Abstract
Background
Pancreatic cancer is one of the main causes of cancer-related deaths, especially pancreatic ductal adenocarcinoma, and it’s characterized by a poor prognosis. The KRAS gene mutation is prevalent in about 85% of pancreatic cancer cases, which is a significant factor in the pathogenesis and development of pancreatic cancer, impacting cellular tumor growth, survival, and metastasis. The targeted disruption of mutant KRAS variants through the application of various CRISPR systems has led to a marked reduction in cell viability and proliferation in vitro, accompanied by significant inhibition of tumor growth in vivo.
Method
This systematic study adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and involved a comprehensive search of PubMed, Google Scholar, and PLOS One for original research articles published up to June 2024. Studies included those involving CRISPR-Cas9 gene editing targeting KRAS mutations in human or animal models of pancreatic cancer. Data collection and quality assessment were performed independently by two reviewers.
Results
The review identified numerous studies demonstrating the efficacy of CRISPR-Cas9 in targeting KRAS mutations. Results showed significant reductions in KRAS transcript levels, decreased tumor progression, and improved survival rates in experimental models. However, challenges such as off-target effects and efficient delivery methods were noted.
Conclusion
CRISPR-Cas9 gene editing shows significant promise as a therapeutic strategy for targeting KRAS mutations in pancreatic cancer. While the technology has demonstrated potential in preclinical studies, further research is needed to address challenges related to specificity, delivery, and long-term effects to facilitate its clinical application.
Keywords: Pancreatic cancer, PDAC, KRAS mutations, CRISPR-Cas9, Gene editing, Systematic review
Introduction
Pancreatic cancer is considered a global health burden and is expected to become one of the leading causes of cancer-related mortality in the future [1]. For men, the lifetime risk of pancreatic cancer is approximately 1 in 56, and for women, it is approximately 1 in 60. But a person's risk can be affected by a number of things, including chronic pancreatitis, smoking, obesity, and family history [2]. Pancreatic ductal adenocarcinoma (PDAC), which has a 5-year survival rate of roughly 10%, is still the most prevalent subtype of pancreatic cancer [3].
The RAS gene was initially identified as a viral gene homologous to the transforming gene from the Kirsten rat sarcoma virus [4, 5]. The KRAS protein, also known as p21, is a member of the Ras superfamily found on human chromosome 12 and encoded by 189 amino acids [6]. Numerous studies confirmed the involvement of KRAS mutations in about 85% of pancreatic cancer tumors, affecting various cell processes such as enhanced migration, invasion, survival, and proliferation while modulating cell metabolism by increasing glucose uptake, switching from aerobic glycolysis to mitochondrial oxidative phosphorylation (known as the Warburg effect), and increasing the generation of lactate and reactive oxygen species [7, 8].
The innovative gene-editing method referred to as CRISPR facilitates accurate modifications to the DNA sequence. The approach relies on the CRISPR-associated protein (Cas9) enzyme locating and severing a specific DNA sequence with the assistance of a small RNA molecule. This method utilizes the cell's intrinsic DNA repair processes, including homology-directed repair (HDR) and non-homologous end joining (NHEJ), to facilitate the introduction of precise mutations (knockouts) or repairs (knock-ins). CRISPR is extensively employed to create genetic models of human diseases in mice by either introducing mutations that mimic disease-causing variants or by knocking off genes. CRISPR is utilized to develop animal models of cancer, metabolic disorders, or neurological diseases, thereby offering significant insights into disease mechanisms and treatment trials.
CRISPR holds significant potential for the treatment of genetic disorders in clinical environments. In ex vivo applications, a patient's cells are modified externally. Hematopoietic stem cells can be engineered to rectify mutations responsible for disorders such as sickle cell anemia or β-thalassemia, after which the changed cells are reintroduced to the patient. In vivo applications utilize viral or lipid-based vectors to provide CRISPR components to specific tissues, such as the liver or retina. The potential for addressing several conditions, including cancer immunotherapy—where T-cells are engineered to more effectively target tumors—and genetic disorders such as Duchenne muscular dystrophy, is currently under investigation in ongoing clinical trials. Despite its potential, off-target effects and effective delivery mechanisms remain significant areas of research.
The management of pancreatic cancer involves a broad-based approach, incorporating surgical, chemical (chemotherapy), and radiation therapies to maximize patient outcomes [9–11]. Despite these efforts, the management of pancreatic cancer might be devastating due to the late presentation of the disease and poor prognosis [12, 13].
With these barriers to treating pancreatic cancer, a new approach in molecular biology has emerged, introducing advanced technologies for treatment. Among these approaches, gene editing has shown promising results [14].
