthologous cleavage systems, is attributed to the REC lobes and another domain, WED. This analysis served to authenticate and elucidate the dynamic mechanism employed by SaCas9 in recognizing the PAM sequences [83]. Subsequently, Xie et al. employed a dual fluorescence reporter system to evaluate the efficiency of SaCas9 in comparison with canonical SpCas9 and fnCpf1, a member of the Cas12 family. The results demonstrated that SaCas9 exhibited superior cleavage activity over the other two enzymes, both in episomal gene and genome contexts [84].

A comprehensible approach to mitigate off-target mutagenesis induced by RNA-guided nucleases during gene editing is to enhance the stringency of PAM requirements, thereby necessitating more intricate and elongated PAM profiles. Müller et al. conducted the first-ever comparison of RGN specificities across different Cas9 proteins. Utilizing the COSMID tool, researchers identified the top 24 potential off-target sites in silico and analyzed off-target mutagenesis through high-throughput sequencing. These findings emphasize the pivotal role of the PAM sequence as a major determinant of RGN specificity. Alternative Cas9 proteins recognizing more restrictive PAMs demonstrated enhanced specificity, with St1Cas9 and St3Cas9 showing minimal off-target effects compared to conventional SpCas9. While longer PAMs reduce genotoxicity, they also narrow the targeting range. Diversifying the CRISPR–Cas9 toolbox with enzymes from various sources provides tailored solutions for specific genome editing needs, offering flexibility and precision for clinical applications [85].

In addition to the previously mentioned Cas9 orthologs, FnCas9 of Francisella novicida was reported to suppress endogenous transcript profile in 2013 [86]. Additionally, the discovery of ThermoCas9 from Geobacillus thermodenitrificans T12 in 2017 has broadened the applicability of gene editing technologies across diverse temperature ranges [87]. The diminutive CjCas9 from Campylobacter jejuni, notable for its efficacy in gene editing via an all-in-one AAV vector, has shown promise in mitigating choroidal neovascularization [88]. NmCas9, originating from Neisseria meningitidis and distinguishable by its unique PAM sequence (5′-NNNNGATT-3′) recognition, enhances the versatility of genomic editing by targeting a broader array of sequences [89]. ScCas9, originating from Streptococcus canis, stands out due to its 5′-NNG-3′ PAM requirements, which is attributed to unique insertions within ScCas9’s sequence that alter its PAM specificity. The advantages of ScCas9 include its ability to expand the scope of DNA sequences feasible to recognize with high efficiency and specificity in both bacterial and human cells [90]. Furthermore, FrCas9’s specificity for unique PAM sequences facilitates targeted interventions in both prokaryotic and eukaryotic cells, especially for diseases linked to the TATA-box. Its refined sgRNA designs enhance specificity and efficacy, minimizing off-target effects [91].

The evolution and development of CRISPR/Cas9 derivatives

The CRISPR interference (CRISPRi), CRISPR activation (CRISPRa) brought out by dCas9, and the base editor, prime editor evolved from nCas9 are the derivatives of canonical CRISPR/Cas9. By applying the basic biological trait of dCas9, CRISPRi can be easily achieved through transient combination with targeted DNA as alluded to earlier [55]. In addition to its role in gene elimination or suppression, the CRISPR/Cas9 system possesses the capability to prompt gene expression by leveraging the exploitation and advancement of dCas9 mutant [92,93,94,95]. Among CRISPR/Cas9 systems for gene activation, the synergistic activation mediator (SAM) consisting of a VP64/dCas9 fusion protein, syncretic sgRNA sequence-MS2 hairpin, and assistant activating complex MCP-p65-HSF1 demonstrated the highest level of gene expression actuation and genetic versatility. Fang et al. exploited a cloning-free method employing CRISPR/Cas9 activators, specifically dSpCas9VPR and dSaCas9VPR systems, which combine a nuclease-null Cas9 with transcriptional activator domains to induce gene expression efficiently. These activators were tested on various genes in human and rat cells, demonstrating their potential in rapidly initiating gain-of-function studies and their utility across different molecular and cell biology disciplines [96]. The present approach tackles the issue arising in the biofunctional investigation of a specific gene of immense magnitude, the synthesis and subsequent transduction of its artificial plasmid being arduous tasks. Additionally, Wang and colleagues developed a method involving coupling microbial single-strand annealing proteins (SSAPs) with dCas9 to enable the insertion of long DNA sequences into mammalian cells without causing DNA cleavage [97]. This state-of-the-art technique, known as the dCas9–SSAP editor, addresses a critical challenge in genome editing: the introduction of unwanted mutations and off-target effects that are often associated with traditional CRISPR/Cas9 editing techniques which rely on creating double-stranded breaks (DSBs) in DNA [97]. The dCas9–SSAP system is demonstrated to be effective in promoting the knock-in of kilobase-scale sequences with up to approximately 20% efficiency, showcasing robust performance across different genomic targets and cell types. Besides, the dCas9–SSAP editor is less sensitive to the inhibition of DNA repair enzymes than traditional Cas9 editors, potentially offering a safer alternative for genome engineering [97]. In addition to the above CRISPRa system, TET-CRISPRa systems [98,99,100,101,102,103], enCRISPRa [104], and CRISPRa-piggyBac transposon [105]. Li and colleagues described a novel genome editing technique that allows for precise, large-scale deletions in mammalian genomes using dCas9 combined CRISPR/Cas9, which circumvents the limitations of canonical Cas9 in generating precise large fragment deletions [106]. The researchers speculated that dCas9 could effectively control the size of deletions by preventing Cas3 from extending them beyond a specific threshold. This was confirmed experimentally, including the precise elimination of the Y chromosome and retention of the Sry gene in mice, establishing models for Turner syndrome and a specific Sry retention phenotype [106]. dCas9-controlled CRISPR/Cas3 provides a method for establishing animal models through chromosome elimination and a potential strategy for treating diseases caused by fragment mutations or aneuploidy [106]. Additionally, dCas9 has the ability to manipulate DNA methylation in mammalian cells through fusion with Tet1 or Dnmt3a enzymes, facilitating precise modifications of the genome’s methylation patterns, which as a result effectively alters gene expression. Demonstrated across various experiments, including in post-mitotic neurons and during myogenic reprogramming, this method shows potential for broad applications in studying epigenetic regulation’s functional impacts [99]. CRISPRoff, a revolutionary dCas9 fusion protein-based epigenome editing tool was designed to create programmable and heritable transcriptional memory across the genome. It allowed for transient expression to initiate enduring gene silencing, which can be reversed through targeted DNA demethylation for dynamic control over gene expression through cell division and differentiation from stem cells to neurons [107]. The proposed approach demonstrates enhanced durability, heritability, specificity, reversibility, multiplexing capability, broad applicability, and versatility for implementing epigenome editing compared to previous methods. The broad potential of the dCas9-mediated toolkit in modulating the epigenome has been well established, qualifying it for investigating the role of DNA methylation in development, disease, and beyond [108,109,110,111,112,113].

