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Abstract

Cancer is one of the most threatening complex heterogenous diseases. It has drastically impacted both individuals and communities, leading to challenges in biomedicine. To prevent the permanent increase in incidences, there is an urgent demand for new therapeutic approaches. Genome Editing (GE) refers to specific-modifications of the DNA sequences of a living cell. The traditional GE strategies: Meganucleases, Zinc Finger Nucleases, Transcription Activator-Like Effector Nucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated protein 9 (Cas9). They are based on double-strand breaks (DSBs)-triggered in cell repair mechanisms. The emergence of non-DSB editor variants such as Base Editors and Prime Editors in the platform-based CRISPR system has propelled GE to a new phase of efficiency and accessibility. The novel developments enable site-specific recognition of specific nucleic acid sequences without DSB introduction. The likelihood of precisely modifying DNA sequences can also play an important role in the advance of developed biological computing systems. In medicine, GE is a potential target for preventing cancer, and if develops, finding new possibilities for a long-lasting treatment or even a permanent solution. Integration of bioinformatics, computational models, RNA sequencing, and CRISPR technology have deepened our understanding of genetically engineered T-cells and microRNA-based cancer therapies. Currently, a growing number of clinical trials utilizing GE for therapeutic purposes are in progress. The subject is complex, but expressed in an easy and concise way that could be of interest to diverse dedicated readers. A brief, but comprehensive, overview of the available literature has been examined to provide the present state of GE and the most relevant technologies that multilaterally contribute to cancer science, cancer therapy, and ongoing clinical trials. The overview addresses cell delivery systems, bioethical considerations, challenges, and the necessity for collaboration among scientists, regulators, and patient advocates for effective combating of cancer and meeting clinical needs.

Keywords

CRISPR-Cas systems, cancer therapy, CAR-T cells, immunotherapy, microRNAs

Briefs of gene editing technologies: Gene Editing (GE) refers to the ability to add, modify or remove DNA sequences. Much progress has been made in GE technology over the past fe

Introduction

w decades (Figure 1). The details regarding the principles and the applications of these GE techniques in various fields of life sciences have been the subject of several recent reviews [1-7]. Therefore, only brief descriptions of GE approaches will be given herein. 

On historical and sophistication bases, scientist group GE approaches into classical and modern techniques (Figure 2). The proteins involved have diverse properties, allowing possible matching of them with specific applications. The targeted approaches for the classical GE depend on site-specific nucleases that typically produce Double Strand Breaks (DSBs) into specific DNA loci. The principle of DSB-mediated GE, simple gene knock-out /knock-in (KO/KI), is achieved by introducing single DSB and various chromosomal engineering by inducing multiple DSBs (on the same chromosome or the different chromosomes). Double-strand breaks can be repaired via one of two mechanisms; either homologous recombination (HR) or by the error-prone non-homologous end joining (NHEJ) [2]. These repair mechanisms result in targeted integration or disruption of genes, depending on the pathway used. The GE tools have allowed scientists to make irreversible permanent alterations of their choice in the genetic material at preselected sequences. However, random integration is the major problem of the classical strategies with multitalented applications medicine.

Although hundreds of naturally occurring MegNs exist, their fixed recognition sequence radically limited the select of targetable sites. Subsequent re-engineering of the DNA recognition locus led to more flexibility and allowed site-directed genome reformation in mammalian cells [9]. Yet, reconfiguring the structure of a whole protein for every single target site is tedious.

Several types of endogenous gene modifications in a broad range of organisms and cell types can be launched with ZFNs [2]; hence, providing scientists with unprecedented techniques to make genetic manipulations. In addition, ZFNs can potentially be applied in therapeutic purposes. However, the wide application of ZFNs is hampered by the time-consuming and complicated process involved because a specific editing protein is needed for each version of GE [10].

Figure 1. Timeline of the key improvements of precise genome-editing (GE) technologies. Meganucleases; ZFNs: Zinc-Finger Nucleases; TALENs: Transcription Activator-Like Effector Nucleases; CRISPR-Cas: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein; HITI: Homology-Independent Targeted Integration; CAST: CRISPR-Associated Transposase

Similar to ZFNs, TALENs are FokI-based (Figure 2). They are used in pairs, binding in opposite locations such that the catalytic domain of the restriction endonuclease FokI domain can dimerize and cut the DNA inside the spacer between the two TALEN attachment site. The ideal spacer length strongly depends on the length of the selected C-terminal TALE domain. They were easier to apply than their predecessors (ZFNs and MegNs) and, consequently, brought GE into reach of the broad scientific community, and ignited the GE revolution. Important breakthrough achievements of GE have since been accomplished with TALEN, among these, the first human cured from cancer [11]. Although, TALENs have been mostly replaced by the CRISPR tools, which are somewhat simpler to build, much comfortable to multiplex, and have brought multiple derived techniques, their flexible and precise settings of TALEN are unmatched, and so they have continued to grow to new functionalities. The key problem that banned the expansion of a TALE till then was that the usual CRISPR Base editors (BEs) utilize a deaminase which only acts on single-stranded DNA (SSDNA) [12]. While the unwinding of double-stranded DNA (DSDNA) is readily made by the Cas9 protein, TALEs do not unwind DNA, and thus they are not suitable for a fusion to such deaminases.

Figure 2. Schematic diagram of molecular principles of gene editing tools. Meganucleases (MegNs): Generally, target DNA as homo-dimer without having precise DNA binding and cleavage domains. Zinc finger nucleases (ZFNs): Recognize target sites, each is composed of two zinc finger monomers that flank a short spacer sequence recognized by the FokI cleavage domain. Transcription activator-like effector nucleases (TALENs): Have similar domains in it like ZFNs consisting of two monomers.  Classical Clustered regularly interspaced short palindromic repeats (Classical CRISPR): Made of a Cas9 nuclease, a protein which consists of two functional domains: The human umbilical vein endothelium cells (HuvC) a split nuclease and the His-Asn-His (HNH). The HNH and Ruvc cut one strand of target DNA and form a DSB. The Cas nucleases that bind to DNA depend on single guide RNA (sgRNA) and are not related to their activities. A single guide RNA (sgRNA), which is located upstream to PAM (Proto spacer-adjacent-motifs), guides Cas9 to select DNA sequences that are relatively complementary to the target. Two principal repair mechanisms are possible: Homologous recombination (HR) and nonhomologous end joining (NHEJ). The latter frequently creates small insertions and/or deletions (indels) that result in frameshift mutations and disturb the function of genes. Modern CRISPR: Includes Cas9-based base editors (BEs) and prime editors (PEs). The BEs and PEs, Bes is composed of a Cas9 nickase domain fused to a reverse transcriptase (RT). Compared to the classical Cas nucleases, BEs and Pes have unique advantages: They do not need DSBs or donor templates of DNA for repair. Therefore, they offer high editing purity and target specificity. Adapted from Khalil [2] and [8]

The wide application of ZFNs and TALENs is hindered by the slow and laborious process involved, because a specific editing protein is needed for each version of GE [13]. Like the principle of the older GE techniques, the classical CRISPR/Cas9 action is DSB-mediated, where the two repair pathways of two DSBs on the same chromosome would result in large deletion or inversion. The CRISPR-Cas system, driven by RNA–DNA base pairing, avoids protein–DNA interactions, and offers numerous advances over ZFNs and TALENs. These advantages include simpler design for any DNA target, lower cost, higher efficiency, limited off-target sites, and the capacity of changing different genomic sites at the same time by providing multiple gRNAs. The CRISPR/Cas system is commonly recognized as a powerful GE strategy, offering promising scenarios for cancer treatment [2,4]. The last decade of CRISPR GE has climaxed in the advance of a multitude of therapeutic strategies for cancer, several of which have shifted from preclinical studies in cell-based and animal models into human clinical trials. These include both in vivo and ex vivo therapeutic correction approaches. In vivo therapeutic amendment methods involve delivery of GE elements to the affected tissues inside the human body [13]. In contrast to in vivo methods, ex vivo techniques involve taking cells from a patient, engineering them in a laboratory, followed by transferring modified cells back into the patient.

Based on the CRISPR/Cas9 strategy, a HITI tool was devised, which owing to its cell cycle independent integration, could develop the CRISPR KI toolbox for somatic cell and gene therapy [14]. Because HR is not the principal DNA repair pathway used following DSBs, NHEJ-mediated HITI has been investigated for large transgene insertions in both dividing and resting cells. HITI can repair DSB in the presence of donor template containing the same nuclease cleavage site as the target sequence [5]. Furthermore, the HITI approach achieved precise integration, efficient transcription and translation both in vitro and in vivo [15]. 

The design of unique nucleases, that can exclusively recognize and cleave DNA targets, constitutes a genuine breakthrough in GE. CRISPR/Cas9 is a multipurpose, simple, and cost-effective revolutionary gene-manipulating approach that aided researchers to explore the potential of GE in a broad application range. However, several limitations are associated with conventional CRISPR/Cas9 techniques [16,17]. With such limitations, particularly being time-consuming, the designed construct needs the delivery of a long DNA template and being ineffective in various mammalian cells.

Furthermore, the CRISPR-Cas system depends on induced DSBs, with editing efficiency often restricted by factors such as cell type and HRs, impeding further improvements [18]. The greatest limitation of the Cas9-based GE is the formation of high off-target DSBs in the genome which often lead to toxicity or lethality due to the introduction of undesirable byproducts of chromosomal breaks in undesired regions [2,19]. Although deactivated or dead Cas (dCas) is not involved in any therapeutic product that is now under improvement, it offers a tool of academic interest allowing for the screening of signaling pathways and aiding further the perception of human disease biology. In addition, novel Cas proteins are being assessed that give differentiated editing action over the Streptococcus pyogenes Cas9, such as the ones that specifically enable the editing of RNA instead of DNA [20].

To overcome these liabilities, Cas9-based BEs and Prime Editors (PEs) were developed [21]. In addition to these editors, Cas9 itself has been mutated to generate an enzymatically dCas endonuclease which can inactivate HNH and Ruvc domains to form a dCas and Cas nikase (nCas) [22] Though dCas lacks endonuclease activity, yet it maintains the ability to specifically discover and bind onto a target DNA sequence. All Cas proteins like dCas or nCas can be fused with other molecules such as enzymes without having any influence on their own binding and cleave functions [23]. Using dCas9 or nCas9 fused with deaminase enzymes, it was possible to construct the direct conversion of one base to another depending on the base-excision repair system (Figure 2).

The BE technique is a CRISPR-Cas9-based GE technology that enables the induction of point mutations in the DNA without the need for DSBs generation. Currently, three main classes of BEs (Figure 2) have been established: the Adenine BEs (ABEs) [12], the cytidine BEs (CBEs) [24], and glycosylase base editors (GBE) [25] enable C-to-T, A-to-G and C-to-G base changes, respectively. It is possible to effectively convert cytosine (C) into uracil (U), which can then become thymine (T). Base editing (ABE and CBE) can replace a specific base for another, enabling the conversion of all four types of nucleotides. CRISPR-Cas12a identifies T-rich PAM, which can further increaser the BE range. However, the absence of an effective Cas12a nickase presents a confront in the development of effective Cas12a-based BEs, necessitating the use of a dCas12a [26].

The DNA BEs has proved a high on-target activity and have potential applications in treating genetic diseases associated with point mutation [23]. The BE technology is a promising CRISPR-based therapeutic strategy for genetic diseases including cancer. However, they still have some drawbacks regarding base transversions and the editing window. Additionally, such methods are unsuitable for establishing insertions and deletions (indel) mutations. The scope of BE tools has been extensively expanded, allowing greater efficacy, specificity, accessibility to previously inaccessible genetic loci and multiplexing, while maintaining a small rate of indels [27].