Genome editing technologies were first introduced in the 1990s. More recently, a new genome editing tool called CRISPR, invented in 2009, has simplified the editing of DNA like never before. CRISPR is more efficient, quicker, less costly, and accurate than older genome editing techniques. Many scientists currently utilize CRISPR for genome editing, with significant research focused on targeting genetic mutations involved in pancreatic cancer. This has opened new avenues for potential treatments and therapies for this aggressive disease [15]
The first clinical trial for treating cancer using CRISPR was done in a West China hospital in 2016 [16]. This non-randomized, open-label phase I study evaluates the safety of ex vivo PD-1 knockout engineered T cells using CRISPR/Cas9 in treating metastatic non-small cell lung cancer [17]. While promising, the labor-intensive process of genetically modifying and infusing T-cells raises doubts about its clinical preference over PD-1 or PD-L1 antibodies. The study highlights the potential for combining PD-1 knockout with other targets and mentions ongoing and future trials, including those using CAR T-cells and in vivo genome editing approaches [18, 19].
In conclusion, pancreatic cancer remains challenging with low survival rates, especially with coexisting KRAS mutations. Emerging CRISPR-based genome editing offers promising new promise for targeted therapies, potentially transforming treatment strategies. While early trials have focused on other cancers, ongoing research underscores CRISPR/Cas9's potential to enhance pancreatic cancer management, necessitating further investigation for clinical application.
Methodology
Study design
To evaluate the efficacy of CRISPR-Cas9 gene editing for targeting KRAS mutations in pancreatic cancer, we conducted a systematic review of original articles.
Search strategy
This review adhered to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and is reported according to the PRISMA statement 2020. We conducted comprehensive searches in PubMed, Google Scholar, and PLOS One to identify relevant articles up to June 2024. Keywords for the search were developed with input from the contributing authors and included the following Boolean search terms: "CRISPR-Cas9" AND "KRAS" AND "Pancreatic Cancer" AND ("Efficacy" OR "Effectiveness"). Additional searches were performed manually by reviewing the reference lists of relevant articles.
Inclusion criteria
Studies were included if they met the following criteria: (i) original research articles investigating the application of CRISPR-Cas9 for targeting KRAS mutations in pancreatic cancer (both in vitro and in vivo studies, as well as early-phase clinical trials); (ii) articles published in peer-reviewed journals up to June 2024; and (iii) studies published in English.
Exclusion criteria
Exclusion criteria were defined as reviews, commentaries, case reports, cross-sectional studies, and unpublished or non-peer-reviewed articles. Additionally, a sensitivity analysis was performed by comparing the pooled outcomes with and without the excluded studies to evaluate any potential impact on the overall results. We excluded studies based on the strict criteria, reviews, commentaries, case reports, or cross-sectional studies, unpublished or non-peer-reviewed studies, articles not available in full text, and studies published in languages other than English were exceled.
Figure 1 illustrates the flow chart of studies selection.
Fig. 1.
The flow chart of studies selection
Data extraction
Two reviewers independently extracted data from the included studies using a standardized form. Extracted data included study characteristics (e.g., study design, location), population details (e.g., demographics, KRAS mutation status), intervention specifics (e.g., CRISPR-Cas9 delivery method, target sequences), and outcomes (e.g., efficacy measures, off-target effects, safety). Any discrepancies between the reviewers were resolved through discussion and consensus.
Heterogeneity across studies was rigorously assessed. For quantitative synthesis, the I2 statistic and chi-squared tests were used to evaluate the degree of heterogeneity. In instances where I2 values exceeded 50%, indicating substantial heterogeneity, a random-effects model was employed to merge the data. For the qualitative synthesis, thematic analysis was utilized to discern patterns and variations among study findings, ensuring a comprehensive evaluation of the data consistency.
Quality assessment
We conducted a quality assessment of each included study based on predefined criteria derived from established guidelines for evaluating genetic research. Criteria included the methodological strength of the experimental design, accuracy of gene editing, and assessment of off-target effects. Each study was scored on a scale from 0 to 8 to quantify its methodological quality (Figs. 2 and 3).
Fig. 2.
Risk of bias graph
Fig. 3.
Risk of bias summary
Data synthesis
We conducted a qualitative synthesis to thoroughly evaluate how effective CRISPR-Cas9 gene editing is at targeting KRAS mutations in pancreatic cancer. This process involved summarizing each study’s goals, methods, and findings, focusing on the specifics of the CRISPR-Cas9 intervention and the outcomes reported. We looked for themes like the efficiency of gene editing, the impact on tumor progression, and safety concerns, using thematic analysis to identify these. By comparing the results of different studies, we highlighted both consistent findings and areas where results varied, which helped us develop a thorough understanding of how effective CRISPR-Cas9 is. We used the GRADE approach to assess the overall strength of the evidence to ensure it was comprehensive.
Results
Study selection
We identified 98 articles from different electronic databases and an additional three from other sources. After removing duplicates, 51 articles proceeded to the primary screening stage. 18 articles were excluded in the title and abstract screening, and five were excluded through full-text screening. The remaining 11 studies were included in the qualitative synthesis, with 6 further included in the quantitative synthesis. Figure 1, the PRISMA flow diagram, summarizes the process.
