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CRISPR/Cas9 gene editing: a new approach for overcoming drug resistance in cancer.
The CRISPR/Cas9 system is an RNA-based adaptive immune system in bacteria and archaea. Various studies have shown that it is possible to target a wide range of human genes and treat some human diseases, including cancers, by the CRISPR/Cas9 system. In fact, CRISPR/Cas9 gene editing is one of the most efficient genome manipulation techniques. Studies have shown that CRISPR/Cas9 technology, in addition to having the potential to be used as a new therapeutic approach in the treatment of cancers, can also be used to enhance the effectiveness of existing treatments. Undoubtedly, the issue of drug resistance is one of the main obstacles in the treatment of cancers. Cancer cells resist anticancer drugs by a variety of mechanisms, such as enhancing anticancer drugs efflux, enhancing DNA repair, enhancing stemness, and attenuating apoptosis. Mutations in some proteins of different cellular signaling pathways are associated with these events and drug resistance. Recent studies have shown that the CRISPR/Cas9 technique can be used to target important genes involved in these mechanisms, thereby increasing the effectiveness of anticancer drugs. In this review article, studies related to the applications of this technique in overcoming drug resistance in cancer cells will be reviewed. In addition, we will give a brief overview of the limitations of the CRISP/Cas9 gene-editing technique.
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Effective use of sequence information to predict CRISPR-Cas9 off-target.
The CRISPR/Cas9 gene-editing system is the third-generation gene-editing technology that has been widely used in biomedical applications. However, off-target effects occurring CRISPR/Cas9 system has been a challenging problem it faces in practical applications. Although many predictive models have been developed to predict off-target activities, current models do not effectively use sequence pair information. There is still room for improved accuracy. This study aims to effectively use sequence pair information to improve the model's performance for predicting off-target activities. We propose a new coding scheme for coding sequence pairs and design a new model called CRISPR-IP for predicting off-target activity. Our coding scheme distinguishes regions with different functions in the sequence pairs through the function channel. Moreover, it distinguishes between bases and base pairs using type channels, effectively representing the sequence pair information. The CRISPR-IP model is based on CNN, BiLSTM, and the attention layer to learn features of sequence pairs. We performed performance verification on two data sets and found that our coding scheme can represent sequence pair information effectively, and the CRISPR-IP model performance is better than others. Data and source codes are available at https://github.com/BioinfoVirgo/CRISPR-IP.
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Rapid genome editing by CRISPR-Cas9-POLD3 fusion.
Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein-Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.
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Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing.
Gene knock-in in mammalian cells usually uses homology-directed repair (HDR) mechanism to integrate exogenous DNA template into the target genome site. However, HDR efficiency is often low, and the co-localization of exogenous DNA template and target genome site is one of the key limiting factors. To improve the efficiency of HDR mediated by CRISPR/Cas9 system, our team and previous studies fused different adaptor proteins with SpCas9 protein and expressed them. By using their characteristics of binding to specific DNA sequences, many different CRISPR/SpCas9 donor adapter gene editing systems were constructed. In this study, we used them to knock-in eGFP gene at the 3'-end of the terminal exon of GAPDH and ACTB genes in HEK293T cells to facilitate a comparison and optimization of these systems. We utilized an optimized donor DNA template design method, validated the knock-in accuracy via PCR and Sanger sequencing, and assessed the efficiency using flow cytometry. The results showed that the fusion of yGal4BD, hGal4BD, hLacI, hTHAP11 as well as N57 and other adaptor proteins with the C-terminus of SpCas9 protein had no significant effect on its activity. At the GAPDH site, the donor adapter systems of SpCas9 fused with yGal4BD, hGal4BD, hLacI and hTHAP11 significantly improved the knock-in efficiency. At the ACTB site, SpCas9 fused with yGal4BD and hGal4BD significantly improved the knock-in efficiency. Furthermore, increasing the number of BS in the donor DNA template was beneficial to enhance the knock-in efficiency mediated by SpCas9-hTHAP11 system. In conclusion, this study compares and optimizes multiple CRISPR/Cas9 donor adapter gene editing systems, providing valuable insights for future gene editing applications.
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Label-free Quantitative Analysis of Protein Expression Alterations in miR-26a-Knockout HeLa Cells using SWATH-MS Technology.