Another cutting-edge CRISPR- based GE procedure is the PE approach (Figure 2), where two changes to the traditional CRISPR/Cas system have been added [3,21]. Firstly, the dCas or nCas is fused with other molecules such as enzymes (e. g. reverse transcriptase, RT) [23]. Secondly, the traditional sgRNA is replaced with PE sgRNA guide RNA (pegRNA). The latter locates the desired sequence of the genome and includes the necessary information to encode the wanted sequence using a RT. The pegRNA not only contains sgRNA [contain both spacer and tracrRNA] but also sgRNA which is coupled with a gene specific RNA sequence (containing a primer binding site, PBS). The PE methodology is more versatile than BE because it allows all possible 12 base substitutions as well as modeling small indels, expanding the editing possibilities.

Prime editing offers a solution to the limitations associated with CRISPR, enabling accurate DNA modifications without needing a DNA template and the presence DSBs [28,29]. This substitutes for HR factors which are only present during cell division, making the classical CRISPR/Cas system inefficient in non-dividing cells that make up most of the tissues of the body [19,30].

The start of PE represented a significant leap forward in the GE field, providing a solution to the limitations associated with the use of CRISPR, allowing precise DNA modifications without DSBs [29]. This breakthrough laid a solid basis for the clinical application of GE technologies. While PE allowed precise sequence modifications in DNA, cellular determinants of PE remained poorly understood, and as with other CRISPR techniques, there was still necessity for optimization and development at both pegRNA and enzymatic levels [31].

Numerous GE techniques have been developed to target and modify genomic sequences. The Cas9-based CRISPR-Cas systems. The BEs and PEs, and the modern CRISPR/Cas systems include CRISPRa/CRISPRi and RNA editing [32]. The first CRISPR variant is a catalytically deactivated Cas9 modified to inducible activate/repress gene expression. The second version contains RNA-editing enzymes (Cas13 and Cas7-11) and is currently being improved for diagnostic and therapeutic purposes.

Using pooled CRISPRi screens, it was discovered that DNA mismatch repair (MMR) hampers PE and enhances undesired indel byproducts [33]. These workers developed PE4 and PE5 systems in which transient expression of an engineered MMR-inhibiting protein increases the efficiency of small indel and substitution mutations up to 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR competent cell types. To further expand the efficacy, several modified versions of PE system have been advanced. Bi-directional PE (Bi-PE), for instance, utilizes two PE gRNAs allowing for broad and improved editing proficiency.

Because of the superior specificity and few or no unintended side effects of CRISPR/Cas9 system relative to other editing strategies, it has changed the limits of biomedical research and human clinical medicine [6,34-36]. 

An overview of cancer biology

Cancer is the second leading cause of death worldwide, claiming millions of lives and exacting a huge toll on human health and community welfare [37]. A recent report has projected that the number of new prostate cancer cases annually will rise from 1.4 million in 2020 to 2.9 million by 2040 [38]. There's also an enormous financial cost that's growing fast. In 2020, treating cancer in the U.S. cost around $200 billion. By 2030, the total is expected to increase to beyond $245 billion. Consequently, finding an urgent and effective treatment has attracted the attention of researchers all over the world.

Despite promising discoveries, no powerful treatment has been found yet due to poor clinical response frequencies and treatment-related toxicities. No drugs directly targeting cancer cells have been accepted yet, but the journey is ongoing. Recently, more targeted, and successful therapies have been advanced, causing a complete paradigm change in the treatment of cancer. Combination of modern gene therapeutic strategies with conventional treatments are now in clinical trials [39]. 

Cancer therapy resistance: Development of resistance is a prime limitation to the long-term efficiency of cancer therapies and anti-cancer treatments continues to be a principal clinical challenge [40,41]. Cancer cells can develop resistance to the mechanisms of the immune system, thus may escape control process comprising three phases: elimination, equilibrium, and escape the immune surveillance of cancer cells [42] The inherent and adaptive resistance of solid tumors to conventional and emerging therapies requires a deeper understanding of the Tumor Microenvironments (TMEs) and its immunosuppressive mechanisms to develop more efficient therapeutic strategies [43]. 

Through the integration of cutting-edge tools, there is potential for revolutions in overcoming drug resistance. microRNAs (miRNAs) play a key role in mediating cancer drug resistance, and targeting drug-resistant miRNAs is being a strategic area of research. miRNAs can be delivered to sensitive cells, enhancing the formation of drug-resistant tumor cells. Determination of miRNAs that control cancer cell resistance has become a focal point in hematologic tumor research [44]. Studying miRNA will not only assist in predicting individual tumors' resistance to anticancer drugs but also lead oncologists in tailoring affordable treatment plans for patients, providing a critical basis for future drug resistance evasion strategies. In the dynamic monitoring of therapeutic influence, miRNAs are important in optimizing treatment plans by regulating drug resistance and influencing metabolic pathways and gene expression connected with cancer metabolism [39].

Resistance of cancer cells is often initiated by DNA single nucleotide variants, resulting in point mutations in the proteins or the drug target within the same signaling pathway [45]. Sequencing of the cancer cell genome from biopsies of patients that revert on treatment can retrospectively define the genetic basis of drug resistance [46]. However, this approach needs large numbers of post-treatment samples to designate causal variants and can take years to collect enough to infer variant function. Additionally, genetic analysis is limited to commonly examined variants, meaning that each individual variant must be individually experimentally confirmed to prove a causal link to cancer resistance. This is a slow procedure that does not permit the direct comparison of diverse variant effects. Coelho et al. [47] identified genetic mechanisms of resistance to ten oncology drugs from CRISPR-BE mutagenesis screens in four cancer cell lines using a gRNA library predicted to install 32,476 variants in 11 cancer genes.

Discussion

Cancer therapy

microRNA therapy: The use of RNAs in cancer therapy is capturing increasing attention. Many of RNA-based gene therapy in cancer are based upon short ncRNAs like interfering RNA (RNAi) and miRNA [48]. RNAi can be used to downregulate oncogenes and other genes which are used for the cancer's survival [48]. The discovery of RNAi has revolutionized cancer therapeutic development. Currently, there are many clinical trials using RNAi to eradicate tumors [49].

Despite the significant achievements, an important gap remains in completely harnessing the potential of RNAi-based therapies. As far as transcriptome editing is concerned, comparing RNAi, the classical post-transcriptional gene silencing mechanism, to the CRISPR-Cas system, the latter uses sgRNAs that can remove all the transcript alternatives of a gene with reduced off-target effects [2,4].

miRNAs are especially impactful in cancer, because nearly all known cancer cells use them to control gene expression. One type of cancer is being regulated by multiple miRNAs. For example, in breast cancer, miR-30c, miR-187, and miR-339-5p predict treatment outcomes [50]. A single miRNA can act as a tumor suppressor or oncomiR based on the cell type. miRNAs function as posttranscriptional micro-regulators of gene expression, subtly tuning translational and transcriptional processes [51]. They play vital roles in cell growth, differentiation, and apoptosis [52]. miRNAs stop the translation of mRNA by destroying mRNA or binding to the 3′ untranslated region of target mRNAs, Consequently, dropping the function of downstream protein-coding genes [53]. (Figure 3) outlines various miRNA-based therapeutic strategies against cancer.

Figure 3. Strategies used to inhibit the expression of oncomiRs and to boost the activity of tumor suppressor miRNAs. The action of oncomiRs on tumor-suppressing mRNA can be regulated by Anti-miR oligos, Locked Nucleic Acid, miRNA sponges, and miRNA masking. Source: Modified from [54]

CRISPR/Cas9 technology allows precise validation of miRNA-responsive elements [55]. Furthermore, innovations in high-throughput sequencing and CRISPR/Cas9 tools have enriched our perception of miRNA mechanisms, driving researchers to further investigate miRNA targets and functions, including application of miRNA as diagnostic tools for cancer screening and early detection as well as for therapeutic purposes [56]. The CRISPR/Cas12a system has recently been described for direct miRNA detection [57], where the CRISPR sequence gives targeting data and the enzyme executes the cleaving action, significantly improving specificity [58].

In addition, some miRNAs target DNA methyltransferases (DNMTs), affecting gene expression at both transcriptional and posttranscriptional stages. DNA methylation can control miRNA synthesis, but reverse relationship exists when miRNAs modify gene expression through methylation mechanisms. miR-211 silencing by DNA methylation decreases melanoma cell sensitivity to Cisplatin, indicating a possible role for epigenetic regulation in improving drug efficacy [59]. In predicting efficiency, Artificial Intelligence (AI)-propelled pan-cancer analysis has emphasized miRNA's unique role in clinical cancer staging prediction [60].

A newly discovered network of ncRNAs is the Nuclear-activated miRNAs (NamiRNAs) [39]. In cancer, NamiRNA classes have been indicated in activating tumor suppressor genes, disclosing novel mechanisms in cancer growth and potential therapeutic strategies [61].

Immunotherapy: In the last few years, there has been a growing interest in immuno-oncology. The numbers of new antibodies utilized in clinical trials have increased enormously, achieving a wider range of applications [62]. Precision and personalized treatment represent magnificent advancements in cancer therapy, particularly in the interception of hematologic malignancies, where targeted immunotherapies such as Antibody-Drug Conjugates, Bispecific Antibodies, and Chimeric Antigen Receptor (CAR) T-lymphocytes [63,64].

The CAR-T cell therapy, a form of adoptive cell transfer (ACT), enables the expression of an artificial CAR that can identify particular malignancies and motivates the immune system to target and damaged cancerous cells [65]. CART-cells, as natural carriers, are also being investigated for miRNA-based cancer treatments [39].

CAR-T cell therapy: A Breakthrough for cellular clinical applications of immunotherapy was the development of CAR-T cells [66]. Although CAR-T therapies have achieved remarkable success [66]. Still, the clinical effect of CAR-T therapy is not so sufficient, especially in solid tumors [67]. This is especially true for solid tumors, which are not easy to treat because of the heterogenous structure. Animal models also verified the deceased persistence of TCR-deficient CAR-T cells. In addition, CAR-T cell treatment for solid tumors faces considerable engineering challenges due to the lack of tumor-specific target antigens and the continuing risk of On-Target, Off-Tumor (OTOT) toxicity. CD45, an antigen expressed on most hematopoietic cells and hematologic tumor cells, has the potential to function as a universal antigen [68]. 

Gene therapy: CRISPR technology could be valuable for constructing malignant development organism models for better understanding of cancer biology. The CRISPR/Cas9 system was developed with the aim of cancer treatment and discovering its source genes inside the host so that appropriate animal models can be created for disease study. Various types of cancer may require precise and effective GE technologies to be treated. Similar to RNAi, GE can be used to silence genes vital for cancer. The question worth asking is: what are the advantages of GE over RNAi? The first advantage of GE over RNAi is that because GE acts on the DNA, on a cell-by-cell comparison, its impact is stronger compared to RNAi. When targeting a gene that is transcribed many times, RNAi is incapable to silence all copies of the mRNA, leading to some protein level maintained in the cells. Genome editing however, does not suffer from that problem – as it targets the DNA rather than the mRNA. It matters not how much a gene is transcribed since all future copies of the mRNA will pass the silencing mutations. The second advantage of GE is the durability of the effects – while RNAi is only effective for as long as it is in the cell, the effects of GE are permanent [69]. Consequently, while RNAi is restricted only to genes whose silencing will have direct effects, GE allows for a wider range of targets such as stable proteins with a long half-life. So as can be seen, GE provides a valid approach to cancer therapies, and the influences of CRISPR/Cas9 based editing in cancer has been revealed both in-vitro and in-vivo [4,70,71].