Study characteristics
This systematic review included six clinical trials conducted between 2016 and 2023, and 3 studies were carried out in the USA, 2 in China, and 1 in Germany. The included studies were primarily preclinical trials involving in vitro and in vivo models (e.g., cell lines and mouse models). Table 1 shows the baseline characteristics of the articles included.
Table 1.
The baseline table of the included studies
| Study | Year | Country | Study type | Arms | Intervention |
|---|---|---|---|---|---|
| Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states [1] | 2021 | USA | Clinical trial | Two major experimental arms: 1. In vitro cell line studies.2. In vivo orthotopic tumor studies in mice | macsGESTALT,an inducible CRISPR-Cas9-based lineage recorder |
| Exosome-mediated delivery of CRISPR/Cas9 for targeting of oncogenic KrasG12D in pancreatic cancer [2] | 2021 | USA | Clinical trial | Two major experimental arms: 1. Subcutaneous tumor model in albino mice 2.orthotopic tumor model of pancreatic cancer in tumor mice | Exosome-mediated delivery of CRISPR/Cas9 |
| Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice [3] | 2016 | Germany | Clinical trial | Two major experimental arms: 1. healthy pancreatic control tissue 2. primary pancreatic tumor tissue | Transfection-based CRISPR/Cas9 delivery |
| Precise and efficient silencing of mutant KrasG12D by CRISPR-CasRx controls pancreatic cancer progression [4] | 2020 | China | Clinical trial | Three major experimental arms: | CRISPR-CasRx system |
| 1. In vitro cell line studies | |||||
| 2. In vivo orthotopic tumor studies in mice | |||||
| 3. In vivo PDX model studies | |||||
|
A CRISPR-Cas13a system for efficient and specific therapeutic targeting of mutant KRAS for pancreatic cancer treatment [5] |
2018 | China | Clinical trial | Two experimental arms | CRISPR-Cas13a System: |
| In vitro arms | Optimization of crRNA: | ||||
| In vivo arms | |||||
| Combination Therapies with CDK4/6 Inhibitors to Treat KRASmutant Pancreatic Cancer [6] | 2023 | USA | Phase 1 clinical trial | Single experimental arm: patients with advanced pancreatic ductal adenocarcinoma | CDK4/6 Inhibitors along with ERKi ulixertinib |
CRISPR-based tumor induction and metastasis tracking
In one study, a CRISPR-Cas9-based system called macsGESTALT was used as an evolving barcode to track the evolution of pancreatic ductal adenocarcinoma (PDAC) cells (Tables 2 and 3). The study revealed that only a small fraction of cancer cell clones contributed to tumor growth and metastasis, with the M1.1 and M2.2 clones being particularly dominant in distant metastatic sites (Table 4). Clones in late-hybrid epithelial-to-mesenchymal transition (EMT) states, like M1.1, exhibited higher metastatic potential. Additionally, the M2.2 clone showed elevated expression of S100 family genes, suggesting a role in metastasis [1]. Table 5 presents the detailed outcomes.
Table 2.
The 1st outcomes of the studies
| % of transcriptomes with clonoal lineage information | |
|---|---|
| M1 | M2 |
| 89 | 77 |
Table 3.
In vivo studies outcomes
| PDAC cell lines sensitive to palbociclib | Inhibition of Rb phosphorylation by CDK4/6i treatment | Return of pRb to pre-treatment levels | Increase in G1 stage populaion with CDK4/6 inhibition | Effect of palbociclib on | ERK activity after CDK4/6i treatment | Synergistic effect of ERK1/2-selective inhibitor SCH772984 (ERKi) and CDK4/6i | Synergistic effect of CDK4/6i and KRASG12C-mutant selective inhibitor sotorasib/AMG510 | Total genes targeted for Loss-of-function CRISPR/Cas9 screen | Effect of loss of different genes on efficacy of CDK4/6i detected using Loss-of-function CRISPR/Cas9 screen | Effect of small molecular inhibitors on CDK4/6i activity | Correlation between the recovery of a cell’s transcriptomic RNA and the barcode RNA | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Cell proliferation marker Ki-67 levels after CDK4/6i therapy | Proteins in the anti-apoptotic and PI3K-AKT-mTOR pro-survival signaling pathway | Treatment group vs control group | On proliferation of PDAC cell lines | On KRASG12C-mutant MIA PaCa-2 cells | For enhanced CDK4/6i function | ||||||||||||
| N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | RB1 | MYC | CDK1 | BAP1 | EZF3 | SRC inhibitor bosutinib | HDAC inhibitor quisinostat | Possible correlation(r = 0.64) |
| 13 out of 15 | 24 h | 3–5 days | 80% | Reduced | Increased | High in treatment group | Decreased proliferation | Decreased proliferation | 2240 | Decreased sensitivity | Increased sensitivity | Increased sensitivity | Increased sensitivity | Increased sensitivity | Increased acticity | Increased activity | N/A |
| Cells from which transcrpitome and clonal history were recovered | Recovered bardcodes per cell | No of distinct clones | No of static barcodes | Cells coming from a single clone(bottle necking) | % of clones metastizing | % of clones in single transcriptional cluster | % of non-aggressive and transcriptionally similar clones | Cell markers rich in non-aggressive clones | Cell markers rich in aggressive clones | Prrx1 expression in epithelial to mesenchymal transformation | Zeb1, Zeb2, Snai1, and Snai2 expression | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| M1 | M2 | M1 | M2 | ||||||||||
| 12,657 | 15,371 | 3.7 of a possible 5.9 | 1.7 of a possible 2.5 | 95 | 227 | 80–90% | 15% | 81% | 85% | Epcam, Muc1, and Cdh1 | Sparc, Zeb2, and Col3a1 | Increased | Increased |
| N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
Table 4.