Base editor and prime editor

Although CRISPR technology has shown great potential in gene therapy, its safety for clinical application remains a big concern. Due to the plenty of random insertions and deletions (indels) at the target site induced by the cellular response to DSBs, non-negligible side effects appear while coming into play. To address this issue, new gene editors have been developed that differ from traditional CRISPR-induced DSBs and allow for precise gene editing at specific sites. Researchers combine different-function enzymes with modified Cas proteins to create base editors (BE) and primer editors (PE), both of which can achieve site-specific point mutations without requiring donor DNA or causing DSBs. Base editors consist of a deaminase and a whole or partial catalytically inactive Cas9 protein to achieve directly site-specific point mutations in a programmable manner. They neither require donor DNA nor produce DSBs. BEs work through special deaminase to catalyze appropriate bases for which the exposure of ssDNA in the Cas protein-DNA ‘R-loop’ region provides opportunities [114]. There are two main types of BEs: cytosine base editor (CBE) and adenine base editor (ABE). Besides, fusing extra uracil DNA glycosylase (UDG) to CBE has developed glycosylase base editor (GBE) [115]. Diverse BEs accomplish C·G and T·A bases free interconversion.

In 2016, team David Liu [114] and team Kondo [116] successively reported the initial version of CBE (BE1-3 and Target-AID) utilizing different cytidine deaminases. These two CBEs through adding Uracil DNA glycosylase inhibitor (UGI) to restrain the removal of U in U:G heteroduplex DNA catalyzed by UNG and replacing dCas9 with nCas9 to stimulates mismatch repair (MMR) which preferentially converts the U:G mismatch into U:A successfully achieved some irreversible Cs to Ts conversion in vivo. As the same strategy, team David Liu used an evolved adenine deaminase TadA* completed As to Gs conversion [117]. And since the excision efficiency of inosine is much farther from uracil in mammalian cells, the product of ABEs is much higher in purity. These above original BEs demonstrate their potential in therapeutic applications, but the editing efficiency, product purity, editing window and genome targeting scope still need further improvement. Since then, base editors have been exploited and optimized rapidly in different aspects.

The first major challenge is improving on-target editing efficiency. On one side, adding nuclear-localization sequences (NLS) and preforming codon optimization can ensure the base editor gets well expressed in the nucleus [118, 119]. On the other side, co-expressing free UGI [120] or fusing more UGI to CBE components [121] for reducing the frequency of uracil excision. In addition, potential DNA and RNA off-targets of BEs represent a considerable issue. Similar to traditional CRISPR editing technology, replacing with high-fidelity Cas9 variants [70, 122] can decrease Cas protein-dependent off-target, and change or evolve to higher activity and targeting specific deaminase [123,124,125,126,127,128,129,130] can reduce the Cas-independent off-target. Applying BEs to disease therapy, the optimal result is to induce accurate base-pair conversion at the specific site, but there have been some unwanted non-C-to-T conversions in CBEs [131,132,133]. Hence, the editing fidelity is also a side to advance. Fusing a bacteriophage Mu dsDNA end-binding protein (Gam) to create the new-generation CBE, BE4, with a higher product purity [121] and utilizing the SunTag system to recruit multiple APOBEC1 (BE-PLUS [134]) that greatly reduces undesirable indels. Different therapeutic applications require different BE editing windows, while BE-PLUS expends the base window to 13 positions of protospacer and availably enlarges genome-targeting scope. Another way to approach a broader targeting scope is to increase PAM sequence diversity, such as optimizing with diverse SpCas9 variants [67, 79, 135,136,137] or replacing other Cas-protein [126, 137,138,139,140]. Interestingly, via inlaying [141] or tethering [142, 143] deaminases to the PI domain also expands the editing window. But for SNP mutants’ treatments, minimizing the editing window to prevent bystander editing is extremely important. Protein engineering of the deaminase domain [137, 144, 145] can alter activity window widths and augment editing precision. An additional strategy to adjust the activity range of the BEs window is related to the spatial location of deaminase [146]. Altogether, these evolving base editors lay the foundation for required screening and therapy in cancers.

Missense/nonsense mutations are widely spread over in tumor-associated genes [147, 148]. Harnessing the unique base conversion capability of BEs can reprogram missense mutations and nonsense mutations of ORFs [149, 150]. Therefore, BEs can be used as a strategy to accurately screen [151,152,153,154,155] and model [156, 157] tumor-associated gene mutations for exploiting the pathogenesis of cancer. Especially, base editing mutagenesis can identify gene pathway variants affecting drugs activity in cancer [158]. It is necessary to utilize BEs to investigate drug-protein interaction and perfect the context of oncology for guiding clinical individualized cancer therapy.