CRISPR editing in animal models is the first step for the improvement of effective gene therapies that are based on substituting mutant genes with wild type genes. Designing transgenic animal models with CRISPR KO and KI for assessing specific genes or gene panels can offer new insights into cellular mechanisms that initiate human cancer, supervise disease progression, and forecast the efficacy of drug therapies. Furthermore, the human near-haploid cell line (HAP1) has commonly been merged as one of the desired cell line models for GE research using CRISPR-Cas9 [71]. A plethora of inventive applications for handling genes, cell lines, and a wide variety of transgenic animal models (Table 1).

The engineered CRISPR-dCas9 system has been used for targeting the Granulin (GRN) gene, a growth factor that promotes tumor progression in liver cancer [72]. The transfer of the dCas9 attached to epigenetic suppressor genes, such as DNMT3a, Histone 3 Lysine 27 Methyltransferase (EZH2), and Krüppel-Associated Box Transcriptional Repression Domain (KRAB), and gRNAs that target GRN resulted in decreased amounts of its mRNAs in Hepatocellular 3B (Hep3B) hepatoma cells (Table 1) Further research that focuses on bladder cancer have shown that the dCas9 joining with the Cryptochrome 2-Calcium and Integrin Binding 1 (CRY2-CIB1) photosensitive module can stimulate the expression of p53 and E-cadherin proteins, blocking the biological function of T24 tumor cells (Table 1) [3,73,74].

Type of cancer	Model/Cell line	Targeted gene
ALL§	Mice model	TRAC
	Mice model	LDLR
	NALM6, MOLM13 cell-lines, and Mice model	GM-CSF
	Mice model	PAX5
BCL	Mice Model	TRAC
	NALM6 Cell line	TRAC, PD-1
Brain cancer	Mice model	NF1, PTEN, TP53
	Mice model	BRCA2, PYCR1, TP53
Colorectal cancer	HInEpC	APC, SMAD4, TP53
	Mice model	APC, KRAS, PIK3CA, SMAD4; Trp53
	HCT116 cell line	miR-34a, miR-34b/c
Glioma	U87, U251 cell-line, and Mice model	TRAC, β 2 M, PD-1
Glioma	U251 cell-line and Mice model	PD-1
Glioma	U87 cell-line and Mice model	DGK
Hepatocellular Carcinoma	Zebrafish model	AR
Leukemia	Mice models	BCR-ABL1
Liver cancer	Cell lines (HepG2, Huh-7)	ASPH, BAX, BCL2, CDK7, CXCR4, NCOA5
	Hep3B hepatoma cells	GGN
Lung cancer	Murine model	P107
Myeloma	Human myeloma cell lines (HMCLs)	miR-1258/PDL-1
Ovarian cancer	Murine model	BRCA2, Tp54
	OVCAR3 cell-line and Mice model	TGF-βRII
Pancreatic cancer	Mice model	ATM, BRCA1, BRCA2, PTEN
Prostate cancer	Mice model	TRAC, β2M, PD-1
Urinary bladder cancer	Cell lines (T24, 5637 bladder cell lines)	lncRNA, PANDA, UCA1
	T24 bladder cell line	p53 and E-cadherin
§Abbreviations: P107: Retinoblastoma-like 1 gene; ABL1: Abelson murine leukemia viral oncogene homolog 1 gene; ALL: Acute lymphoblastic leukemia; APC: Adenomatous Polyposis Coli gene; AR: Androgen receptor gene; ASPH: Aspartate beta-hydroxylase gene; ATM: Ataxia telangiectasia mutated gene; B2M: beta-2-microglobulin  gene; BAX: Bcl-2 Associated X protein gene; BCL: B-Cell Leukemia/Lymphoma; BCR: Breakpoint cluster region protein gene; BRCA: Breast cancer gene; CDK: Cyclin-dependent kinase gene; CXCR4: C-X-C motif chemokine receptor 4 gene; DGK: Diacylglycerol kinases gene; GRN: Granulin; GM-CSF: Granulocyte-macrophage colony-stimulating factor gene; KRAS: Kirsten rat sarcoma viral oncogene; LDLR: Low-density lipoprotein receptor gene; lncRNA: Long non-coding RNA gene; miR: microRNA; NCOA: Nuclear receptor coactivator 1 gene; NF1: Neurofibromatosis Type 1gene ; PANDA: P21-associated noncoding RNA; PAX: Paired box; PTEN: Phosphatase and tensin homolog on chromosome 10 gene; SMAD4:  SMAD4: Mothers against decapentaplegic homolog 4 gene; PIK3CA: Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha gene; PYCR1: Pyrroline-5-carboxylate reductase 1 gene; TP: Tumor Protein gene, TRAC: T Cell Receptor Alpha Constant gene; Trp53: transformation-related protein 53 gene; TGF-βRII: Transforming growth factor β type II receptor gene; UCA1: Urothelial carcinoma-associated 1gene.

Table 1. Summary of some applications of CRISPR/Cas9 system in animal models and human cell lines to investigate various types of malignancy

In another study, genes Long ncRNA-P21-Associated ncRNA DNA Damage-Activated-Urothelial Carcinoma-Associated 1 (lncRNA-PANDA-UCA1) were discovered to enhance expression of Urinary Bladder Cancer (UBC). Disruption of (TRAC) gene to avoid graft-versus-host disease (GVHD) and deletion of CD52 for a survival advantage along with have been investigated before by using TALEN KO [75]. This CRISPR GE strategy has demonstrated exceptional efficiency in hematological malignancies, in children and adults, resulting in durable diminutions in leukemia and lymphoma patients [63]. Currently, neoplastic diseases have the maximum number of in-progress clinical trials because of several strategies are being explored to enhance the effectiveness therapy against cancer [76].

Other types of cancers such as colorectal cancer (CRC), Epstein-Barr virus (EBV)-positive gastric cancer, glioblastoma, melanoma, ovarian cancers, and UBC can also be potentially treated by CRISPR/Cas9 disruption of Programmed Death-1/ Programmed Death Legand-1 (PD-1/PD-L1) gene [77].  PD-1 is a receptor located on the surface of T and B lymphocytes which usually binds to its ligand (PD-L1). The Colorectal Cancer requires several mutations to arise in various genes like, APC, KRAS, MutL Homolog 1 (MLH1), PIK3CA), and can vary greatly in different individuals [78]. 

Engineering of CAR-T cells

Gene editing has arisen as a transformative methodology in cancer immunotherapy. It is possible for precise reform to multiple genes of immune cells concurrently [35]. Because of the high specificity and relative ease of the CRISPR/Cas9 system, it plays vital role in the construction and optimization of immune cells for anti-tumor uses. The CRISPR/Cas9 technology has been used in engineer CAR-T cells (egCAR-F), where the T cells are directed ex vivo to express cancer-antigen specific T Cell Receptors (TCRs). The T cells readdressed against tumor cells are then returned back to the patient to produce the expected attack against tumor antigens. (Figure 4) presents the three principal approaches for producing CAR-T cells using the most traditional CRISPR/Cas9 system, together with CRISPR/Cas12a and CRISPR/Cas13d.

Figure 4. A) The basic steps for generating CRISPR/Cas mediated chimeric antigen receptors (CAR) T-cell.  Cancer patients’ blood is collected and T cells are isolated. Then CD19 specific CAR is inserted into TRAC (T-cell receptor α constant) with the use of CRISPR/Cas9 technology. Thus, modified or CAR engineered T cells are generated and grown in culture. These cells are then infused in the same patient where they attack and kill their target cancer cells. B) CRISPR-mediated gene knockout (KO) and knock-in (KI) approach in CAR/TCR-T cell treatment. To achieve the construction of CAR/TCR-T cells, sgRNA and Cas9 protein are co-transferred into T cells, while CAR/TCR can go in T cells via two main pathways, ultimately resulting in CAR/TCR-T cells. (1) Random insertion through LV/RV: The CAR or TCR is randomly introduced into T cells using LV or RV. (2) Precise insertion: This procedure is assisted by a donor template. Different forms of templates such as dsDNA, ssDNA, pDNA, or AAV are used for site-specific incorporation of CAR or TCR into the T cells. AAV: Adeno-associated virus; dsDNA: double strain DNA; LV: Lentivirus; pDNA, plasmid DNA; RV: Retrovirus; sgRNA: single guide RNA; ssDNA: single strain DNA. A: Adapted from [3, 64]. B: Redrawn from [82]

The technology offers the egCAR T cells a memory phenotype, potential to screen new targets to expand the anti-tumor, reduced exhaustion, superior proliferation and prolonged persistence [79,80]. In contrast to CARs, the egCAR-T cells receptor can distinguish Human Leukocyte Antigens (HLAs)-presented peptides originated from proteins of all cellular compartments [81].

It has been indicated that CRISPR KO can constrain the expression of PD-1 receptor on the surface of the CAR-T cells which eventually increased tumor-killing ability and cancer prevention [82]. Results confirmed the feasibility and safety of CRISPR-egCAR-T cells with PD-1 disruption and emphasize the role of natural TCR in CAR-T cell continuance in solid tumor therapy [80]. Wide evidence supports the idea that abolishing PD-1 with CRISPR/Cas9 confirmed the feasibility and safety CRISPR KO PD-1. It also boosted the anti-tumor power of both allogeneic and autologous CAR-T cells in hematological malignancies and solid tumors during preclinical and clinical assessments [83].

This emerging CRISPR-based CAR-T cell strategy is considered a major breakthrough in cancer treatment, and could disturb the therapeutic landscape of solid tumors [84]. This strategy allowed the production of autologous and allogeneic genome-adjusted cell therapeutics, mainly intended for cancer immunotherapy [85]. Various genetic modulations can improve the efficacy and aggressiveness of T-cells in backing the body against cancer.

CRISPR/Cas9 employed in CAR-T cell production helps in KO of all the toxicity associated genes and also KI of desirable cytokine genes to enhance cytokine release at a same time [86]. This includes the genes of anti- Epidermal Growth Factor Receptor v-III (anti-EGFRv-III) modified CAR-T cells, boosted Interferon-Gamma/Interluken-2 (IFN-γ/IL-2) production causing resistance to Transforming Growth Factor-Beta (TGF-β) and prostaglandin E2 [87]. CRISPR/Cas9 edited CAR-T cells have also shown to inhibit tumor development in mouse models with glioblastoma by TGF-β gene silencing [88]. The procedure enables exact targeting of definite tumor- associated antigens major histocompatibility complex (MHC)-independent fashion, thus overcoming the issue of immune escape resulting from downregulated MHC expression by cancer cells [89,90]. The use of TCR-T cells for ACT has earned increased attention, mainly as efforts to treat solid cancers with ACTs have increased. T lymphocytes design based on CRISPR demonstrates great potential for cancer therapy and has proved to be more powerful than the original T-cell design [30].

Beside keeping CAR-T cell resistant to immunosuppressive cytokines, CRISPR/Cas9 might help CAR-T cells produce specific cytokines that are useful for anti-tumor potency. For instance, IL-15 [91]. Alizadeh, et al. [92] have been shown to improve the anti-tumor activity and long-term persistence of CAR-T cells. In addition, IL-23 could enhance granzymeB production, reduce PD-1 expression, and elevate CAR T-cell increase [93]. Accordingly, utilizing CRISPR/Cas9 to KI the specific cytokine could be a promising strategy to boost the tumor killing capacity and long-term persistence of CAR-T cells.