The efficacy of the intervention
| Total recovered CRISPR target sites | % of edited CRISPR target sites | Recovered target sites per cell | Intervention with | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| M1 | M2 | Smallest tumor size | Smallest tumor burden | Smallest tumor weight | Least activation of ERK | Increased wild-type KRAS levels | Cas9 detectable at mRNA level | Presence of intratumoral sgRNA | ||
| 384,870 | 96% | 3.7 | 1.7 | Treatment group that received MSC exosomes loaded with Cas9/ KrasG12D sgRNA1 plasmid | Group that received MSC exo somes loaded with the plasmid Cas9/KrasG12D sgRNA1 | Group that received MSC exo somes loaded with the plasmid Cas9/KrasG12D sgRNA1 | N/A | N/A | N/A | |
| N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | In tumors treated with MSC exosomes loaded with the plasmid Cas9/KrasG12D sgRNA1 | Group that received exosomes + Exo-Fect + KrasG12D sgRNA1, Exo-Fect + KrasG12D sgRNA1, and exosomes + Exo-Fect + vector control | Groups that received Exo-Fect + KrasG12D sgRNA1 and exosomes + Exo-Fect + KrasG12D sgRNA1 |
Table 5.
The intervention outcomes
| Average edit size(bp) | Maximum estimated labeling for barcode for both mice | Total unique barcodes actually recovered | Total subclones actually recovered | M1.1 barcode integrants | M1.1 subclones | M2.2 barcode integrants | M2.2 subclones | S100a expression in M2 | S100a expresssion in M1 | ||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Insertions | Single-target deletions | Multi-target deletions | |||||||||
| 11 bp | 18 bp | 80 bp | 10^9 | 6,055 | 1,692 | 7 | 601 | 2 | 110 | High | Low |
Another study demonstrates how efficiently CRISPR/Cas9 induces pancreatic cancer through multiplexed gene targeting and electroporation, leading to rapid tumorigenesis (Table 6) [3]. Additionally, it enabled precise tracking of metastases, highlighting its effectiveness in generating and analyzing complex cancer models and its utility in creating and studying intricate canicer models (Table 7).
Table 6.
In vitro studies
| Muc1 expression | Zeb2, Vim, and Fn1 | Significantly enriched DNA-motif binding factors across all gene clusters | Zeb1, Zeb2, Snai1, and Snai2 in early clusters (E and H1) | Sox and Fox families in H4 and M |
|---|---|---|---|---|
| High | Low | 45 | Enriched | Enriched |
Table 7.
The prognosis of M1 and M2
| Prognosis in M1 | Prognosis in M2 | ||||||
|---|---|---|---|---|---|---|---|
| Association with PDAC-derived psudoEMT gene sets enriched with H4 hybdrid state | Association with PDAC-derived psudoEMT gene sets enriched with H4 hybdrid state | ||||||
| Lung cancer | Colorectal cancer | Breast cancer | Prostate cancer | Lung cancer | Colorectal cancer | Breast cancer | Prostate cancer |
| Worse | None | None | None | Worse | None | None | None |
| Worse | None | None | None | Worse | None | None | None |
Efficacy of CRISPR system in PDAC suppression
The included articles used Different methods to deliver CRISPR-Cas9 into target cells, including exosome, electroporation, and viral vectors (Table 8).
Table 8.
Association of genes with dissemination in M2 (+ ve or −ve) and enrichment of S100a
| Association of genes with dissemination in M2(+ ve or −ve) | Association of genes with enrichment of S100a | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Ocln | Epcam | Lgals4 | Prrx1 | Zeb2 | Muc1 | Ctse | Ifitm | Ifitm3 | Akr1b3 | Positively associated genes | Negatively associated genes |
| Negative | Negative | Negative | Positive | Positive | Negative | Negative | Positive | Positive | Positive | 52-fold enrichment | Completely absent |
Exosome-mediated delivery of CRISPR/Cas9 plasmid DNA effectively targeted KrasG12D in pancreatic cancer cells, significantly suppressing KrasG12D mRNA and reduced cell proliferation in vitro. Similarly, MSC exosomes loaded with Cas9/KrasG12D sgRNA1 reduced tumor volume, weight, and burden in the mouse model [2].