Prime editor, a sophisticated gene editing technique, enables precise modifications such as base changes, insertions, and deletions directly within the genome, bypassing the need for DSB and the following HDR [159]. This method is seen as a significant rival to the traditional CRISPR/Cas9 system because it combines the targeting accuracy of CRISPR/Cas9 with the template-based editing capability of reverse transcriptase. Prime editing differentiates itself through the use of specialized prime editing guide RNAs (pegRNAs). These pegRNAs are similar to conventional single guide RNAs (sgRNAs) but are uniquely designed with an additional primer binding site (PBS) and a reverse transcription template sequence at their 3′ end [159]. The prime editor itself is a fusion protein that merges the precision of nCas9 (Cas9-H840A variant) with the functionality of a reverse transcriptase (M-MLV RT), facilitating the direct rewriting of genetic information. PE3 and PE5 represent enhancements of the foundational prime editing technique. PE3 achieves this by creating an additional single-strand nick, located 50 base pairs from the pegRNA-induced nick on the strand opposite to the edit, designed to prevent double-strand breaks and promote the use of the edited strand for DNA repair. On the other hand, PE5 advances the PE3 strategy by integrating a mutated form of the mismatch repair (MMR) protein (MLH1dn), which is functionally impaired, to boost the efficiency of the editing process [160]. Li and colleagues notably increased the base editing efficiency of Prime Editing (PE) in human cells by integrating synonymous mutations at various sites within the pegRNA’s reverse transcription template, creating spegRNA. They also designed apegRNA by adding C/G bases or converting non-C/G to C/G within pegRNA’s secondary structure, which reduced the rate of insertion and deletion edits. By combining spegRNA and apegRNA with PE3 and PE5, they further enhanced editing efficiency and enabled the efficient modification of previously uneditable sites in human cells [161]. Escalating modifications have been made to the PE system in order to enhance its practicality and applicability [162, 163].

In conclusion, the collective efforts in modifying and engineering the CRISPR system have created a harmonious symphony through its improvement endeavors. This includes optimizing sgRNA, crafting virtuoso instruments with Cas9’s variants and orthologs that resonate with precision and availability. These scholarly revelations not only offer hope for refining genome editing but also exemplify the resilience of scientific inquiry in unraveling molecular complexities. The orchestration of engineering SpCas9 within the scientific narrative unfolds as a pivotal chapter, marking significant progress towards achieving precise genomic manipulation (Fig. 2). As research progresses, the discovery and engineering of novel Cas9 proteins and variants are likely to further enhance the precision, efficiency, and flexibility of gene editing technologies.

Fig. 2

Cas9 family and its derivatives. SpCas9, as the central component of the Cas9 family, functions as a nuclease that induces DNA double-strand breaks by the RuvC and HNH domains. Subsequently, the damaged genes are repaired through non-homologous end joining and homologous recombination mechanisms. The orthologs of SpCas9 significantly enhance cleavage coverage and precision. By introducing mutations in the Cas9 protein to generate dCas9 (non-cleaving Cas9) and nCas9 (Cas9 nickase) variants, these defective Cas9 proteins serve as foundational tools for specific gene labeling, epigenetic editing with specific effector domain and two novel approaches: base editor and Prime editor, thereby expanding gene editing capabilities

Exploiting CRISPR/Cas9 in the realm of cancer

Cancer is a category of disease that has historically presented formidable challenges to the medical community [164]. Its therapeutic strategies and practices stand on the cusp of a significant paradigm shift owing to gene editing technology. Heckl et al. pioneered to utilization of the CRISPR/Cas9 genome editing system to surmount the inherent limitations of mouse models, which traditionally struggle to accurately replicate complex mutations found in malignant tumors [165]. This innovation implied that CRISPR was potentialized to make a breakthrough in the establishment of a cancer investigation model. Afterwards, the evolution of cell engineering technologies has facilitated the emergence of ex vivo prepared immune cell infusions as a highly promising avenue for cancer immunotherapy [166]. A venture was undertaken in 2016 with its first application in treating metastatic renal cell carcinoma (NCT02867332). This advancement was notably propelled by a groundbreaking phase I clinical trial in Sichuan, China, where CRISPR/Cas9 edited PD-1 T cells were used to treat advanced non-small-cell lung cancer, symbolizing a significant leap in medical science [167]. Moreover, the advent of chimeric antigen receptors (CARs) has revolutionized the field of cancer immunotherapy, and the integration of CRISPR technology for CAR modification has proven to be a feasible approach [168,169,170,171]. Besides, the clinical application of this technology is extensively employed in the field of cancer diagnosis as well. However, concerns arise when genomic editing is associated with obscure outcomes, as off-target events are typically unavoidable. Furthermore, ethical considerations have been raised regarding the multiplicity of these concerns [172]. In conclusion, CRISPR technology represents a paradigm shift in cancer diagnosis and therapy, offering innovative strategies for early detection, understanding cancer mechanisms, and devising targeted treatments. As research progresses, CRISPR is expected to play an increasingly pivotal role in the fight against cancer, heralding a new era of precision oncology.

Exploiting CRISPR/Cas9 screening in cancer

According to empirical medical theories, the prioritization of prevention precedes all interventions for disease. Evidence suggests that up to half of cancer cases could be averted through the identification and management of known risk factors [173]. The hinge of cancer prevention is implicated by the primary, secondary and tertiary prevention strategies, encompassing risk reduction or elimination, early detection and surveillance, as well as rehabilitation from morbidity [174,175,176]. First and foremost, the primary prevention can be underpinned by CRISPR screening, a high-throughput technique based on ‘loss-of-function’ (CRISPR knocked-out), ‘defective function’ (CRISPR interference) or ‘gain of function’ (CRISPR activation). CRISPR is proven to supersede obsolete siRNA screening and ectopic overexpression plasmid, and play a multifaceted role in biology and cancer exploration [177,178,179,180,181]. By leveraging CRISPR screening technology, it becomes feasible to interrogate potential genomic sites exerting specific biofunction in the oncogenesis, tumor progression and therapeutic resistance, and elucidate their impact on specific phenotypes. For instance, Malina et al. demonstrated the pioneering use of an all-in-one lentiviral CRISPR system linked with GFP to conduct functional in situ assays, specifically targeting the p53 gene as a proof-of-concept for gene disruption and positive selection screening, both in vitro and in vivo. This study highlights the immense potential of CRISPR/Cas9 as a powerful tool for genetic screening and functional genomics [182]. Additionally, genome-scale CRISPR screening clarified NF1, NF2, MED12 and CUL3 as pivotal genes impart melanoma cells resistance to vemurafenib [177]. Additionally, incorporating CRISPR with other novel technologies provides a more profound insight into the screening of cancer. The integration of CRISPR CRISPRi screening with multi-omics approaches has provided an intricate understanding of the posttranscriptional regulatory circuits that promote GBM tumorigenesis and radioresistance. Depleting DARS1-AS1 inhibits GBM cell proliferation, impairs homologous recombination-mediated double-strand break repair, enhances radiosensitivity, and prolongs survival in orthotopic GBM models [181]. The oncologic function of noncoding regulatory elements, apart from genes, can potentially be identified using these methods [183]. The precise determination of genomic domains associated with oncogenesis and oncology can be utilized to excogitate genetically predicting panel-tailored cancer.