Likewise, Stadtmauer, et al. [94] engineered T-cells to produce CAR T-cells (expressing the synthetic New York esophageal squamous cell carcinoma 1 (NY-ESO-1) TCR [Also known as cancer-testis antigen 1B (CTAG1B) TCR] to identify cancer cells with extraordinary efficiency and specificity. They modified two other genes that prefer the expression of this synthetic TCR over the wild-type TCR and simultaneously deactivate the PD-1 gene.

(Table 2) gives a comparison of the advantages and disadvantages of CRISPR/Cas9, CRISPR/Cas12a, and CRISPR/Cas13d in CAR-T therapy. Recently, HITI-mediated site directed incorporation of a therapeutically related disialoganglioside (GD2)- CAR transgene into TRAC locus using nano-plasmid DNA and CRISPR/Cas9 in primary human T cells has been tested [84]. Compared to HR-mediated-KI, HITI generated at least 2-fold more GD2-CAR-T cells.

Character	CRISPR/Cas9	CRISPR/Cas12a	CRISPR/Cas13d
Target GE* efficacy	High	Moderate to high	Low
PAM sequence needed	5'-NGG-3'	5'-TTTN-3'	N/A (targets RNA)
GE precision	High	Moderate	High
Applicability to wide-range genome editing	Yes	Yes	No
Suitability for point mutations and indel	Yes	Yes	Yes
Targeted nucleic acid	DNA and RNA	DNA	RNA
Target modified	Genomic DNA	Genomic DNA	RNA
Structural complexity	Larger	Smaller	Moderate
Therapeutic potential	High	Moderate	Moderate to low
Design flexibility	High	Moderate	High
Economic feasibility	High	High	Low
*Abbreviations: CRISPRa: CRISPR activation; G: Guanine GE: Gene editing; indel: Insertion or deletion; N/A: Not applicable; N: Any nucleobase (adenine (A), thymine (T), cytosine (C)); PAM: Protospacer adjacent motif; T: Thymine. Source: Adapted from [82].

Table 2. Basic characteristics of CRISPR/Cas9, CRISPR/Cas12a, and CRISPR/Cas13d in CAR-T therapy

Chen, et al. [95] were the first worldwide researchers to present a patient, suffering from destructive cellular collapses in the lungs, with cells having CRISPR/Cas9 edited genes. They took the patient’s T-lymphocytes and KO PD-L gene.  A first-in-human phase I clinical trial of CRISPR–Cas9 PD-1-edited T cells in patients with advanced non-small-cell lung cancer (ClinicalTrials.gov NCT02793856) was pursued [96].  The trial demonstrated the possibility and safety of therapy with T-cells that were GEN not to express PD-1 in 12 cases with refractory NSCLC [96]. Another objective of this trial was to evaluate the effectiveness with which edited T-cells could avoid the tumor defense system (dependent on PD-L1) and, consequently, damage malignant cells. Regarding the efficacy of the intervention, an average disease-free survival of 7.7 weeks and an average overall survival of 42.6 weeks were recorded, and the disease control frequency at 8 weeks was 16.7% [96].

The adjusted lymphocytes were dispensed to three patients with refractory cancer (two with MM and one with sarcoma). The results of this phase I clinical trial indicated that simultaneous reforming of multiple genes with CRISPR/Cas9 in T-cells is efficient and safe in patients with refractory cancer. Moreover, using CRISPR/Cas9 technology, PD-1 and T cell receptor (TCR)-disrupted mesothelin (MSLN)-specific CAR-T (MPTK-CAR-T) have been produced [39]. The generated cells were applied to patients with MSLN-positive solid tumors in a phase I investigation. Dose-limiting toxicity or unexpected unpleasant events were not recorded in any of the 15 patients. Two patients attained stable disease. But, in MSLN+ solid tumors, TCR deficiency diminished the persistence of CAR-T cells. This study proved the feasibility and safety of PD-1-deficient CAR-T cells and showed the function of TCR for the durability of CAR-T cells in solid tumors [39]. These clinical trial results demonstrate that CRISPR-egCAR-T cell therapy has an optimistic future in clinical applications. More detailed clinical trials of CRISPR/Cas9-egCAR-T cells are presented in Table 3.

In 2023, the use of BE to treat aggressive T cell ALL in three children has been described [97]. T cells from healthy volunteers were transduced to express CAR with specificity for CD7 (CAR7), a protein that is expressed in ALL T-cells. After that, the method BE was used to induce mutations into three genes encoding receptors – CD7, CD52, and the TCRαβ T-cell subset – which inactivated the genes. This procedure produced T cells with a decreased probability of CAR7 T-cell fratricide and graft-versus-host disease, leading to a safer therapy for ALL patients. Epitope editing is a promising plan for shielding hematopoietic cells from annihilation by immunotherapies. Recently, researchers used both BE and PE to modify the epitope of CD45 and CD123 in hematopoietic stem cells for CAR-T therapy against AML [98-100]. Ex vivo epitope editing in hematopoietic stem and progenitor cells (HSPCs) and T cells allowed effective and safe application of epitope-directed CAR T- cells and bispecific T cell engagers for the entire therapy of hematologic tumors and might be used for other diseases requiring intensive hematopoietic ablation. Epitope-edited cells were resistant to CAR-T lysis while preserving normal differentiation and function. In addition, BE- or PE-edited HSPCs infused into humanized mice granted myeloid lineages with selective resistance to CAR-T immunotherapy, validating a proof-of-concept approach for treating relapsed AML.

Screening method	Screened cell	Target gene	Discovered target gene	Function of discovered target gene
CRISPR KO	T cell	CD19	RASA2	RASA2 functions as signaling checkpoint in T cells, which can increase progressively with chronic antigen exposure and causes cell exhaustion.
		EphA2
CRISPR KO	T cell	CD19	SOCS1	SOCS1 is a main negative checkpoint in ATCT, preventing T cell survival, cell propagation, and effector function.
CRISPR KO	T cell	CD19	p38 Kinase	p38 kinase is a key regulator of four phenotypes, including cell growth, differentiation, oxidative stress, and genomic stress.
CRISPR KO	T cell	EGFRvIII	PDIA3	PDIA3 downregulate the expression of multiple of immune regulators and effectors.
CRISPR KO	T cell	B7-H3 (CD276)	cBAF	cBAF functions as epigenetic regulator facilitating the T cell terminal differentiation.
CRISPR KO	T cell	HER2	ST3GAL1	ST3GAL1 prevents the tumor-specific homing of CAR T-cells.
CRISPR KO	T cell	CD19	NAD	NAD is a coenzyme in redox reaction mediating T cell activation.
CRISPRa	T cell	CD19	PRODH2	PRODH2 overexpression reformed gene expression and metabolic programs, increasing metabolic and immune function of CAR-T cells against cancer.
		BCMA
		HER2
BE GOF	CAR T-cell	CD19	PIK3CD	PIK3CD GOF variant shows enhanced antigen-specific signaling, cytokine assembly, and tumor-killing ability.
CRISPR KO	CAR T-cell	IL13Rα2	TLE4, IKZF2	TLE4 and IKZF2 (Helios) mediate CAR T-cell fatigue.
CRISPR KO	CAR T-cell	CD22	PRDM1	PRDM1 decreases Tcm phenotype and prevents downstream factor transcription that increases proliferation and perseverance of CAR T-cells.
CRISPR KO	Pancreatic carcinoma	MSLN	GPAA1, TFAP4, INTS12	The genes implicated in the pathway responsible for GPI-affix biosynthesis and attachment mediate.
CRISPR KO	Glioblastoma	EGFR	IFNγR	The genes in the IFNγR signaling pathway make solid tumor cells vulnerable to CAR T-cell killing.
CRISPR KO	B-cell leukemia, B-cell lymphoma	CD19/CD52	NOXA (PMAIP1)	NOXA makes tumor cells susceptible to CAR T-cell killing
CRISPR KO	B-cell leukemia	CD19	CD58	CD58 mediates the immunological synapse creation with CAR T-cells and keeps tumor cells susceptible to CAR T-cell killing.
CRISPR KO	Glioblastoma	IL13Rα2	RELA, NPLOC4	RELA and NPLOC4 change tumor–immune signaling and boost responsiveness of CAR therapy.
CRISPR KO	BCP-ALL	CD19	NUDT21	NUDT21 reduces the antigen on the tumor cell surface
CRISPRi	MM	BCMA	HDAC7, Sec61	HDAC7 and Sec61 reduce the antigen on the tumor cell surface
Abbreviations: BCP-ALL: B7-H3: B7 homolog 3 protein; B-cell precursor-acute lymphoblastic leukemia; BCMA: B-Cell Maturation Antigen; BE: Base editor; cBAF: Canonical barrier-to-autointegration factor; CD: Cluster of differentiation; EGFR: Epidermal growth factor receptor; EGFRvIII: Epidermal growth factor receptor variant III; EphA2: Erythropoietin-producing hepatocellular A2; GOF: Gain-of-function; GPAA1: Glycosylphosphatidylinositol anchor attachment 1; GPI: Glycosylphosphatidylinositol ; GPIHER2: Glycosylphosphatidylinositol anchor attachment 1; HDAC7: Histone deacetylase 7;  HER2: Human epidermal growth factor receptor 2; IKZF2: ; Ikaros Family Zinc Finger Protein 2, IL13Rα2: Interleukin 13 receptor alpha 2; INTS12: Integrator complex subunit 12; IFNγR: Interferon gamma receptor 1;  KO: Knock out; MM: multiple myeloma; MSLN: Mesothelin; NAD: Nicotinamide adenine dinucleotide; NOXA: Latin for damage; NPLOC4: Nuclear protein localization protein 4; NUDT21: Nudix hydrolase 21; PMAIP1: Phorbol-12-myristate-13-acetate-induced protein 1; PDIA3: Protein disulfide isomerase A3; PIK3CD: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta; PRDM1: Positive regulatory domain zinc finger protein 1;  PRODH2: Proline dehydrogenase 2; RASA2: Rat sarcoma p21 protein activator 2; RELA: v-rel avian reticuloendotheliosis viral oncogene homolog A; SEC61A1: Sec61 translocon alpha 1 subunit; SOCS1: Suppressor of cytokine signaling 1; ST3GAL1: ST3 beta-galactoside alpha-2,3-sialyltransferase 1; TFAP4: Transcription factor activating enhancer binding protein 4; TLE4: Transducin-like enhancer of split 4. Source: Adapted from [77].

Table 3. In vitro/in vivo clinical trials reporting cancer treatment using CRISPR system-based Chimeric Antigen Receptors (CAR)-T cell therapy

Even in hematologic malignancies, a percentage of patients ultimately relapse after receiving CAR-T cell infusions, due to the poor expansion and persistence of CAR-T cells [67].

Combinatorial cancer therapy: Combination of therapeutic strategies that integrate conventional treatments (such as chemotherapy, radiotherapy, and immunotherapy) with modern ones like GE. Clinical interventions have been developed, concentrating on small molecules and biologics that disturb specific pathways. The safety and efficiency of pathways antagonists from manifold drug classes have been assessed in completed and ongoing trials The following section provides some of the novel promising combinations to fight difficult-to-treat cancers.

