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Next-generation lung-cancer-on-a-chip: Toward personalized therapy, AI, and CRISPR-driven models.
Lung-cancer-on-a-chip (LCOC) technologies have advanced rapidly, yet most models evaluate mechanical strain, patient-derived tumors, multi-organ interactions, artificial intelligence (AI) analytics, and clustered regularly interspaced short palindromic repeats (CRISPR) editing in isolation. In this review, we uniquely integrate these emerging components into a unified framework centered on the breathing LCOC. We highlight how embedding patient-derived lung tumor fragments into cyclically stretched microenvironments, then linking them to downstream organ compartments, enables patient-specific mapping of metastatic routes under physiologically relevant mechanics. We further describe how continuous high-resolution imaging from these platforms can feed AI pipelines for automated drug-response prediction and metastatic trajectory simulation, and how on-chip CRISPR editing enables accurate investigation of metastatic drivers within dynamic, strain-modulated microenvironments. By synthesizing these technologies, we outline a next-generation, personalized multi-organ-on-chip architecture capable of predicting individual disease progression without direct patient risk. We also address practical barriers, including tumor fragility under strain, imaging domain shift, and gene-editing delivery challenges, and how to overcome such barriers.
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Hallmarks of cancer: The CRISPR generation.
The hallmarks of cancer were proposed as a logical framework to guide research efforts that aim to understand the molecular mechanisms and derive treatments for this highly complex disease. Recent technological advances, including comprehensive sequencing of different cancer subtypes, have illuminated how genetic and epigenetic alterations are associated with specific hallmarks of cancer. However, as these associations are purely descriptive, one particularly exciting development is the emergence of genome editing technologies, which enable rapid generation of precise genetic and epigenetic modifications to assess the consequences of these perturbations on the cancer phenotype. The most recently developed of these tools, the system of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), consists of an RNA-guided endonuclease that can be repurposed to edit both genome and epigenome with high specificity, and facilitates the functional interrogation of multiple loci in parallel. This system has the potential to dramatically accelerate progress in cancer research, whether by modelling the genesis and progression of cancer in vitro and in vivo, screening for novel therapeutic targets, conducting functional genomics/epigenomics, or generating targeted cancer therapies. Here, we discuss CRISPR research on each of the ten hallmarks of cancer, outline potential barriers for its clinical implementation and speculate on the advances it may allow in cancer research in the near future.
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Different Methods of Delivering CRISPR/Cas9 Into Cells.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is comprised of repetitive bases followed by short fragments of DNA from a previously invading organism that provide immunity to the most prokaryotic organisms. An RNA-dependent spacer is required for CRISPR/Cas9 to recognize the target DNA. Delivery of the CRISPR/Cas9-guide RNA (gRNA) complex to any cell results in modification of the target sequence. The CRISPR/Cas9-mediated genome editing technique is currently in the spotlight and has several research interests, including molecular medicine and agriculture. There are several factors that hinder the delivery of this complex, such as the large size of the plasmid or high dosage of the chemical agent. There are several methods available to deliver CRISPR/Cas9 and its components to the target cells. It includes viral, non-viral and physical methods to deliver plasmid or ribonucleoprotein (RNP) of CRISPR components. But in vivo CRISPR/Cas9 delivery remains challenging to the researchers due to insertional mutagenesis, targeted delivery, immunogenicity, and off-targets. However, studies suggesting that the CRISPR/Cas9-RNP delivery can overcome these hurdles. Here, we review the various methods for delivery of CRISPR/Cas9 and gRNA to several cell lines, highlighting the limitations of each approach, and suggest possible alternative methods.
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Selection and Validation of Spacer Sequences for CRISPR-Cas9 Genome Editing and Transcription Regulation in Bacteria.
RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems have recently been adapted as sequence-programmable tools for various purposes such as genome editing and transcriptional regulation. A critical aspect of the system is the selection and validation of spacer sequences that allow precise targeting of the guide RNA-Cas9 complex. We describe a procedure involving computational and experimental steps to identify and test potentially interesting spacer sequences in bacterial genomes.
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Small Molecules for Enhancing the Precision and Safety of Genome Editing.
Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome-editing technologies have revolutionized biology, biotechnology, and medicine, and have spurred the development of new therapeutic modalities. However, there remain several barriers to the safe use of CRISPR technologies, such as unintended off-target DNA cleavages. Small molecules are important resources to solve these problems, given their facile delivery and fast action to enable temporal control of the CRISPR systems. Here, we provide a comprehensive overview of small molecules that can precisely modulate CRISPR-associated (Cas) nucleases and guide RNAs (gRNAs). We also discuss the small-molecule control of emerging genome editors (e.g., base editors) and anti-CRISPR proteins. These molecules could be used for the precise investigation of biological systems and the development of safer therapeutic modalities.
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Genome engineering using the CRISPR-Cas9 system.
Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.
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Clustered regularly interspaced short palindromic repeats tools for plant metabolic engineering: achievements and perspectives.
The plant kingdom represents the biggest source of feedstock, food, and added-value compounds. Engineering plant metabolic pathways to increase the phytochemical production or improve the nutraceutical value of crops is challenging because of the intricate interaction networks that link multiple genes, enzymatic steps, and metabolites, even when pathways are fully elucidated. The development of clustered regularly interspaced short palindromic repeats - CRISPR-associated (CRISPR-Cas) technologies has helped to overcome limitations in metabolic engineering, providing efficient and versatile tools for multigene editing. CRISPR approaches in plants were shown to have a remarkable efficiency in genome editing of different species to improve agronomic and metabolic traits. Here, we give an overview of the different achievements and perspectives of CRISPR technology in plant metabolic engineering.
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Harnessing CRISPR/Cas9 to overcome targeted therapy resistance in non‑small cell lung cancer: Advances and challenges (Review).
Targeted therapy has markedly improved outcomes for patients with non‑small cell lung cancer (NSCLC). However, the emergence of drug resistance remains a major clinical challenge, limiting long‑term treatment efficacy. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, a revolutionary gene‑editing technology, offers precise and efficient genetic modifications, providing new insights into the mechanisms of drug resistance in NSCLC. The present review explored the application of CRISPR/Cas9 in overcoming resistance associated with key oncogenic drivers, including EGFR, KRAS, ALK, ROS1, MET and BRAF. It summarized recent advances in CRISPR‑based strategies to reverse resistance, enhance targeted therapy effectiveness and develop potential therapeutic interventions. Additionally, it discussed current limitations, including off‑target effects, delivery challenges and ethical concerns, while highlighting future directions for clinical translation. Using CRISPR/Cas9 technology may pave the way for novel, personalized treatment approaches in NSCLC, ultimately improving patient outcome.
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas.
The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.
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Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein and hairy roots: a perfect match for gene functional analysis and crop improvement.
Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) gene editing has become a powerful tool in genome manipulation for crop improvement. Advances in omics technologies, including genomics, transcriptomics, and metabolomics, allow the identification of causal genes that can be used to improve crops. However, the functional validation of these genetic components remains a challenge due to the lack of efficient protocols for crop engineering. Hairy roots gene editing using CRISPR/Cas, coupled with omics analyses, provide a platform for rapid, precise, and cost-effective functional analysis of genes. Here, we describe common requirements for efficient crop genome editing, focused on the transformation of recalcitrant legumes, and highlight the great opportunities that gene editing in hairy roots offers for future crop improvement.
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