It’s well established that many biological factors can engender oncogenesis by various means [184,185,186]. Virus-associated malignancies, such as hepatic cell cancer entangled with hepatitis B virus (HBV) and cervical cancer linked to human papillomavirus (HPV), orientating corresponding viral DNA poses a new prospect for etiologic detection, therapeutic intervention or amelioration of these conditions [187, 188]. Hepatocellular carcinoma is commonly attributed to persistent infection with HBV, a cunning adversary that usually manifests as an occult infection [189]. Therefore, accurate detection of HBV DNA is of importance. Researchers introduced a rapid, portable, and sensitive diagnostic tool for HBV DNA detection, particularly catering to patients with low-level viremia (LLV), using the CRISPR/Cas13a system combined with recombinase-aided amplification (RAA) technology [190]. The persistence of HBV covalently closed circular DNA (cccDNA) is the most intractable issue for the treatment of chronic hepatitis [191]. A primary mathematical model was designed to probe the potential of CRISPR/Cas9 in chronic HBV infection and identify parameters affecting treatment outcomes [192]. Revolving around this theoretical framework, researchers employed the CRISPR/Cas9 system to disrupt HBV DNA and accomplished the eradication of persistent HBV infection both in vivo and ex vivo [193]. The feasibility of using an AAV vector carrying SaCas9 to eliminate HBV in a humanized mice model was demonstrated [194]. Wang et al. developed a novel near-infrared (NIR) responsive system using biomimetic nanoparticles (UCNPs-Cas9@CM) for the targeted delivery of CRISPR/Cas9. This method allows for precise spatiotemporal control over the delivery and release of the Cas9 ribonucleoprotein complex, enhancing its efficiency and specificity for HBV cccDNA disruption [195]. Hepatocellular carcinoma can also arise as a consequence of hepatic C virus (HCV) infection, wherein FnCas9 derived from Francisella novicida was employed for the eradication of HCV [196].

Besides, cervical cancer predominantly arises from persistent HPV infection, leading to subsequent malignant transformation [197, 198]. The underpinning hinges on the integration of the HPV genome, which not only perpetuates tumorigenic factors but also destabilizes the human genome at certain vulnerable hotspots [199,200,201,202]. Moreover, the absence of HPV in the resulting tumor cells plausibly indicated the occurrence of hit-and-run tumorigenesis mechanisms [203]. A tumor model of HPV-driven oropharyngeal cancer was utilized to validate the putative theory through CRISPR deletion and long-term observation of recurrence [204]. In 2017, therefore, Wang et al. spearheaded tapping a multiplex method for the detection of human papillomavirus (HPV) using Cas9 protein targeting L1 and E6/E7 viral genes amplified through PCR (ctPCR) [205]. More methods have been dedicated to elevating sensitivity and specificity [206, 207]. However, type V CRISPR was more potent and prospective in the realm of HPV infection detection [208,209,210,211,212]. In the context of cervical cancer treatment, CRISPR/Cas9 emerges as a more reliable and utilizable tool facilitating extensive application. Cervical cancer is characterized by perpetuated ontogenetic factors, E6 and E7 proteins caused by HPV integration. As early as 2014, several researchers utilized CRISPR/Cas9 to eradicate integrated HPV viral DNA and episomal DNA in cervical cancer [213,214,215]. Additionally, the application of FrCas9 in targeting HPV genomes, has been shown to significantly outperform SpCas9, offering a precise and effective tool for oncological genetic interventions [91]. Furthermore, the synergistic effect of CRISPR/Cas9 and other regimens, such as chemotherapy or immunotherapy combinational treatment was exhibited both in vitro and vivo [216, 217]. Additionally, HPV has been established to be implicated in the pathogenesis of anal carcinoma and head and neck carcinoma [218,219,220]. CRISPR technology showcases promise in reversing these malignancies as well [221, 222]. The subsequent perspective focused on the vehicle employed for delivering CRISPR/Cas9 into the HPV host. The first attempt was adapting high-capacity adenoviral vectors (HCAdV) for CRISPR/Cas9 delivery, presenting a method that improves the efficiency, specificity, and safety of gene editing of HPV18 E6. This system realized fast transfer of Cas9 and gRNA expression units into the HCAdV genome and offers options for constitutive or inducible Cas9 expression and gRNA multiplexing [223]. Then researchers from Columbia University developed a self-assembled micelle optimized for delivering the Cas9 plasmid, which allows for effective disruption of the HPV E7 oncogene [224]. Afterward, researchers manufactured a poly (β-amino ester) (PBAE)-based nanoparticles (NPs) incorporating HPV16 E7-targeting CRISPR/short hairpin RNA (shRNA), aiming to attenuate the expression levels of HPV16 E7 as an initial therapeutic approach for managing HPV infection and associated cervical malignancy [225, 226]. Moreover, endogenous exosomes, PEGylated liposomes, and AAV can also be applied to deliver CRISPR system [227,228,229].