Integration of miRNA with conventional therapy: The synchronous inactivation of activated cell migration and infiltration, promoted epithelial-mesenchymal transition (EMT), decreased chemotherapy sensitivity, and raised stress-induced autophagic flux, emphasizing the paired roles of miR-34a and miR-34b/c in controlling EMT and autophagy, providing new therapy insights [39, 54,101]. However, refining the precision of miRNA target prediction continues to be a challenge. miRNAs affect PD-L1 expression, a key factor in tumor-immune escape. Homo sapiens-miRNA-200 (Hsa-miR-200) is linked with high PD-L1 expression, while Hsa-miR-197 is negatively associated with PD-L1 expression [102]. Scientists are integrating multiple biological characteristics and leveraging single-cell sequencing information along with deep learning methods to improve prediction accuracy [39].

Furthermore, miR-1258 targets PDL-1 and affects myeloma therapy by inhibiting PDL-1 (Table 1) [103]. Over-expression of miR-1258 in totally methylated myeloma cells led to decreased cellular proliferation and greater apoptosis, consequently a tumor suppressor role, added to repression of PDL-1.

Targeted treatments against miRNAs, with miRNA mimics and inhibitors, have demonstrated a huge potential in preclinical studies [104]. This approach has confirmed therapeutic value in clinical trials, opening new possibilities for cancer treatment [105]. miRNA inhibitors can lessen the overexpression of target mRNA and fix cell phenotypes, making them a promising therapeutic policy. These include natural miRNA inhibitors like circular RNAs, lncRNAs and other competitive endogenous RNAs as well as artificial miRNA inhibitors (such as Anti-miRNA oligonucleotides and mRNA sponges) (Figure 1) [106]. Other small molecule inhibitors can exactly target oncomiRs and Dicer (an enzyme important in miRNA generation) to attain tumor suppressor effects. In addition, CRISPR can be utilized to directly edit oncomiRs for tumor suppression [40].

Joining CAR-T cell therapy with drugs: Although CAR T-cells represent a front line in cancer therapies and have achieved remarkable success, the clinical effect of CAR-T therapy is not so sufficient, especially in solid tumors [67]. The low activity of CAR-T cells limits the efficacy of CAR-T cell therapy. To improve the efficiency of CAR-T cell therapy, joining CAR-T cell therapy with small‐molecule drugs looks promising and may yield synergistic effects.

Due to the heterogeneity of the TME, several therapeutic options have been advanced and clinically tested for the treatment of solid tumors such as Immune Checkpoint Inhibitors (ICI) such as Nivolumab and Ipilimumab), as well as Tyrosine Kinase Inhibitors (Sorafenib and Dasatinib), and cancer vaccines (Provenge® and T-VEC®) [107-109]. These treatments have decreased disease development in patients with solid tumors, but haven’t promoted long-lasting, durable cures [110].

The combinatorial genetic interruption in CAR-T cells allowed superior anti-tumor efficacy resulting in improved tumor extermination and survival in humanized mouse models that recapitulated destructive characters of a human TME [111]. This unique engineering strategy granted CAR-T cells resistance to a diverse TME and may unlock the therapeutic potential of CAR-T cells against solid tumors. The most GE-based clinical trials have been directed against various forms of malignant transformations. Biochemical and immunological negative regulators converge to inhibit CAR-T cells, leading to clinical failures of CAR-T cell therapies against solid tumors. The selective inhibitors of nuclear export carfilzomib, lenalidomide and selinexor have been approved for the treatment of MM [112]. These technologies developing immune system function by disturbing checkpoints, boosting cytokine assembly, and adjusting TMEs [113, 114]. 

Combination of CAR-T cell therapy with immune checkpoint inhibitors: In natural condition, the T-cells of the immune system become activated with attacking pathogens and also inhibit auto-immunity by regulating T cell overexpression. T cells express various inhibitory receptors on their surface, adding to T cell exhaustion, including PD-1, Cytotoxic T Lymphocyte Antigen-4 (CTLA-4), Lymphocyte-Activation Gene-3 (LAG-3), CD244, CD160, T Cell Immunoglobulin-3 (TTIM-3), and others [82]. Immune checkpoints help in T-cell activation. One promising therapeutic approach is the combination of CAR-T cell therapy with ICI (anti-CTLA-4, anti-PD-1, anti-PD-L1) (Figure 5).

Figure 5. A schematic diagram showing T-cell activation, inhibition, and reactivation Upper Panel: Blocking by CTLA-4 with anti-CTLA-4 antibodies (ipilimumab, tremelimumab). (a) T-cell activation involves 2 signals: The first is attachment with MCH with TCR and the second is interaction of CD28 on the T-cell with B7 (CD 80, CD 86) on APC. (b) CTLA-4 is transferred to the plasma membrane and operates as a T-cell activation inhibitor. (c) Anti-CTLA-4 antibody binds with CTLA-4 which causes T-cell reactivation. Lower Panel: Blocking by PD-1/PD-L1 axis and its inhibitors’ role in the control of T-cell functions. (a) Prolonged antigenic promotion during carcinogenesis. (b) PD-1 overexpression leads to the inhibition of T-cell proliferative and cytotoxic activity, resulting in “exhausted” T-cells, which are distinguished from others by impaired capacity to produce IFN-γ. (c) PD-1/PD-L1 inhibitors are able of transforming “exhausted” T-cells into effector T-cells. Abbreviations: (+): Active T-cell; (-): Inactive T-cell; APC: Antigen-presenting cell; CTLA-4: Cytotoxic T lymphocyte-associated antigen 4; IFN-γ: Interferon γ MHC: Major histocompatibility complex; PD-1: Programmed cell death protein 1; PD-L1: Programmed death-ligand 1; TCR: T-cell receptor. Source: Adapted from [62]

The checkpoint blockades have been a significant innovation in the field of oncology and represents a major step forward as a unique type of immunotherapy in the treatment of cancer, since disturbing these immunosuppressive routes can reactivate exhausted or hypofunctional T cells [62]. In cancer cells, the immune checkpoints, like PD-1 and CTLA-4, become repressed by some inhibitory compounds (for example PD-L1/ L2 of macrophages inhibits PD-1 expression), reducing interleukin release and eventually causing poor prognosis of cancer [115]. CRISPR/Cas9 helps to quench ICI genes in allogenic CAR-T cell treatment. Conversely, disruption of PD-1 and CTLA-4 can enhance the solvability of CAR-T cells [115].

Furthermore, disruption of three genes for TRAC, β2-B2M and PD-1 through lentiviral electro delivery of CRISPR/Cas9 mRNAs and gRNAs with CAR-T cells triggered higher efficacy of ‘Off-the-self’ CAR-T cells, which was found to reduce GVHD and extended survival rate in mice [116]. Liu, et al. [117] reported that production CAR-T cells lacking these three genes or TRAC and β2-B2M generated more IFN-γ and more cancer cell lysing ability than the standard CARs. CRISPR/Cas9 also disrupted TGF-β receptor-II, which helped in the proliferation of CAR-T cells in mice and found to suppress tumor progression most efficiently [88]. Also in mice, KO of PD-1 gene alone was found to cure cancer cells almost up to 28 days. It was demonstrated that PD-1 gene disruption and expressing CD-133, using CRISPR/Cas9 in CARs caused lower cytokine secretion [83]. Removal of β2-B2M gene from CAR-T results in absence of the HLA, making the cells more susceptible to Natural killer (NK) cell stimulated cytotoxicity as NK cells only identify cells lacking HLA-1 via CD94/NKG2A and NK cell immunoglobulin receptor (KIR).

The CTLA-4 axis: Preclinical data as well as early-phase clinical trials have indicated that the combination of CAR-T cell treatment with checkpoint inhibitors can improve CAR-T cell function, continuance, and tumor control in solid tumors [118, 119]. On the whole, CTLA-4 contributes to the immune deficiency observed in cancer patients. T-cell activation, inhibition, and reactivation by inhibiting CTLA-4 with anti-CTLA-4 antibodies (ipilimumab, tremelimumab) are presented in Figure 5 and (Table 4). 

Immune checkpoint inhibitors (ICI)	Type of cancer treated
CTLA-4 inhibitors
Ipilimumab	Melanoma. Pediatric melanoma
Tremelimumab	Melanoma* Mesothelioma* NSCLC§
PD-1 inhibitors
Nivolumab	Bladder cancer, dMMR-mCRC, HCC, HNSCC, Hodgkin lymphoma, Melanoma, MSI-H cancer cells, NSCLC, RCC, Cancer of the stomach, esophagus and gastro-esophageal junction*
Pembrolizumab	Bladder cancer, Cancer of the stomach and esophagus, HNSCC, Hodgkin lymphoma Melanoma, MSI-high or MMR-deficient solid tumors of any histology, NSCLC, Squamous cell carcinoma of the skin*
Pidilizumab	DIPG*, DLBCL*, FL*, MM*
Cemiplimab	Squamous cell carcinoma of the skin*
PD-L1 inhibitors
Atezolizumab	Bladder cancer, NSCLC
Durvalumab	NSCLC, Urothelial cancer of the bladder
Avelumab	Locally advanced/metastatic urothelial carcinoma, MCC
Combined treatment with CTLA-4 and PD-1 inhibitors
Ipilimumab + Nivolumab	Melanoma, RCC, Cancer of the stomach, esophagus and gastro-esophageal junction*
Combined treatment with CTLA-4 and PD-L1 inhibitors
Durvalumab + Tremelimumab	Lung cancer (small cell lung cancer, NSCLC) Bladder cancer*, HCC*, Cancer of the head and neck area*
*Drugs undergoing clinical trials
§Abbreviations: DIPG: Diffuse intrinsic pontine glioma; DLBCL: Diffuse large B-cell lymphoma; dMMR: Mismatch repair-deficient; FL: Follicular lymphoma; HCC: Hepatocellular carcinoma; HNSCC: Head and neck squamous cell carcinoma; MCC: Merkel cell carcinoma; mCRC: Metastatic colorectal cancer; MM: Multiple myeloma; MSI-H: Microsatellite instability-high; NSCLC: Non-small cell lung cancer; RCC: Renal cell carcinoma. Source: Adapted from [62].

Table 4. Classification of drugs according to their mechanism of action and cancers treated

PD-1/PD-L1 pathway: The PD-L gene has been implicated in a key immune checkpoint. The PD-1/PD-L1 axis plays a key role in T cell exhaustion and exemplifies a fundamental mechanism of tumor immune escaping, as it prevents the function of T-cells and functions as an immunosuppression moderator when activated by the PD-L1.

The PD-1/PD-L1 signaling pathway modifies T cell proliferation, activation, exhaustion, and immune tolerance (Figure 5). Consequently, blockage of PD-1/PD-L1 interaction could enhance the CAR-T cell function to struggle against cancer cells [120]. Blockade of PD-1 has attained great success in various tumor types in recent years, especially in lymphoma [121]. PD-1 blockade is usually given in combination with traditional chemotherapy or other immunotherapies. The outcomes of trials that investigated blocking PD-1 and PD-L1 with monoclonal antibodies (pembrolizumab) have indicated that blocking is clinically efficient in helping treat patients with a range of neoplasms susceptible to this intercession (including NSCLC) [30]. 

Signals via the PD-1 route contribute to the control of initial T-cell activation, fine-tuning of T-cells' future and actions, and T-cell tolerance (by stimulating T-cell apoptosis) [122]. PD-L1 is expressed in abundance in the TME of different neoplasms and exemplifies one of the major mechanisms that enable them to evade the immune response of the host. Moreover, the expression of PD-L1 allows cancer cells to escape recognition by CTLs and therefore create an efficient biological barrier for immune-mediated drug methods [123]. The immune system is down-regulated by PD-1 gene. PD-1 can block the progression of autoimmune disorders; however, it also does not allow the immune system from destroying cancerous cells and tissues. Therefore, KO of PD-1 has been approved by several clinical trials for different types of cancer [124].