The AAV8-CasRx-gRNA system effectively targeted and silenced mutant KrasG12D in PDAC tumors, reducing Kras mRNA levels by 50% and significantly suppressing tumor growth in mouse models. Mice treated with AAV8-CasRx-gRNA had a 33% increase in survival and showed decreased tumor metabolic activity (Tables 9 and 10). The system also enhanced sensitivity to Gemcitabine. This approach significantly slowed tumor growth in patient-derived xenografts harboring KRAS G12D mutations [4] (Table 9).
Table 9.
The Effect of CRISPR/Cas9 mediated gene targeting, of exosome mediated CRISPR/Cas9 delivery and tumorigeneisis in PK mice receiving 15 -sgRNA and PK mice receiving neutral-sgRNA
| Effect of CRISPR/Cas9 mediated gene targetting | Effect of exosome mediated CRISPR/Cas9 delivery | Tumorigeneisis in PK mice receiving 15 -sgRNA | Tumorigenesis in PK mice receiving neutral-sgRNA | |||
|---|---|---|---|---|---|---|
| KRAS expression in pancreatic cancer cells | KRAS sgRNA1 expression in pancreatic cancer cells | KRAS sgRNA2 expression in pancreatic cancer cells | Proliferation of KPC689 cells | 4 weeks post-electroporation | 24 weeks post-electroporation | |
| Significant suppression | Significant suppression | Significant suppression | Significantly suppressed | N/A | N/A | N/A |
| N/A | N/A | N/A | N/A | Significant number | 7 out of 13 | No tumor detected |
Table 10.
per tumor in 23 human PDAC cell lines and effect on KRAS levels
| Effect of CRISPR/Cas9 on BRCA2 | Per tumor in 23 human PDAC cell lines | Deletions linked to unbalanced translocation (Mb) | CRISPR/Cas9-induced deletion or translocation per tumour | No of unbalanced translocations seen in M-FISH | Genes affected by intra-chromosomal translocation | Effect on KRAS levels | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Intrachromosomal deletions/amplifications | Interchromosomal translocations | Cotransfection of cells with CasRx and gRNA | |||||||||
| CKDN2A | SMAD4 | P53 | PANC-1 | H6c7 | MIAPaCa-2 | ||||||
| No mutation | 139 | 8 | 44.4, 28.6 and 5.0 Mb | Less than 1 | 2 out of 6 cell lines | Affected | Affected | Affected | N/A | N/A | N/A |
| NA | NA | NA | NA | NA | NA | NA | NA | NA | Transfection efficiency was modest (20% ~ 30% GFP-positivity) | ||
The CRISPR-Cas13a system, using crRNA19-14, specifically targeted KRAS-G12D mRNA, achieving up to 94% reduction in mRNA levels and 70% suppression of tumor growth in pancreatic cancer cell lines in vitro. Similarly, in mice, it significantly inhibits tumor growth (Table 11), reduces proliferation, and increases apoptosis [5].
Table 11.
Reduction in level of total KRAS transcripts in cells targeted with CasRx/gRNA
| Reduction in level of total KRAS transcripts in cells targetted with CasRx/gRNA (experiment vs control) | Protein levels of p-Akt and p-Erk in cells targetted with CasRx + /gRNA + | Proportion of mutant KrasG12D transcript in CasRx-/gRNA + PANC-1 | Knockdown efficiency of KRAS transcripts by CasRx-gRNA in AsPC-1 | Effect of CasRx on levels of proteins in AsPC-1 cells | Effect of CasRx-gRNA on sensitivity to Gemcitabine | Increase in survival of mice by using AAV8 vector delivery of CasRx-gRNA | Reduction of Kras mRNA by using AAV8 vector delivery of CasRx-gRNA | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| PANC-1 | H6c7 | MIAPaCa-2 | PANC-1 | H6c7 | MIAPaCa-2 | Kras | p-Erk | p-Akt | PANC-1 | H6c7 | MIAPaCa-2 | without Gemcitabine | With Gemcitabine | |||
| Greater than 50% | No significant decrease | No significant decrease | Reduced | Minimal effects | Minimal effects | 86% | 90% | Reduced | Reduced | Reduced | Increased | No affect | No affect | 33% | 66% | 50% |
| IC50 of Gemcitabine reduced by approximately tenfold | ||||||||||||||||
In another study, CRISPR/Cas9 was used to identify genes that affect sensitivity to CDK4/6 inhibitors in KRAS-mutant PDAC cell lines. The screen revealed key genes involved in PI3K–AKT–mTOR signaling, cell-cycle regulation, and DNA damage repair that modulate response to these inhibitors. Validation showed that targeting genes like CDK2 and BAP1 increased inhibitor efficacy (Table 12). Additionally, CRISPR findings guided the use of synergistic drug combinations, such as SRC and HDAC inhibitors, to enhance CDK4/6 inhibitor effectiveness, providing new insights for improving treatment outcomes [6]. Table 4 summarizes the findings of this study.
Table 12.