In addition to virus-associated cancers, most malignancies exhibit common features, such as specific gene loss, amplification, or mutations. CRISPR can accurately and promptly identify these abnormalities and further utilize these traits for drug discovery, exploitation and development [230]. Fulco et al. introduced a high-throughput CRISPR interference (CRISPRi) strategy for systematically mapping functional enhancer-promoter connections, focusing on the MYC and GATA1 gene loci in chronic myeloid leukemia K562 cells. By using a pooled CRISPR screen with CRISPRi, which utilizes a KRAB domain fused to dCas9 to modify chromatin state at targeted loci, the study identifies regulatory elements affecting gene expression and cellular proliferation. This approach revealed nine distal enhancers controlling MYC and GATA1 expression and cellular proliferation [231]. For an in-depth understanding of the genome-scale landscape, Replogle and colleagues introduced a genome-scale Perturb-seq technique, leveraging CRISPRi to map functional genomic elements across over 2.5 million human cells. The study’s objective is to utilize transcriptional phenotypes for predicting the functions of poorly characterized genes, thereby revealing new regulators across various cellular processes. This method also motivated an in-depth examination of complex cellular phenomena by identifying genetic drivers and consequences of aneuploidy and discovering stress-specific regulation of the mitochondrial genome, offering a multidimensional portrait of gene and cellular function [232]. By virtue of this technology, investigators parsed out melanoma whole-genome sequencing samples to identify highly recurrent regions (HRRs) associated with enhancers that may play roles in melanoma cell growth. Through genome-scale CRISPRi screening on HRR-associated enhancers, researchers identified 66 significant hits with potential tumor-suppressive roles. These functional enhancers displayed unique mutational patterns independent of classical significantly mutated genes in melanoma, and target gene analysis revealed both known and novel mechanisms underlying melanoma growth. Among the findings, a super enhancer element was shown to modulate melanoma cell proliferation and apoptosis by targeting MEF2A, and another distal enhancer was found to sustain PTEN’s tumor-suppressive potential in melanoma cells. The study establishes a catalog of crucial enhancers and their target genes, shedding light on new mechanisms of dysregulation for melanoma driver genes and suggesting new therapeutic targeting strategies [233]. CRISPRi-based screening enables a detailed analysis of transcriptional, epigenetic, and chromatin conformational networks and the interpretation of noncoding genetic variations’ impact on cells, showcasing its potential to enhance our comprehension of gene regulation mechanisms in cancer development and prognosis [234,235,236,237,238,239]. Besides, CRISPRa is also a feasible tool used to screen essentialities in tumorigenesis, tumoral progression and cancer treatment resistance [240,241,242] (Fig. 3).

Fig. 3

The CRISPR/Cas9 technology was employed to tumor cells screening. By selectively manipulating specific genes or non-coding genetic material in different regions of the entire genome, particularly focusing on the interaction between tumors and drugs, the impact on the phenotype of tumor cells can be comprehensively studied through knock-out (CRISPR/Cas9), knock-down (CRISPR interference), or activation (CRISPR activating)

Furthermore, multiplexed transcriptomic regulation through CRISPR/Cas9 has garnered more and more attention than single genetic manipulation for unraveling tumorigenesis and early diagnosis that is conducive to cancer prophylaxis [243,244,245]. In addition to focusing solely on cancer cells, the screening of the TME can also provide valuable insights into the prognostic direction of these formidable diseases, given their inherent immune evasion characteristics [246].

Utilization of the Cas9 system for cancer modeling investigations

The animal tumor model simulates the occurrence, development, invasion, and metastasis of human tumors in intracorporal ambience, accurately reproducing the biological characteristics of tumors. This provides crucial insights for clinical research on precision treatment [247,248,249]. The utilization of CRISPR/Cas9 technology has significantly streamlined the development process of cancer animal models, resulting in cost reduction and accelerated modeling cycles [2, 35, 250]. Ulteriorly, through incorporating with Cre-loxP system, targeted genes’ editing with spatiotemporal specificity, especially organic specificity in the animal body, was accomplished in the investigational profiles of cancer [251, 252]. As early as 2014, Maddalo and colleagues developed an efficient method to induce specific oncogenic EML4 and ALK chromosomal rearrangements in vivo using CRISPR/Cas9 technology delivered via Adv vectors to somatic cells of adult animals. The resulting tumors mimicked the histopathological and molecular characteristics of human ALK1-positive non-small cell lung cancers (NSCLCs), demonstrating the model’s validity [253]. Then, by comparing the recognition scope and precision of editing in various Cas9 variants, Robertson et al. found that the SaCas9 can be identified as alternative for null lethal phenotype in mouse model [254]. In 2017 the VEGFA in retinal pigment epithelium cells was knocked out by SpCas9 enveloped by the lentivirus shed light on the in vivo gene editing in the central nerve system [255]. In 2018, therapeutic genome editing for Myotonic Dystrophy Type 1 (DM1) using CRISPR/Cas9 was conducted. The research focused on two main strategies: targeted deletion of expanded CTG repeats and targeted insertion of polyadenylation signals upstream of these repeats to eliminate toxic RNA CUG repeats. As a result, the latter spurred a phenotype reversal in pathologic cells [256]. This study clarified the anfractuosity and sophistication in the factual implementation of manipulation of the human genome. In the context of drug metabolism and pharmacokinetics (DMPK), CRISPR/Cas9 is qualified to facilitate the creation of rat models with targeted gene modifications such as knockouts of Cyp, Abcb1, and Oatp1b2. These models are instrumental in researching drug metabolism, chemical toxicity, carcinogenicity, and understanding the mechanisms related to DMPK [257]. The CRISPR/Cas9 system can also efficiently induce tumor formation, resulting in the generation of spontaneous tumor models that exhibit more consistent and reliable characteristics. This provides a superior platform for cancer treatment and drug development [258]. In addition to the canonical CRISPR system, as alluded earlier, BEs are qualified as useful tools to construct appropriate models for cancer essentialities identification and therapeutic effect inspection. Base editing of oncogenic KRAS and TP53 mutations was successfully achieved in patient-derived cancer organoids, indicating the potential development of base editor approaches for precise investigation of cancerous susceptibilities in a personalized manner [156]. Another tumoral organoid molding was crafted from human adipose stem cells which are subsequently engineered by the BE system. Cytosine and adenine BEs in introducing CTNNB1 mutations in liver tissue organoids. Furthermore, investigators utilized C>T base editing to induce stop mutations in the PTEN gene within endometrial organoids, revealing tumorigenic potential even in a heterozygous state. Drug sensitivity assays conducted on organoids with PTEN or combined PTEN and PIK3CA mutations elucidate the foundational mechanisms of endometrial tumorigenesis. Moreover, researchers demonstrated that multiplexing BEs enabled the modeling of colorectal tumorigenesis by targeting five cancer genes simultaneously, complementing the vacancy of complex tumor models [157]. These preliminary studies underscore the potential of BE as a feasible and effective strategic paradigm for cancer investigational modeling. However, while the editable genome spectrum of BE technology remains unsatisfactory, PE technology has emerged as a prominent tool for oncologists due to its enhanced accessibility to human and mouse genomes. In 2023, Tyler Jacks’s team from MIT devised a Cre inducible prime editor-based approach that facilitates the targeted design of specific cancer-associated gene mutations in mouse models. Utilizing this technique, the team successfully generated models harboring diverse oncogene K-Ras mutations across various organs. Furthermore, this method exhibits versatility in modeling nearly any other type of cancer-related genetic mutation, thereby aiding researchers in identifying and evaluating novel drugs targeting these mutations [259].