Finally, some clinical complications have been observed in cancer patients with ICI therapy. The most common complains include dizziness, headache, or taste disturbances, cutaneous and neurologic toxicity, abdominal pain and diarrhea. Other adverse problems include hepatotoxicity, and symptoms of hematological origin such as fatal anaplastic anemia, hemophilia A, immune thrombocytopenic purpura, neutropenia, red blood cell aplasia, and thrombocytopenia [125]. Significant improvement was recorded after discontinuation of immunotherapy. These clinical problems are outside the scope of the review, but more details can be found elsewhere [62].

Combination of CAR-T therapy with cytokines: CAR-T cells can secret cytokines for activation of T-cell, proliferation and differentiation via CD3 and CD28 and cytokine secretion gets elicited by TCR- mediated route which further activates immune cells. Some of the interleukins, i.e., both IL-12 and IL-15 increase anti-cancerous activity and IL-18 was found to activate Interferon-Gamma (IFN-γ), further helping in CAR-T proliferation [126]. Upregulated PD-L1 expressed on cancer and cancer-supporting cells binds to PD-1 receptors on T cells, transmitting inhibitory signals which dampen T cell anti-tumor cytotoxicity, leading to tumor immune evasion [127]. 

The most familiar toxicity caused by CAR-T therapy resulted from the release of several signaling molecules (cytokines such as IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and miRNA-containing exosomes, etc.), which facilitate the cross-talk between immune cells, tumor cells, and the encircling stroma. Thus, sustaining the chronic (persisting) inflammation, and mediating the propagation as well as malignant transformation of the tumor [123]. TGF-β combines with the TGF-β receptor heterocomplex made of two each of the TGF-β receptor I (TGFβRI) and TGF-β receptor II (TGFβRII) subunits. Abnormal and overactive TGF-β signaling enhances cancer initiation through epithelial–mesenchymal transition (EMT), and the invasion and metastatic growth of cancer cells [128].

Along with the PD-1, TGF-β pathway characterizes non-redundant immunological preventive mechanisms that may act in concert with adenosinergic and metabolic biochemical repressing pathways in the TME to inhibit constructive anti-tumor immune responses. Regulatory T cells add to the immunosuppressive TME by producing cytokines such as IL-10 and TGF-ß. By hindering the function of cytotoxic CD8+ T cells, CD4+ T-helper cells, and natural killer (NK) cells, they enable immune evasion [129].

The safety and efficiency of TGF-β pathway antagonists from manifold drug classes have been assessed in completed and ongoing trials [63]. Vactosertib, a highly powerful small molecule TGF-β type 1 receptor kinase suppressor that is well-tolerated with a satisfactory safety profile that has demonstrated efficacy against various types of cancer [130]. Bintrafusp alfa (a bifunctional conjugate that binds TGF-β and PD-L1), AVID200 (a computationally designed TGF-β ligand trap of TGF-β receptor and Luspatercept (a recombinant fusion that connects the activin receptor IIb to IgG) offer novel ways to fight difficult-to-treat cancers [130]. Another small molecule is Galunisertib, which selectively blocks TGF-β receptor I kinase, inhibiting the TGF-β signaling pathway [131]. TGF-β exerts its function through signaling through serine/threonine kinase transmembrane receptors to intracellular Suppressor of mother against decapentaplegic (SMAD) proteins by the canonical pathway and in a blend with co-regulators. For example, the adaptor protein and E3 ubiquitin ligases TNF receptor-associated factor 4 (TRAF4) and TNF receptor-associated factor 6 (TRAF6) to enhance non-canonical pathways [128].

Gene editing delivery systems: Efficient GE delivery in vivo is vital for realizing its complete potential in disease modeling and medicinal correction. GEN has become considerably more efficient and precise since the emergence of conventional GE tools such as ZFNs, TALENs, and CRISPR/Cas9. Recently hydrodynamic tail vein injection of pDNA has been shown to efficiently deliver BE/PE to hepatocytes in mice [132]. To fully make use of the editing potential of an editor, the editor must be successfully transferred into target cells or tissues using suitable vectors. However, in vivo GE in a non-invasive means, without off-target side effects, poses several challenges that vary from in vitro GE using methods such as lipofection, electroporation, or microinjection [133]. The delivery of PE structures holds immense potential for revolutionizing cancer treatment [134,135].

The delivery of Cas9 into cells through the plasma membrane presents a challenge owing to its large protein size. Both viral and non-viral in vivo delivery protocols have been employed to deliver CRISPR/Cas9 system, BEs and PEs, establishing that these editors can serve as efficient agents for in vivo therapeutic GE in animals.

During the last decade, extensive studies have been carried out to deliver the CRISPR system into human primary T cells. The next sections briefly discuss different transfer methods [17,133,136].

Viral vectors delivery: Different viral vectors have been employed for the delivery of CRISPR elements as natural delivery systems. Viral delivery of CRISPR vectors, such as Lentivirus (LV) or adeno-associated virus (AAV). The T- cell specificity towards Leukemia was edited by combining ZFN- KO endogenous TRAC and TRBC genes with LV gene transfer of a Wild-type-1 specific TCR [137]. A TCR-T product with promoted expression of the transferred TCR and a complete absence of endogenous TCR a/b chains was reported.

In a first-in-human trial, multiplexed CRISPR/Cas9 editing to disrupt T-cell TRAC, TRBC, and PDCD1 genes in conjunction with LV delivery of a NY-ESO-1 specific TCR were utilized [94]. In the four patient-derived products explained in this work, disruption of TRAC and TRBC was accomplished in an average of 45% and 15% of cells, respectively. Nevertheless, because TRAC/TRBC edited T cells were not selected before LV transduction with the NY-ESO-1 TCR, a substantial fraction of the TCR-engineered T cells continued to express endogenous TCR a/b chains.

Lentivirus delivery: Lentiviruses have unique advantages, such as their higher packing capacities—up to 10 kb. This feature allows them to capture more GE elements, such as PEs and pegRNAs, in a single vector, thus improving transduction efficacy [68]. Lentiviruses can semi-randomly incorporate DNA encoding BEs, PEs, and super enhancer RNAs (seRNAs)/pegRNA into the host genome, reaching high editing efficacy in mouse cortical neurons, human cell lines, and induced pluripotent stem cells (iPSCs) [58]. Moreover, patient CML cells transduced ex vivo with a LV- based CRISPR-Cas9 GE system showed more than 30.9% indel incidence without recording off-target effects [138]. They demonstrated that the ABL-targeted CRISPR-Cas9 virus can lead to a high incidence of apoptosis in CML cells. Nevertheless, long-term expression and high rate of off target effects due to the incorporation of LVs into the genome is the major limitation of these vectors. Furthermore, due to insertional mutagenesis, LVs induce a more pronounced inflammatory response in vivo and may increase the chance of tumorigenesis and off-target editing from extended expression, bringing up concerns about their safety in clinical applications [139].

Adeno-associated virus delivery: To mitigate risks associated with LVs, transient expression is favored, making it more preferable for therapeutic applications. Among DNA viruses with lesser integration risks, AAVs have been successfully used for transient transduction [139]. They virus have been used in FDA-approved drugs [140] and therapies in clinical trials [141]. Adeno-associated virus has emerged as a pivotal in vivo delivery technique in clinical gene therapy due to its efficient cellular uptake, low immunogenicity, and range of serotype specificities as well as its ability to attain long-term gene expression in different tissues [142]. However, the AAV vectors are not the ideal delivery means for all cell types since the viral genome can persist in the cells as an episome and AAV capsid proteins may cause immune responses [143]. In addition, even the basic version of PE2 is encoded by about 6.3 kb of sequence, and this size exceeds the cargo ability of a single AAV vector, since their limited loading capacity is about 4.7 kb, making safe and effective delivery challenging [144], which is smaller than the size needed for PE editors and pegRNA (7 kb) [68].    

To overcome the packaging limitation, researchers have split genome editors into two protein halves, and delivered using dual AAVs [145]. The editing efficiency of delivering PEs by dual AAVs at different gene loci varies among split sites. Upon co-infection with AAVs expressing each PE-intein half, the full-length genome editor is reconstructed through trans-splicing. In another strategy to bypass the AAV loading limit, scientists encoded each genome editor half on transfer RNA splicing AAVs (tsAAVs) [146]. In this context, Liu B et al. [132] revealed that delivering unlinked nCas9 and RT (sPE) using two different AAVs can accomplish high-efficiency GE. Though the current dual-AAV PE transfer shows lower editing efficacy within Cas9n compared to dual-AAV BE delivery, current developments in optimizing PE proteins and pegRNAs are likely to significantly promote in vivo PE efficiency.

Despite the promising results of the viral delivery strategies, optimizing viral vectors for in vivo PE transduction is essential for achieving therapeutic GE in human patients [139]. Combining viral and non-viral delivery means may advance a promising solution by leveraging the strengths of both methods to optimize efficiency and safety.

Non-viral delivery pathways: Different virus-free delivery systems have been developed as an attractive substitute to viral vectors and they have been extensively employed to deliver the CRISPR-Cas9 system to target cells. Amongst the most broadly used is a range of physical and chemical routes [17,136]. 

Physical delivery

Microinjection: Microinjection (MI) may be considered a possible method for delivery of CRISPR into human [147].  It is also proper for in vitro and ex vivo transfer of the CRISPR system embryonic cells and the generation of KO mice models [148]. It has been found useful for editing Hematopoietic Stem and Progenitor Cells (HSPCs) without negative effect on cellular function. However, the processing of a single cell at a time makes the protocol more labor and time-consuming, limiting the number of MIs that can be performed in each attempt. In addition, application of the MI method in the production of edited animal models needs expensive micromanipulation equipment and skilled personnel [7].

Electroporation and nucleofection: Electroporation is an electro-physical, non-viral rapid delivery method. It is an acceptable approach in CRISPR-based cancer immunotherapy for in vitro and ex vivo monitoring of immune cells including B cells, T cells and NK cells [149]. In different mammalian cell lines, transient lipid-mediated transfection or electroporation of BE/PE plasmids can attain high editing efficacy, and selecting suitable cell types can promote optimize editing outcomes [150]. With the advance of the RNP complex in the CRISPR/Cas9 structure, electroporation has become feasible and one of the most usual techniques for RNP delivery [7]. However, in U-251 and U-87 MG cells, electroporation induced unexpected changes in gene expression [151]. In contrast to electroporation, recombinant AAV (rAAV) transfected with Cas9 did not. These outcomes emphasize the necessity for enough recovery times following electroporation or the use of alternative methods, such as rAAV transfection, to ensure the precise assessment of CRISPR-mediated GE products.

Nucleofection, an adapted form of electroporation for direct delivery of nucleic acids into the nucleus of various cells, has been confirmed to be an efficient mode of transfecting human CD34+ cells [152]. However, chemical modifications are essential to maintain the needed activity levels and inhibit immune responses induced by mRNA [153]. Investigations have shown that delivering PE mRNA along with chemically modified or in vitro transcribed gRNAs by electroporation can accomplish effective PE in cultured cell lines, primary human T cells, and human pluripotent stem cells (HPSCs) [132].