MicroRNAs (miRNAs) bind to the 3'-untranslated region of target mRNAs in a sequence-specific manner and subsequently repress gene translation. Human miR-26a has been studied extensively, but the target transcripts are far from complete. We first employed the CRISPR-Cas9 system to generate an miR-26a-knockout line in human cervical cancer HeLa cells. The miR26a-knockout line showed increased cell growth and altered proliferation. Proteomics technology of sequential window acquisition of all theoretical mass spectra (SWATH-MS) was utilized to compare the protein abundance between the wild-type and the knockout lines, with an attempt to identify transcripts whose translation was influenced by miR-26a. Functional classification of the proteins with significant changes revealed their function in stress response, proliferation, localization, development, signaling, etc. Several proteins in the cell cycle/proliferation signaling pathway were chosen to be validated by western blot and parallel reaction monitoring (PRM). The satisfactory consistency among the three approaches indicated the reliability of the SWATH-MS quantification. Among the computationally predicted targets, a subset of the targets was directly regulated by miR-26a, as demonstrated by luciferase assays and Western blotting. This study creates an inventory of miR-26a-targeted transcripts in HeLa cells and provides fundamental knowledge to further explore the functions of miR-26a in human cancer.
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CRISPR-Cpf1-mediated genome editing and gene regulation in human cells.
Clustered regularly interspaced short palindromic repeat (CRISPR) system is being championed as a robust and flexible tool for genome editing. Compared with CRISPR associated protein 9 (Cas9), the CRISPR from Prevotella and Francisella 1 (Cpf1) protein has some distinct characteristics, including RNase activity, T-rich protospacer adjacent motif (PAM) preference and generation of sticky cutting ends. The extremely low propensity of off-target effects and relatively high editing efficiency represent prominent advantages of Cpf1 over Cas9. CRISPR-Cpf1, alone or fused with function domains, has broadly expanded the applications such as multiplex gene knockout, transcriptional repression or activation and epigenome editing in a drug controlled way. Meanwhile, the modification of CRISPR RNAs (crRNAs) with aptamer RNA achieves great promotion on genome editing. Moreover, disease-associated gene manipulation in mice, tumor mutation detection in patients with cancers, and more yet to come, represent growing demands of CRISPR-Cpf1 in clinical genome therapy. In this review, we summarized the unique properties of Cpf1 and the molecular mechanisms underlying CRISPR-Cpf1 on gene editing and regulation in human cells.
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Inconclusive studies on possible CRISPR-Cas off-targets should moderate expectations about enzymes that have evolved to be non-specific.
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Public acceptance and stakeholder views of gene edited foods: a global overview.
The increasing popularity of gene editing in plants has prompted research on stakeholder views. Gene edited foods are often more accepted than genetically modified foods, though differences occur within target groups, regions, and products. Nevertheless, marketing challenges related to a lack of familiarity with the technology, labeling, and risk perception remain.
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A new 'mini-CRISPR' flexes its editing power in monkey muscles.
The downsized DNA-slicing machinery may reach more tissues to take aim at more diseases.
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Inducible CRISPR/Cas systems in precision oncology: Current applications and future perspectives.
BACKGROUND: Inducible CRISPR/Cas systems enable spatiotemporal control of genome editing in response to chemical, optical, biological, or physical stimuli. By restricting genome-editing activity to defined conditions, these systems may reduce off-target exposure and immune burden while improving tumor-selective control, making them attractive tools for precision oncology. MAIN BODY: This review summarizes the molecular mechanisms, design principles, and current applications of inducible CRISPR/Cas systems in cancer research and therapy. These platforms are classified into chemically inducible, optogenetic, tumor microenvironment-responsive, physically triggered, and logic-gated systems. Regulatory strategies are discussed at multiple levels, including transcriptional control, post-translational regulation, guide RNA engineering, and stimulus-responsive delivery. Key applications include functional genomic screening, cancer modeling, therapeutic gene editing, immunotherapy enhancement, and combinatorial treatment strategies. We also examine current delivery approaches, including viral vectors, lipid nanoparticles, stimulus-responsive nanocarriers, and biomimetic platforms. CONCLUSION: Inducible CRISPR/Cas systems represent a promising platform for next-generation precision cancer therapy. However, substantial optimization and rigorous preclinical validation remain necessary to address challenges related to leaky expression, induction efficiency, tissue penetration, immunogenicity, and long-term safety before clinical translation can be realized. KEY POINTS: Inducible CRISPR/Cas systems enable conditional genome editing in precision oncology. Chemical, optical, TME-responsive, physical, and logic-gated systems offer distinct control features. Delivery, leakiness, immunogenicity, and safety remain key translational barriers. Ex vivo immune-cell engineering and locoregional delivery may offer nearer-term clinical routes.
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