Gene editing employed by CRISPR/Cas9 targeting subsistent cancers

In certain scenarios, the eradication of pathogenesis-facilitating cancer prevention is tantamount to intervening in subsistent cancer, particularly when dealing with virus-associated cancers, as elaborately elucidated in the aforementioned sections. In addition to directly targeting cancer cells, engineering the components of the TME through CRISPR/Cas9 provides a promising approach to circumvent tumor invasion. Nonetheless, how to deliver the CRISPR/Cas9 system accurately into the targeted locus is the next issue warranted to embark on. Different vehicles have been developed to deliver CRISPR/Cas9 components monolithically or separately into target cells, such as electroporation, recombinant adenoviral vectors, endogenous exosomes, nanoparticles, PEGylated liposomes, AAV, virus-like particles (VSV-G-enveloped vesicles) [260], transient non-integrating retrovirus-based CRISPR/Cas9 all-in-one particles [261], and cell‐penetrating peptides [262] (Table 1). These delivery strategies exhibit respective peculiarities, yet also possess inherent limitations. Viral vectors, such as lentiviruses, adenoviruses (Advs), and adeno-associated viruses (AAVs), are efficient in delivering CRISPR/Cas9 components into cells. They can integrate into the host genome (lentiviruses) or maintain episomal forms (Advs and AAVs), ensuring sustained expression of CRISPR components. Different serotypes of AAV vectors characterized by distinct tissue-chemotaxes can also transmit CRISPR system to targeted tissues [263, 264]. However, Immunogenicity is a major concern, as viral vectors can trigger immune responses in patients [265, 266]. The integration of lentiviral vectors into the human chromosome may pose uncontrolled risks, despite ongoing efforts to avert their chromosomal integration [267]. Besides, in many circumstances, the package gauge of AAVs encumber their further applications [268]. As for nanoparticles, they offer non-viral delivery options, potentially reducing immunogenicity compared to viral vectors. Through specifical amelioration, they can encapsulate CRISPR/Cas9 components and deliver them specifically to target cells [269, 270]. Nevertheless, efficient delivery across biological barriers, such as the blood–brain barrier or solid tumors, remains a challenge [271, 272]. Ensuring sufficient uptake by target cells without inducing cytotoxicity or off-target effects is another hurdle [273, 274]. Electroporation is validated to direct the delivery of CRISPR/Cas9 components into cells using electrical pulses, particularly for ex vivo applications. The issue is warranted to address as this method cannot be used in the whole entity thwarting its further clinical employment [275]. Furthermore, techniques such as microinjection or cell-penetrating peptides are currently being investigated for localized delivery with somewhat advantages over traditional approaches; however, they possess certain technical limitations and are still in the early stages of development [276,277,278,279,280].

Table 1 Delivery strategies of CRISPR/Cas9 systems for cancer therapy

Recently, investigators introduced a novel biomimetic mineralized CRISPR/Cas9 RNA validated for efficient tumor-specific multiplex gene editing, which utilizes magnesium pyrophosphate biomimetic mineralization to co-encapsulate Cas9 mRNA and multiple sgRNAs within a single nanoparticle, enabling precise control over the co-encapsulation ratio [281]. Harnessing a sgRNA and a catalytically inactivated CjCas9-merged adenine base editor (CjABE), researchers successfully corrected the − 124C>T mutation in the TERT promoter back to − 124C. This alteration hindered the E26 transcription factor family’s attachment to the TERT promoter, diminishing TERT transcription and protein levels, thereby prompting cancer cell senescence and halting proliferation. Further, local administration of AAVs encoding sgRNA-directed CjABE curtailed glioma growth in models with TERT-promoter mutations [282]. The use of AAV with CjABC targeting TERT promoter in liver cancer has also demonstrated significant anti-cancer effects [283]. Moreover, AAV6 is proven to efficiently deliver templated DNA donors into human pluripotent stem cells (hPSCs) employing CRISPR–Cas9 ribonucleoproteins (RNPs), enabling the introduction of precise modifications, from single-base changes to insertions larger than 3 kb. This approach avoids the use of antibiotic selection markers or fluorescent tags, which are commonly employed in other techniques but can potentially interfere with gene function and are not suitable for clinical applications [284]. It is well known that induced hPSC is an important source for the engineered chimeric antigen receptor (CAR)-immune cells [285,286,287,288].