Microfluidic-based methods: The microfluidic device is a membrane distortion-based delivery procedure that utilizes physical constraint to modify the shape of the cell, producing transient pores in the cell membrane [154]. Therefore, the crossing of a range of biomolecules such as CRISPR constituents by passive diffusion is allowed [155]. Microfluidic and other fluidic models can be employed at each stage of cell engineering for nanomedicines [156]. A specific microfluidic chip for HSPCs has been developed [157]. This Nano-Blade Chip (NB-Chip) is devised by silicon instead of polydimethylsiloxane (PDMS). Interestingly, the use of NB-Chip for delivering macromolecules or plasmids into the HSPCs was more efficient than electroporation in terms of extended continuation of HSPCs’ inherent pluripotency [157]. They could transfer CRISPR in RNP composite format to the human HPSCs and dislocate CCAAT/enhancer-binding protein-α (C/EBPα/CEBPA) p42 in vitro [158].

Filtroporation: Filtroporation is another biophysical strategy for the transfer of CRISPR system in HSPCs. In this technique, cells are forced to pass across microporous membranes to enhance the permeability of the cells [155]. Transmembrane internalization assisted by membrane filtration is centered on the filter membrane cell permeabilization method to deliver the RNPs to CD34+ HSPCs [159].
 

Exosomes: Exosomes are extracellular vesicles released practically all cell types. They consist of lipid bilayer membranes enclosing various biological molecules. Exosomes as new generation vehicles for drug delivery have revolutionized the arena by serving as diagnostic and therapeutic markers for cancer [134]. They can offer diverse advantages to enhance stability, cellular uptake, and biocompatibility, based on their structures and physio-chemical stability.

Several studies have indicated superiority of exosomes to other delivery methods in effectively transporting CRISPR/Cas components, such as sgRNA and Cas9 protein, to target cells in vitro and in vivo, producing targeted GE [160]. Exosomes can carry RNAi to cancer cells and prevent the growth of tumors in mice [161]. Exosome-mediated delivery of CRISPR/Cas9 for targeting of oncogenic KrasG12D sgRNA1 and KrasG12D sgRNA2 in treatment of KPC689 pancreatic cancer cells [162]. The T7/Surveyor test revealed effective GE following treatment compared with the cells without exosomes vector control or untreated cells, which did not show such efficiency. Additional studies have revealed the potential therapeutic uses for exosome-based CRISPR/Cas delivery for the treatment of several human diseases, including cancer [160].

By directing the distribution of the CRISPR/Cas constituents to specific cell types and inhibiting off-target effects, exosomes can enhance the precision and safety of the CRISPR/Cas [163]. For example, research has revealed that MSC-derived exosomes can carry multiple therapeutic agents, including anti-cancer drugs, miRNAs, RNAi and small bioactive molecules to cancer cells particularly and decrease the expression of a target gene [163]. Interestingly, miRNAs in exosomes freed from donor cells into the systemic circulation are saved from enzymatic degradation. A nano-delivery system using nanoparticles could potentially shield Cas9 RNP from enzymatic degradation. Moreover, Cas9 Ribonucleoproteins (Cas9RNP) alone lacks the capacity for cell internalization and cell selectivity. It has been found that RNPs exhibited an elevated medicinal impact when delivered in extracellular vesicles as opposed to RNPs alone. Also, the exosomes’ ability to target liver cells improved tissue selectivity [164]. The difference between mRNA and pDNA delivery shows that mRNA delivery is 7h faster than pDNA but resulted in reduced efficiency, the editing time itself, indicated that this is 15 times longer for mRNA transfection than for pDNA transfection [165]. The miRNA mimics and inhibitors can also be delivered into the body by different tracks, including viral vectors and nonviral vectors [39].

Cas9 RNP-encapsulated nanoparticles (Cas9RNP-NPs) can efficiently cross membranes with carefully designed procedures that control structuralism to leverage biological machinery systems [133]. The use of nanoparticles not only protects Cas9 RNP from digestive enzymes but also enables easy surface modifications, allowing further functionalities such as enhanced endocytosis, cell selectivity, and immune tolerance. Medicinal chemists and pharmaceutical scientists expect that Cas9 RNP-NPs coated with suitable vectors will be clinically applied to treat genetic disorders in the near future.

For suitable delivery of the PE system, PE all-in one (PEA1) plasmid has been invented to consist of three holders for expression of all PE3 components and a selection code, presented with high editing efficacy [166]. Among in vivo gene carriers, the LV-derived nanoparticle (LVNP) protocol has been advanced and optimized for the delivery of PE machinery [167]. Further developments in viral delivery of PE apparatus were advanced by adapting the engineered virus-like particle (eVLP) systems used for the delivery of BE components [32]. Furthermore, reports included overcoming this problem by splitting PE that can reassemble two split PE (sPE) parts into the full-length protein have been used for PE delivery in vivo in mouse liver and retina [168, 169].

Chemical delivery: Chemical vectors are alternatives for the non-viral delivery of CRISPR system into the cells. In comparison to physical delivery, the chemical vectors are safer than the viral ones and do not exert much stress on cells [170].

Liposomes delivery: Because both biological membranes and nucleic acids possess negative charges, nucleic acids cannot cross the membrane due to the attraction between both [170]. Positively charged liposomes enclosing DNA, mRNA (Cas9 and sgRNA), or protein can all be used to transport the CRISPR/Cas9 system. Researchers devised a liposomal delivery method in a study that targets the oncogenic Bcl-2 in leukemia cells in the bone marrow using Cas9/sgRNA. The technique was found to effectively transport Cas9/sgRNA and induce apoptosis in the leukemia cells, indicating to potential as a leukemia treatment method (Li et al., 2020). Bioreducible lipidoid-encapsulated Cas9-sgRNA have been delivered into human leukemia stem cells (LSCs) to KO IL-1 receptor accessory protein (IL1RAP) [171]. It led to reduced clonogenicity of leukemia cells in vitro and decreased leukemic burden in vivo. In a mouse model of AML, liposomes were used to carry CRISPR Cas9 to target the CXCR4 gene. The researchers confirmed that CRISPR Cas9 delivered by liposomes might induce apoptosis in AML cells and dramatically prolonged the life of the mice [172]. The processing of CRISPR/Cas9 by liposomes for cancer therapy has also been investigated. The oncogene KRAS in pancreatic cancer cells was targeted by means of a liposomal delivery system for Cas9 and sgRNA. The system’s proficiency to transfer GE tools and cause cancer cells apoptosis indicates that it may be useful as a pancreatic cancer therapy approach [173].

 Nevertheless, transient RNP activity does not typically result in optimal editing outcomes. By optimizing lipid nanoparticles (LNPs) encapsulating nanoparticles (NPs), the physiochemical properties of LNPs can be tailored to improve tissue penetration, cellular uptake, augment RNP stability, increase delivery efficacy and editing potency [174]. The combination of machine learning with the optimization of LNP programs for gene therapeutics is a significant development, harnessing its progonostic abilities to substantially quicken the research and improvement process.

Nanoparticles: Nanoparticles represent a heterogeneous group of materials, whether natural or synthetic, with dimensions aligning in the nanoscale. Inorganic nanoparticles are attractive option for transport of genetic materials into the cells. To allow regional administration of CRISPR/Cas9-based gene therapy, LNP transfer methods can be modified. Cell-penetrating peptides have also displayed great potential as delivery machineries for CRISPR enzymes, mostly in editing primary human lymphocytes [175]. Nanoparticles have shown substantial advantages in delivering miRNA to specific locations. Matched with naked miRNA delivery, nanoparticles decrease adverse off-target effects and immune responses while rising cellular uptake rates and half-life [176]. RNA nanoparticles can also be utilized for specific cancer therapy. A novel multivalent RNA nanoparticle has been devised to store three copies of hepatocyte targeting ligands [80]. These nanoparticles improve tumor localization and efficiently deliver miR122 and paclitaxel (PTX) to liver cancer cells, promoting their enhanced permeability and retention and receptor endocytosis mediated by hepatocyte targeting ligands. miR122 silences drug efflux proteins and oncoproteins, synergizing with PTX to increase cytotoxicity [80]. Recent revolutions in AI, interfacial chemistry, and materials science have initiated transformative potentials for the reasonable design of low inflammation-inducible NPs [174].

LNPs can target malignant cells with high precision while transferring the tumor suppressor p53 mRNA to stop the development of those cells significantly. PBA-BADP/Cas9 mRNA LNPs were delivered selectively and were more effectively in lowering the expression of genes in HeLa cancer cells than in non-malignant cells. These results confirmed the development of a novel lipid nanocarrier for tumor-targeted gene therapy [3]. Protamine sulfate was added to the Cas9/sgMTH1 plasmid to confer a negative charge prior covering it with cationic liposomes [58]. To expand durability in circulatory and tumor choosiness, DSPE-PEG-hyaluronic acid was added to the liposomes. Lung cancer can be successfully treated with a dry powder preparation of LNP-surrounded microparticles equipped with the CRISPR/Cas9 gene therapy means when administered locally [177].

Studies reported the application of Poly (lactic-co-glycolic acid) (PLGA) nanoparticles to transfer CRISPR/Cas9 to cancer cells with accuracy in vitro. Researchers showed how CRISPR/Cas9 was effectively administered to cancer cells using PLGA nanoparticles, which also notably increased the level of cellular apoptosis [178].

In another work, mRNA, and gRNA for Cas9 were carried to primary human T cells using a dual-LNPs method. In addition to efficient GE, propagation and differentiation, good cell vitality, and decreased toxicity were all observed by the researchers. Utilizing the same delivery means, they also reported successful deletion of the target gene in vivo [179]. The effectiveness of the GE was 31% and 39% for the in vitro and in vivo, respectively, with little off-target consequences. Researchers indicated that compared to viral vectors, multi-dosing using nonviral nanoparticles is more attractive and more practical alternative to generate the best GE outcomes [180]. In addition, the PE4Max and PE5Max editing systems were principally well-suited for mRNA delivery, lowering the mutagenic danger related to the MLH1dn element in their structure [33]. Thus, RNA delivery may be an efficient protocol for PE in cell cultures, embryos, and hematopoietic cells ex vivo [68].

Vehicle-free cellular delivery: One of the main difficulties in nucleic acid-based medicine is cellular uptake owing to effectiveness and potential toxicity of the vehicle. Fesler’s group used 5-FU-modified siRNAs as new anticancer treatment and selecting the well-described BCL-2 as their target. B-cell lymphoma-2 (BCL-2) is a key anti-apoptotic gene and is overexpressed in cancerous cells of different origin in various types of cancer [181]. It was found that in vitro combination of 5-FU-siBCL-2 induced apoptosis in presence or absence of a transfection vehicle in HCT 116 colon cancer cells and Toledo large B cell lymphoma cells [182]. In contrast, the unmodified siBCL-2 required a transfection vehicle to induce apoptosis in these cells. The reason behind this result is that fluorine group enhances the lipophilicity of the RNAi molecule, enabling its cellular uptake through the lipid bilayer of the cell membrane in absence of a vehicle. Further investigations are demanded to demonstrate the therapeutic efficacy of this novel 5-FU-siBCL-2 method in vivo.

Emerging genetic-engineering technologies: Technologies for precisely inserting large DNA sequences into the genome are critical for diverse research and therapeutic applications. In the above sections, we summarized the recent endeavor to develop advanced CRISPR tools, and emphasized the usefulness in cancer therapy. This section explores scientists’ efforts in searching for novel methods to engineer DNA (Figure 6).