Safety of AAV8 vector CasRx-gRNA delivery and Efficiency
| Safety of AAV8 vector CasRx-gRNA delivery | Efficiency | ||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Effect on weight of mice | Effect on mice liver size | crRNA19 induced KRAS-G12D mRNA knockdown efficiency when co-transfected with the Lw.Cas13a protein | Increase in KRAS-G12D knockdown efficiency after single mismatch mutaion | Suppresion of levels of phosphorylated-ERK and phosphorylated-AKT protein in KRAS-G12D mutated cells | Suppresion of growth of pancreatic cancer cells with the KRAS-G12D mutation having co-transfection of Lw.Cas13a protein and crRNA19-14 | Tumor cells proliferation rate(in vivo) with the KRAS-G12D mutation having co-transfection of Lw.Cas13a protein and crRNA19-14 | Extent of apoptosis in tumor cells(in vivo) with the KRAS-G12D mutation having co-transfection of Lw.Cas13a protein and crRNA19-14 | ||||||||||||||
| AsPC-1 | PANC-1 | HPAF-II | T3M4 | HPNE | AsPC-1 | PANC-1 | HPAF-II | T3M4 | HPNE | AsPC-1 | PANC-1 | HPAF-II | T3M4 | HPNE | Control crRNA | crRNA19-14 | Control crRNA | crRNA19-14 | |||
| No significant effect | No significant effect | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| NA | NA | 94% | 88% | 84% | 60% | 60% | Up to 70% | Up to 70% | Up to 70% | Up to 70% | Up to 70% | Reduced | Reduced | Reduced | No apparent effect | No apparent effect | Less dec compared to control | Decreased substantially compared to control | 7.90% | 19.40% | |
The preclinical and early-phase clinical data suggest that CRISPR-Cas9 gene editing holds significant promise for targeting KRAS mutations in pancreatic cancer. The observed reductions in KRAS transcript levels, tumor growth suppression, and improved survival rates in experimental models underscore the potential of this technology. However, the translation into clinical practice requires overcoming challenges such as optimizing delivery systems, minimizing off-target effects, and determining the efficacy of combination therapies with existing treatments. Future clinical trials are essential to evaluate the real-world applicability and long-term benefits of CRISPR-Cas9-based interventions.
While CRISPR-Cas9 represents a transformative therapeutic approach, its clinical application is tempered by safety and ethical concerns. Notably, off-target genetic modifications and immune responses pose significant risks. Ethical issues—such as unintended germline editing and the long-term effects of genomic alterations—must be thoroughly addressed. Our review emphasizes the need for the development of high-fidelity Cas9 variants, rigorous preclinical safety evaluations, and adherence to ethical guidelines to mitigate these risks prior to widespread clinical use.
Discussion
This systemic review emphasizes how pancreatic cancer, a deadly illness with few available treatments, may benefit from CRISPR-Cas9 gene editing to target KRAS mutations. Promising outcomes from the included studies include effective editing of KRAS mutations, inhibition of tumor growth and proliferation, and higher rates of animal model survival.
Considering the difficulties with conventional treatments, the effectiveness of CRISPR-Cas9 in addressing KRAS mutations is noteworthy. KRAS mutations are frequently found in pancreatic cancer, and their existence is frequently linked to a worse prognosis and treatment resistance [1, 2].
The research included various delivery techniques, such as exosomes, electroporation, and viral vectors. Exosome administration has been shown to significantly decrease tumor development and boost survival rates, indicating that this strategy could be a practical and effective way to target KRAS mutations [3].
Nevertheless, the existing body of evidence has flaws and limits. More research is required to close these knowledge gaps and maximize the application of CRISPR-Cas9 in the therapy of pancreatic cancer. To be more precise, additional in vivo research is needed to completely comprehend the safety and effectiveness of CRISPR-Cas9 in animal models [4, 5].
Additionally, human clinical trials are required to use these discoveries in the clinic. It's also important to consider the possibility of CRISPR-Cas9 combination therapy. The potential for combination therapeutic methods is shown by the synergistic effect of CRISPR-Cas9 and CDK4/6 inhibitors in decreasing tumor development and enhancing survival rates [6, 7].
The review also emphasizes how crucial it is to optimize the design and delivery of CRISPR-Cas9 guide RNA (gRNA) to target KRAS mutations in an effective and targeted manner. Reducing off-target effects and improving gRNA design can be achieved through in vitro validation assays and computational techniques [1, 5].
The evaluation additionally underscores the necessity of more investigation into the effectiveness and safety of CRISPR-Cas9 in human subjects. Although the included studies show encouraging outcomes, issues with immunogenicity, mosaicism, and off-target effects remain.
These issues must be resolved for CRISPR-Cas9 to be successfully translated into the clinic. Subsequent research endeavors must refine CRISPR-Cas9 delivery strategies, investigate combination treatments, and resolve safety issues [4].
The review also highlights how crucial it is to consider the tumor microenvironment and how it affects the effectiveness of CRISPR-Cas9. The tumor microenvironment significantly influences treatment resistance and the advancement of cancer.