Regarding CAR regimen, it signifies a groundbreaking advancement in cancer treatment, particularly by orchestrating the interaction between artificially engineered immune cells and tumor cells. Unprecedentedly, it offers enhanced therapeutic durability, adaptability, and customization, and is distinguished for circumventing cancer’s immune evasion strategies [289,290,291,292]. Unlike the traditional method of randomly integrating engineered CAR sequences into the genome of targeted cell, which is fraught with the risk of compromising genomic integrity [293,294,295], CRISPR/Cas9 validates a more tailored and precise genomic positioning of CAR within immune cells sourced from disparate origins. Eyquem et al. utilized the HDR feature of the CRISPR/Cas9 tool to achieve targeted gene insertion of large DNA sequences, successfully integrating a CD-19 specific CAR into the TRAC locus, which encodes the T-cell receptor (TCR) α chain [169]. This precision enhances CAR expression uniformity, improves T cell potency, and may reduce risks associated with random integration methods, thereby optimizing the effectiveness and safety of CAR-T therapies. Subsequently, Zhang et al. directed a non-viral CAR DNA cassette, comprising the CD19 antigen recognition domain and the 4-1BB-CD3ζ signaling domain, to the AAVS1 site via CRISPR/Cas9. This endowed the resultant CAR-T cells with cell proliferation and tumor-killing characteristics similar to those of CAR-T cells produced using lentiviruses. Furthermore, targeting the same cassette to the PD-1 site via CRISPR/Cas9 enhanced the CAR-T cells’ proliferation traits and their efficacy in suppressing tumor growth [296]. Subsequently, the researchers conducted a clinical trial to assess the safety and effectiveness of CRISPR/Cas9-targeted PD1 CAR-T cells in patients with relapsed or refractory B-cell non-Hodgkin lymphoma. Researchers employed ribonucleoprotein complexes and a non-viral CAR DNA template to electroporate T cells derived from eligible patients. The outcomes indicated that the refined CAR-T cells not only averted to provoke severe adverse events but also successfully induced complete remission in two representative patients [296, 297]. More delving was conducted to enhance the HRD rate and improve the yield of CAR cells by utilizing a modified ssDNA HDR template [298] (Fig. 4).

Fig. 4

Refining the process flow for CAR-T cell therapy development via CRISPR/Cas9-HDR. The development of CAR-T cell therapies using CRISPR/Cas9-HDR technology begins with identifying specific genomic sites in immune cells for the precise insertion of the CAR gene, aimed at targeting and destroying tumor cells. Following site identification, the CAR sequence is designed and synthesized. Concurrently, the CRISPR/Cas9 system is prepared and introduced into T cells via electroporation, allowing the entry of this genome-editing tool. The CAR sequence is then incorporated into the T cells using either Adeno-Associated Virus (AAV) vectors or non-viral methods, followed by precise insertion at the target site through homology-directed repair (HDR). This ensures the CAR gene integrates accurately within the genome, prompting cells to express the CAR protein. Post-integration, a sorting and purification process selects cells with correct CAR incorporation, resulting in a pure batch of CAR-immune cells. These engineered cells are designed to specifically recognize and eliminate cancer cells, offering a powerful treatment option for both hematological malignancies and solid tumors. By focusing on cancer cells, this approach minimizes harm to healthy tissues, marking a significant advancement over traditional cancer treatments

Additionally, CRISPR/Cas9 is conducive to recognizing potential factors or directly reprogramming the genome of immune cells to enhance the cytotoxicity of CAR transfusion towards cancer cells [299, 300]. Nevertheless, the efficacy of the CAR regimen may be compromised by concomitant immune responses in the patient [301]. In such circumstances, BEs provide novel and secure strategies to effectively tackle this issue. In 2019, Webber et al. demonstrated that base editors could induce multiplex human T cell engineering in support of an allogeneic CAR-T platform [302]. Subsequently, Georgiadis et al. obtained fratricide-resistant T cells by orderly removing TCR/CD3 and CD7 with CBE [303] and Diorio et al. developed 7CAR8, a quadruple-edited allogeneic CART cells [304]. In 2023, three children with relapsed T-cell acute lymphoblastic leukemia (T-ALL) in Great Ormond Street Hospital were treated with base-edited CAR7 therapy which showed potent activity in leukemia remission [305]. The saliently remarkable progress signifies the indispensable application potential of BEs in immunotherapy, thus generating great excitement.

In limited instances, organ transplantation represents one of the limited therapeutic options available for managing end-stage tumors [306,307,308]. Nevertheless, organ transplantation is positioned unsubstitutably in multitudes of end-stage diseases. Despite the potential of xenotransplantation to offer novel insights into refractory diseases in humans, the challenge of rejection due to interspecies heterogeneity hinders progress in this field [309]. The utilization of CRISPR/Cas9 in the nematode Caenorhabditis elegans for genetic engineering laid the groundwork for manipulating genetic material in more complex organisms [310]. The porcine derivate enjoys exceptional advantages. Specific genes, such as a gene expressing galactose-alpha-1,3-galactose, which are uniquely expressed in pigs, have presented challenges to the success of xenografts derived from porcine sources [311]. The utility of CRISPR/Cas9 in the field of organ transplantation is justified for these targeted gene knockout [312]. Until 2020, United Therapeutics corporation had utilized genetically modified s lacking these specific genes surmounting the obstruct, with the innovation approved by the FDA [313]. Following this, Montgomery et al. pioneered a groundbreaking initiative that translated the kidneys of the transgenic pig into two brain-dead, observing urine outputted and no obvious rejection reaction [314]. Reinforced evidence from escalating cases validates the application of CRISPR/Cas9-mediated bioengineered xenotransplantation [315,316,317]. Recently, clinicians from China have successfully performed an engineered pig-to-human liver transplantation surgery on a deceased patient, which demonstrated no signs of rejection reaction and maintained normal bile secretion for 96 h [318]. Another modality applying CRISPR/Cas9 in the context of cancer is to wake dormant tumor suppressor genes up [319]. Through upregulating MASPIN in lung cancer and REPRIMO in gastric cancer, impaired proliferation and phenotypic reprogramming induction has been achieved.