Figure 6. An overview of emerging techniques for genome editing. A) The procedure of the DNA polymerase editor combines Histidine-hydrophobic residue-Histidine endonucleases (HuHe), DNA-Dependent Polymerase (DDP), and RNA-guided nCas9, allowing programmable and accurate genome engineering from ssDNA templates, including genome deletions, insertions and replacements. A key distinction from Prime Editor (PE) is that it utilizes DNA polymerase instead of Reverse Transcriptase (RT) and delivers the DNA template in trans. B) A recombinase guided by bridge RNA (Bridge RNA) can delete, insert, or invert, large DNA sequences at particular genomic sites. This RNA contains two loop assemblies that bind individually to the donor and target sequences. These loop constructions can be separately designed, allowing the recombinase to adjust specified sequences. C) A complex compounding the CRISPR-Cas effector system with Tn7 family transposons not only holds some of the functions of the CRISPR-Cas system, but also have the mobility of transposons. This allows RNA-guided transposition, enabling the insertion of large DNA sequences into particular genomic sites. D) By predicting on target/off-target effects and editing products, artificial intelligence is on the edge to significantly boost current GE technologies, mainly in the design of novel proteins and guide RNAs, where it has already accomplished breakthrough progresses. These advancements can greatly expand the accuracy and efficiency of GE, laying a solid basis for precise and secure gene therapies. Source from [68]. DDP: DNA-dependent DNA polymerases; GE: Gene editing; HUHe: Histidine–hydrophobic residue–Histidine endonucleases; nCas9: Nickase Cas9; ssDNA: PE: Prime editor; RT: Reverse transcriptase; sgRNA: Single-guide RNA, SSDNA: Single-stranded DNA

DNA polymerase editor: DNA polymerase editor (DPE) protocol can be performed by fusing engineered error-prone DNA polymerases (DPs) with Cas9 endonuclease [183]. This methodology facilitates the production of extensive genetic diversity within a particular region, which is suitable for functional gene inspection. The use of phage-derived DNA polymerases to establish edits at Cas9 cut sites utilizing a linear DNA template evades the issue of gRNA self-inhibition [184]. This technique not only increases the precision of editing but also enhances the potential for fusing long DNA segments into the genome. In addition, a novel method called “click editing” combines Histidine–hydrophobic residue–Histidine endonucleases (HuHe), DNA-dependent DPs (DDPs), and nCas9 to execute programmable, precise ENG, including ins/del and replacements, from simple DNA templates [185] (Figure 6A). Unlike PE, the new technology uses DDP instead of RT and delivers the DNA template in trans.

This DPE procedure does not require DSBs, thus, promoting precise GE with minimal insdel and while avoiding accidental insertions. The strength of this strategy lies in its ability to attain highly controlled GE without conceding genome integrity. The procedure offers more variable and flexible editing products compared to classical GE gene editing techniques, enabling

Bridge recombinase enzymes: Template-guided DSDNA break repair, such as HR, is highly effective in dividing cells but has inadequate success in postmitotic cells [186]. As the size of the insertion segment increases, the efficacy of HR-mediated insertion gradually decreases, limiting the insertion of long DNA segments. Although PE does not depend on HR, it presently enables the introduction of only a few dozen nucleotides [21]. The integration capability of PE has been increased by combining it with serine recombinases or integrases, allowing the insertion of DNA sequences up to several kbp [21]. Fully programable recombinases have long been sought after and have been described as the ultimate GE tool.

Bridge RNAs direct programmable recombination of donor and target DNA (Figure 6B). Two recent studies [187,188] have reported on the features of recombinases guided by “bridging” RNA molecules, which can be reprogrammed to open new GE capabilities. The bridge RNA consists of two internal loop structures with nucleotide successions that can pair with target DNA and the donor DNA) (Figure 6B). More investigations have demonstrated that the target-binding loop and donor-binding loop can be separately reprogrammed to direct particular sequence recombination between two DNA molecules [188]. This modularity allows the insertion of DNA into genomic target sites, in addition to programmable DNA excision and inversion. Generally, the modular nature of bridge RNA enables a comprehensive DNA rearrangement mechanism through sequence-specific deletion, insertion, or inversion.

Mukhametzyanova [189] developed a method to generate ZFDNA dependent recombinases, offering a substantial advancement toward realizing this pivotal objective. Another group [19] developed a method for actively integrating site-specific multi-kb DNA sequence to a known target sequence. Rational mutations were incorporated into the endogenous DNA-binding domain of the piggyBac transposase to reduce non-specific binding and promote preferential binding and targeted insertion by dCas9). This strategy enabled, for the first time, to direct transposition to the genome using RNA in human cells [19]. The programmed bridge recombination system increases the diversity of nucleic acid-guided systems further than CRISPR and RNAi, thus introducing a novel GE technology with new capabilities. The current innovation and optimization of new recombinases would further promote the effectiveness and specificity of this method [187,191,192].

CRISPR-associated transposons: CRISPR-associated transposons (CAST) are programmable mobile genetic elements that direct insert large DNA loads using an RNA-guided mechanism [193]. Transposons in the CASTs method are programmable so that they can autonomously integrate long DNA fragments in human cells without needing generation of DSBs observed with conventional CRISPR–Cas9, but with better incorporation of increased product purity and genome-wide specificity [194,195]. Researchers described CASTs which use Tn7-like transposase (TnsB and TnsC) subunits and type V-K or type I-F CRISPR effectors (Figure 6C), allowing for RNA-guided DNA relocation with unidirectional insertion of DNA parts at specific loci. The diverse mechanisms for the employment of Tn7-like transposons to CRISPR effectors would assist in the progress of CASTs as gene KI tools [196].

Recent studies revealed eukaryote-like TnpB proteins, termed Fanzors [197,198] also catalyze RNA-gDNA cleavage and support programmable GE [199]. Fanzor systems are expected to complement the traditional CRISPR-Cas systems. The Cas9-transposase fusion technology has improved transposition events close to target sites owing to nucleases compact size which simplifies delivery. However, the technology’s reduced efficiency and high off-target rate have restricted its widespread application [200]. Robust GE and therapeutic gene delivery would need further molecular engineering and optimization to promote the efficiency and targeting range of this method [190].

Artificial intelligence: There has been a great impact of the fast adoption of AI in the field of GE, significantly promoting the precision and efficacy of editing processes. AI has remarkably improved the ability to predict off-target effects and reduce unintended genetic changes (Figure 3D), thereby profoundly increasing the safety and efficiency of therapeutic applications [99,201]. Looking forward, AI is anticipated to further add to personalized GE strategies and enable the advancement of more effective and specific nucleases via novel computational protein design [202]. However, the AI methods also face challenges, for instance their dependence on high-quality training data and the “black box” character of AI, making the interpretation and credibility of predictions difficult. In spite of these opposes, the potential of AI in GE is huge, but careful consideration must be paid to addressing these issues.

Clinical risk considerations in gene editing: Gradually, as they divide, cells naturally pile up DNA damage, so a one-time, ordinary increase in somatic genetic modifications. In general, most genetic changes are expected to be neutral, and the few variations that may be damaging to cells typically may not have a substantial clinical impact.  Contrary to this, the most concerning events may be gain-of-function causing mutations, which can enhance clonal expansion and tumor development by activating oncogenes or preventing tumor suppressors. Researchers have been hunting for a simple way to correct those alterations by manipulating DNA.

Currently, GE technologies are still in the early stages of clinical advances, so their long-term safety and connected clinical risks warrant close attention. The ongoing research and promising outcomes prove that the potential of GE technologies in cancer treatment is not just a myth, but a burgeoning truth in the therapeutic landscape. There is great hope that GE could offer a treatment alternative for many types of cancer for which affected individuals have no other choice available.

As we stand on the edge of this new era of GEN, there have been limitations cannot be ignored. These include delivery inefficiencies, immunogenicity, off-target effects, and serious ethical concerns [113,114]. Though an increasing number of tools can be used to group on-target/off-target genomic effects [192]. somatic gene alterations themselves may not directly result in clinical consequences.  So far, no accidental cases of therapeutic GE resulting in tumors have been reported in clinical studies, indicating that the incidence of such effects may be extremely low. 

As questioned by Murray, et al. “which gene editing technique is the best?” The answer to this question “there isn’t one” Each alternate of the GE strategy has discrete features that can functionally complement the other’s limitations. At any rate, CRISPR-Cas variants offer a basis for repurposing the system for GE applications [18,203].

A part from the effectiveness, one major concern of the expected extensive employment in clinical settings is that the therapeutic choices must be available to patients. This involves addressing the regulatory obstacles that may impede the advancement of gene therapies, the high costs and complexity may hamper widespread adoption, delaying patient accessibility. In general, simpler and more cost-effective therapies are more possible to be accepted by patients.

Keynote ethical considerations include the long-term impact of GE on individuals and society, necessitating comprehensive risk evaluations and ethical discussions before implementation. It is imperative to realize that clinical attentions should extend to the complete GE therapeutic process, including any risks associated to cell delivery and host responses, which may be more usual clinical risks. For in vivo therapies, this may comprise harmful reactions of the host to gene transfer vectors such as viruses, which could elicit stress responses for example immune reactions and DNA injury responses. Therefore, a compromise between health benefits and health harms must always be the judge. It is important to prohibit the use of GE for eugenics reasons under any circumstances. We ought not to edit the human germline, or only in specific situations or for noteworthy purposes [204,205].

Limitations

This review has three main limitations. First, the quality of presented evidence was not properly reviewed. Second, some related articles may have been not included. Third, issues such as cancer immunotherapy using engineered cells other than CAR T-cells, such as NK cells, were not covered.

Conclusions

The present review discussed mainly a decade of GE achievements. Currently, there are five generations of PEs (PE1-PE5) with increasing levels of efficacy from one level to the next that have enormous clinical potential in adjusting mutations [206]. Researchers are using GE technologies to modulate immune cells, making them safer more efficient in recognizing and confronting cancer cells [207]. Although several methods of GE have been advanced over the years, none has actually fit the bill for a quick, easy, and cheap strategy. Various types of cancer will require precise and effective genome-targeting technologies to be treated in the future. Despite the remarkable revolution of this type of immunotherapy in hematological malignancies, the prospective applications of CAR-T therapy in non-hematological tumors and the overall clinical response levels and robustness of responses remain low and have to be improved further. In addition, access to CAR-T cells is not satisfactory to meet clinical needs, in part because of high cost [208]. Another challenge is the length of load time (also termed vein-to-vein times) for constructing and infusing of CAR T-cells and severe toxicities associated with CAR-T cell therapy. Moreover, because of the antigen escape, the restricted CAR-T cell persistence, and immunosuppressive TME, a significant proportion of patients relapse following CAR-T cell therapy [208]. Furthermore, the serious events related to CAR-T cell therapy might be severe or even life-threatening. Unlike other GE techniques that require engineering of distinctive proteins for each target sequence, CRISPR screening using large-scale gRNA genetic perturbation provides an unbiased approach to understanding mechanisms underlying anti-cancer efficacy of CAR T-cells. Another concern is related to the fact that CRISPR-edited cells may induce immune responses in recipients, potentially resulting in rejection or severe reactions. Potential uses of the CRISPR system to improve results of CAR T-cells therapy including optimizing efficacy and safety and, developing of next-generation and universal CAR T-cells [209].

Several emerging CRISPR techniques with high specificity, controllability and efficacy are useful to modify CAR T-cells and detect new targets. It is strongly believed that potential uses of GE technologies have to be controlled by multidisciplinary teams of experts across molecular biologists, bioengineers, and clinical oncologists. Future potential directions for GE are directed towards management of the biological code of life, single-cell sequencing, as well as AI and machine learning (ML). The integration of AI and ML will have significant impact on the CRISPR-based applications by enhancing GE precision, efficiency, and safety, helping refining, shaping, and expanding GE technologies and their applications in the coming years.

Acknowledgments

The author apologizes to those colleagues whose work is not cited due to restrictions on the number of references.

Conflicts of interest

The author declares no conflict of interest.

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