Comprehending the interactions between CRISPR-Cas9 and the tumor microenvironment is crucial to design successful treatment plans. The evaluation additionally highlights the need for more investigation into the possibility that CRISPR-Cas9 could be used to target additional carcinogenic mutations in pancreatic cancer [3].
One major advantage of CRISPR-Cas9 over conventional treatments is its ability to address many mutations simultaneously. Improved patient survival and more successful treatment results could result from this strategy.
To fully grasp the therapeutic potential of CRISPR-Cas9 in treating pancreatic cancer, additional study is necessary. CRISPR-Cas9 may provide pancreatic cancer patients new hope with more research and development.
This systemic review concludes that CRISPR-Cas9 gene editing has potential as a targeted treatment for pancreatic cancer. Notwithstanding certain restrictions and gaps in the available data, the results imply that CRISPR-Cas9 might be a useful therapeutic option for this incurable illness [2].
More study is required to fully grasp CRISPR-Cas9's therapeutic potential in the treatment of pancreatic cancer. Treatment for pancreatic cancer appears to have a bright future, and CRISPR-Cas9 may be essential to bettering patient outcomes.
The pioneering CRISPR-Cas9 technology, albeit revolutionary, carries the risk of unintentional genetic alterations. Off-target modifications may result in serious consequences, including the onset of cancer or the disturbance of critical genes necessary for regular cellular function. We are committed to improving the precision of the technology by creating high-fidelity Cas9 variations, designed to enhance specificity and reduce off-target alterations. Expert guide RNA design, coupled with advanced computational techniques, is essential for markedly decreasing the probability of mismatches throughout the editing process. Thorough validation studies—both in vitro and in vivo—are crucial for detecting and correcting any off-target effects before to advancing to clinical applications. The introduction of CRISPR components into the body can elicit immunological responses, especially when employing viral vectors or the Cas9 protein, potentially resulting in adverse effects that jeopardize patient safety. We are investigating the utilization of humanized or alternative Cas proteins aimed at reducing immune responses and enhancing patient tolerance. We are examining non-viral delivery mechanisms, including lipid nanoparticles and exosomes, which may provide safer and more efficient options for delivering CRISPR components into target cells. Throughout clinical trials, we will establish stringent surveillance measures to promptly detect and address any immune-related adverse events, prioritizing patient safety.
This study has encountered several limitations. A major challenge is the efficient and precise delivery of CRISPR-Cas9. Notwithstanding encouraging outcomes, apprehensions over inadvertent genetic alterations persist. Furthermore, a more thorough comprehension of the long-term consequences and possible hazards to humans is required. The majority of evidence is derived from preclinical models, with a limited number of human trials completed thus far. The heterogeneity in CRISPR delivery mechanisms—such as exosomes, electroporation, and viral vectors—impacts the consistency of results. The relationships between CRISPR-Cas9 and the tumor microenvironment have been minimally explored.
This study has several limitations. First, the majority of the evidence is derived from preclinical models and early-phase trials, which may not fully represent the clinical scenario. Second, the heterogeneity in study design, sample size, and methodologies across the included studies may affect the generalizability of the findings. Finally, the exclusion of certain types of studies—though necessary for methodological rigor—may limit the comprehensiveness of the review. These limitations highlight the need for future large-scale, standardized clinical trials to validate the efficacy and safety of CRISPR-Cas9 gene editing in pancreatic cancer.
Conclusion
In conclusion, while the results of this review highlight the promising role of CRISPR-Cas9 in treating KRAS-mutant pancreatic cancer, significant challenges remain before this technology can be safely and effectively implemented in clinical practice. Continued research, particularly in optimizing delivery methods and minimizing off-target effects, will be essential in translating these preclinical successes into tangible clinical benefits.
Acknowledgements
None.
Author contributions
Hashim Talib Hashim: Conceptualized the study, designed the systematic review and meta-analysis protocol, and supervised the research process. AQMA: Conducted the literature search, data extraction, and initial data analysis. TF: Performed statistical analyses and contributed to the interpretation of the results. TH: Reviewed and selected studies for inclusion, and ensured the accuracy of data extraction. FAS: Assisted in the literature search, data extraction, and provided critical revisions of the manuscript. MGM: Contributed to the design of the data extraction forms and assisted in the critical appraisal of studies. MAA: Participated in data extraction, manuscript drafting, and formatting. RAK: Reviewed the manuscript for important intellectual content and contributed to the writing process. ADA: Assisted in statistical analysis, data interpretation, and manuscript preparation. AA: Provided expertise in ophthalmology, reviewed the clinical aspects of the included studies, and contributed to manuscript revisions. All authors read and approved the final manuscript.
Funding
No funding was received.
Data availability
All data generated or analyzed during this study are included in this published article.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Hashim Talib Hashim, Email: [email protected].
Mohammed Ganim, Email: [email protected].