However, THERE’S no such thing as perfect. The hindrance to clinical application arose due to the immunocompetence of both the delivery vector and Cas9 itself. Specifically, T cell response to SpCas9 could impact the efficiency and safety of CRISPR-based therapies. Among all kinds of T cells, the presence of regulatory T cells (Tregs) within the reactive T cell population is emphasized to possess a potential mechanism to mitigate adverse immune responses against SpCas9, thereby influencing its therapeutic application and tolerance in gene editing procedures [320]. The clinical applications of CRISPR/Cas9 upon cancer treatment have witnessed intensified efforts (Table 2).

Table 2 The ongoing clinical trials on CRISPR/Cas9 in cancer therapy

Ethical implications of gene therapy using CRISPR/Cas9

CRISPR technology offers new hope for genetic diseases, tumors, or diseases with no fundamental cure, but the clinical application of gene editing is accompanied by a series of ethical considerations. Up to now, the medical community has been cautious about heritable (germline) gene therapy, in most countries, germline modification is illicit [321]. However, modifying nonheritable (somatic) genome within a favorable benefit-to-risk ratio, especially in cancer, is ethically acceptable. Thus, the application of current ethical and regulatory standards mainly focuses on somatic gene editing [322, 323].

At present, the ethical controversy of cancer gene therapy mainly includes the following aspects. One of the primary concerns is the uncertainty of gene therapy. The selection of in vivo delivery vectors, target cells and target genes, gene modification efficiency and potential off-target effects all affect the safety of tumor gene therapy [324]. Moreover, the long-term effects of such unintended modification are still uncertain. The second concern is the underlying problem of genetic inequality. Advanced gene therapy (such as CAR-T) is expensive, and only benefits minorities, which exacerbates the unequal distribution of medical resources [325]. At the same time, there is uncertainty about whether such therapeutic effectiveness feedback matches the socioeconomic input. The third point is that CRISPR technology not only affects human genes, but may also have an impact on natural evolution [326]. On the one hand, its modification of tumor genes is limited and cannot eliminate all defective genes. While it can help a patient to extend their life span, there is the risk of defective genes inherited by offspring, which may increase the harmful genes in the human gene pool. On the other hand, widespread use of gene editing may lead to a reduction in genetic diversity, which might have unforeseen consequences on the resilience of human populations to future diseases or environmental changes. Both are likely to have unforeseen consequences on future ecosystems.

In addition to the parts that remain controversial, tumor gene therapy should also follow current ethical principles: respect for patient autonomy, beneficence, nonmaleficence, and justice [327]. Ensuring that patients fully understand the risks and benefits of CRISPR/Cas9 therapies is crucial. The treatment process should avoid as much as possible to the patient’s physical and mental harm. All aspects of the treatment process should protect the privacy and security of the patient’s genetic information. The rapid advancement of CRISPR/Cas9 technology requires interdisciplinary discussion and regulation, involving scientists, ethicists, jurists, and social scientists. Ensuring that clinic trials and treatments are conducted ethically and safely.

Continued ethical scrutiny and regulation are essential for CRISPR/Cas9 to strike a balance between potential benefits and risks in cancer research and treatment.

Conclusions and perspectives

The CRISPR/Cas9 system, a cornerstone of modern genetic engineering, has ushered in a new era of precision in gene editing, marking a seminal advancement in the biosciences. Its versatility extends from unraveling fundamental biological mechanisms to pioneering therapeutic interventions for complex diseases, notably cancer. Through the strategic engineering of pluripotent variants, the exploration of Cas9 orthologs, and the development of next-generation Cas9 derivatives, CRISPR/Cas9 has transcended previous technological limitations, expanding the horizons of genetic and epigenetic research, and clinical applications. In the domain of oncology, CRISPR/Cas9 has illuminated paths toward understanding the molecular intricacies of cancer, fostering the development of novel diagnostics [328] and targeted therapies. Its role in reprogramming immune cells, exemplified by advancements in CAR-T cell therapy, underscores its potential to revolutionize cancer immunotherapy, enabling the precise modulation of immune responses against malignancies. Additionally, the application of gene-engineered xenotransplantation has heralded the rise of cloning technology to develop nonantigenic biological organ substitutes, beneficial for the treatment of peculiar cancers. Looking forward, the amalgamation of CRISPR/Cas9 with emerging technologies like single-cell sequencing and artificial intelligence holds the promise of enhancing our comprehension of cancer at an unparalleled resolution. This synergy is poised to facilitate the discovery of novel therapeutic targets, the tailoring of personalized treatment strategies, and the advancement of precision oncology. Moreover, ongoing efforts to refine CRISPR/Cas9’s specificity and genome-scale accessibility, minimize off-target events, reshape the current identified PAM sequence, and improve delivery efficiency are critical for optimizing its applicable profile. However, the journey towards the clinical translation of CRISPR/Cas9-based therapies is not without challenges. Addressing immunogenicity concerns, ensuring ethical considerations, and navigating regulatory landscapes are imperative to harness the full potential of this technology in clinical settings. The quest to mitigate these hurdles is vital for realizing the transformative impact of CRISPR/Cas9 on various intractable diseases, especially on cancer therapy.

In summary, the CRISPR/Cas9 technology stands at the forefront of a scientific revolution, offering new avenues for research and therapeutic interventions previously deemed unattainable. Its continued evolution and integration with other scientific breakthroughs herald a future where genetic engineering not only demystifies the complexities of life but also empowers humanity in its enduring battle against cancer. As we navigate the ethical, regulatory, and scientific complexities of its application, the promise of CRISPR/Cas9 in crafting personalized, effective, and safer treatments for cancer patients shines as a beacon of hope in the ongoing quest to conquer this formidable disease (Fig. 5).

Fig. 5

Harnessing CRISPR/Cas9 system in cancer investigations and treatment. Commencing from the fundamental principle of double-stranded DNA cleavage, this paper provides a comprehensive overview of diverse CRISPR/Cas9 variants and homologs, alongside their engineered derivatives. Equipped with these gene-editing tools, scientists and clinicians are propelling a new era of gene therapy in the domains encompassing tumor identification, modeling, screening, and treatment