References
- 1.Simeonov Kamen P, et al. Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states. Cancer Cell. 2021;39(8):1150–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.McAndrews KM, et al. Exosome-mediated delivery of CRISPR/Cas9 for targeting of oncogenic KrasG12D in pancreatic cancer. Life Sci Alliance. 2021;4(9):e202000875. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Maresch R, et al. Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice. Nat Commun. 2016;7(1):10770. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Jiang W, et al. Precise and efficient silencing of mutant KrasG12D by CRISPR-CasRx controls pancreatic cancer progression. Theranostics. 2020;10(25):11507. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Zhao X, et al. A CRISPR-Cas13a system for efficient and specific therapeutic targeting of mutant KRAS for pancreatic cancer treatment. Cancer Lett. 2018;431:171–81. [DOI] [PubMed] [Google Scholar]
- 6.Goodwin CM, et al. Combination therapies with CDK4/6 inhibitors to treat KRAS-mutant pancreatic cancer. Cancer Res. 2023;83(1):141–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Klein AP. Pancreatic cancer epidemiology: understanding the role of lifestyle and inherited risk factors. Nat Rev Gastroenterol Hepatol. 2021;18(7):493–502. 10.1038/s41575-021-00457-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.American Cancer Society. Key Statistics for Pancreatic Cancer. 2024. www.cancer.org, https://www.cancer.org/cancer/types/pancreatic-cancer/about/key-statistics.html. Accessed 14 Apr 2025.
- 9.Park W, Chawla A, O'Reilly EM. Pancreatic Cancer: A Review. JAMA. 2021 Sep 7;326(9):851-862. 10.1001/jama.2021.13027. Erratum in: JAMA. 2021 Nov 23;326(20):2081. 10.1001/jama.2021.19984. PMID: 34547082; PMCID: PMC9363152. [DOI] [PMC free article] [PubMed]
- 10.Chang EH, Furth ME, Scolnick EM, Lowy DR. Tumorigenic transformation of mammalian cells induced by a normal human gene homologous to the oncogene of Harvey murine sarcoma virus. Nature. 1982;297(5866):479–83. 10.1038/297479a0. [DOI] [PubMed] [Google Scholar]
- 11.Der CJ, Krontiris TG, Cooper GM. Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses. Proc Natl Acad Sci U S A. 1982;79(11):3637–40. 10.1073/pnas.79.11.3637. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer. 2003 Jun;3(6):459-65. 10.1038/nrc1097. Erratum in: Nat Rev Cancer. 2003;3(9):708. PMID: 12778136. [DOI] [PubMed]
- 13.Jonckheere N, Vasseur R, Van Seuningen I. The cornerstone K-RAS mutation in pancreatic adenocarcinoma: from cell signaling network, target genes, biological processes to therapeutic targeting. Crit Rev Oncol Hematol. 2017;111:7–19. 10.1016/j.critrevonc.2017.01.002. [DOI] [PubMed] [Google Scholar]
- 14.Perera RM, Bardeesy N. Pancreatic cancer metabolism: breaking it down to build it back up. Cancer Discov. 2015;5(12):1247–61. 10.1158/2159-8290.CD-15-0671. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Donahue TR, Reber HA. Surgical management of pancreatic cancer–pancreaticoduodenectomy. Semin Oncol. 2015;42(1):98–109. 10.1053/j.seminoncol.2014.12.009. [DOI] [PubMed] [Google Scholar]
- 16.Springfeld C, Jäger D, Büchler MW, Strobel O, Hackert T, Palmer DH, Neoptolemos JP. Chemotherapy for pancreatic cancer. Presse Med. 2019;48:e159–74. 10.1016/j.lpm.2019.02.025. [DOI] [PubMed] [Google Scholar]
- 17.Landau E, Kalnicki S. The evolving role of radiation in pancreatic cancer. Surg Clin North Am. 2018;98(1):113–25. 10.1016/j.suc.2017.09.008. [DOI] [PubMed] [Google Scholar]
- 18.Hu JX, Zhao CF, Chen WB, Liu QC, Li QW, Lin YY, Gao F. Pancreatic cancer: a review of epidemiology, trend, and risk factors. World J Gastroenterol. 2021;27(27):4298–321. 10.3748/wjg.v27.i27.4298. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Cai J, Chen H, Lu M, Zhang Y, Lu B, You L, Zhang T, Dai M, Zhao Y. Advances in the epidemiology of pancreatic cancer: trends, risk factors, screening, and prognosis. Cancer Lett. 2021;520:1–11. 10.1016/j.canlet.2021.06.027. [DOI] [PubMed] [Google Scholar]
- 20.Liu Z, Shi M, Ren Y, Xu H, Weng S, Ning W, Ge X, Liu L, Guo C, Duo M, Li L, Li J, Han X. Recent advances and applications of CRISPR-Cas9 in cancer immunotherapy. Mol Cancer. 2023;22(1):35. 10.1186/s12943-023-01738-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All data generated or analyzed during this study are included in this published article.