Advances on Stimulus-Responsive Gene Editing Tools Delivery System

Gene Editing Tools

Gene editing tools are sophisticated instruments utilized to modify the genetic makeup of organisms. Their primary function is to introduce precise alterations into DNA sequences, including the addition, deletion, or modification of specific genes. The most prominent gene editing tools employed in contemporary molecular biology include:


The CRISPR Cas9 system stands as a revolutionary gene editing tool, distinguished by its remarkable precision and versatility. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences, coupled with the Cas9 protein, facilitate targeted modifications within the genome. CRISPR Cas9 has emerged as a preferred choice for its simplicity, efficiency, and ability to edit genes across a wide range of organisms.

Oligo Labeling ModificationsIllustration of CRISPR Cas9 genome editing enzyme.

TALENs (Transcription Activator-Like Effector Nucleases)

TALENs represent another class of gene editing tools adept at genome manipulation. They function through the fusion of transcription activator-like effector proteins with nucleases, enabling precise DNA cleavage at specific target sites. TALENs offer researchers a customizable approach to gene editing, allowing for tailored modifications to suit diverse experimental requirements.

ZFNs (Zinc Finger Nucleases)

Zinc Finger Nucleases (ZFNs) constitute an earlier generation of gene editing tools, characterized by their modular structure composed of zinc finger domains and nucleases. By harnessing the DNA-binding capabilities of zinc finger proteins, ZFNs can induce targeted DNA cleavage, facilitating precise genetic modifications. While not as widely used as CRISPR Cas9, ZFNs continue to be employed in specific applications where their unique attributes are advantageous.

These gene editing tools collectively empower researchers to manipulate the genetic blueprint of organisms with unprecedented precision, fostering advancements in genetic engineering, biotechnology, and biomedical research.

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CRISPR Cas9 Gene Editing Delivery System

CRISPR Cas9 is a revolutionary gene editing technology, but its delivery system has always been one of the challenges in research and application. The delivery system is responsible for guiding the CRISPR Cas9 complex (including Cas9 protein and RNA guide sequence) into target cells. Here are some common classifications of CRISPR Cas9 delivery systems.

Direct Delivery of CRISPR Cas9

  • Plasmid Delivery: This method involves introducing plasmids encoding Cas9 protein and sgRNA into cells, allowing the cells to express the CRISPR Cas9 system autonomously. Plasmid delivery offers good stability and enables long-term gene editing. However, it has drawbacks such as slow onset, low editing efficiency, high off-target effects, and the risk of genomic integration.
  • mRNA Delivery: By transfecting mRNA encoding Cas9 protein and sgRNA into cells, the CRISPR Cas9 complex can be produced using the cell's natural translation mechanism. mRNA delivery avoids the risk of DNA insertion but may reduce overall editing efficiency due to mRNA's instability and susceptibility to degradation.
  • RNP Delivery: Pre-assembled complexes of Cas9 protein and sgRNA (RNPs) are directly introduced into cells to achieve gene editing. RNPs offer advantages such as high stability, rapid action, low off-target effects, and low immunogenicity, without the risk of genomic insertion mutations. However, RNP delivery is associated with higher costs and shorter editing durations.

Delivery Using Vectors

  • Viral Vectors: Examples include adeno-associated virus (AAV) and lentivirus (LV), which are commonly used for efficient delivery of gene editing tools into cells. While viral vectors offer efficient delivery, they are limited by payload capacity, immunogenicity, and the risk of inducing mutations. Prolonged editing within cells via viral vector delivery may lead to serious off-target effects.
  • Non-viral Vectors: These include synthetic nanoparticles, liposomes, exosomes or other compounds used to deliver CRISPR Cas9 components into cells. Non-viral vectors avoid safety concerns associated with viruses but still face challenges such as lower delivery efficiency and the need to overcome multiple barriers both extracellularly and intracellularly.

The delivery system of CRISPR Cas9 still requires further research and optimization to improve its targeting, delivery efficiency, specificity, etc., in basic research and applications.

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Stimulus-Responsive CRISPR Cas9 Delivery System

Stimuli-responsive carrier systems for the delivery of CRISPR Cas9 tools represent a novel delivery system aimed at achieving efficient and precise genome editing. These carrier systems possess the ability to sense and respond to specific stimuli, thereby releasing CRISPR Cas9 tools under particular conditions to accomplish gene editing. These stimuli can be endogenous biological factors, such as pH, enzymes, redox factors, etc., or exogenous factors, such as sound, light, electricity, small molecules, magnetic fields, etc. This responsiveness allows for precise control over the release of CRISPR Cas9 tools in both time and space, enabling precise editing of the genome. These stimuli-responsive carrier systems represent the future direction of CRISPR Cas9 delivery, offering new possibilities for the precision and efficiency of gene editing technology. Their design principles and biological application directions are receiving increasing attention and research.

Endogenous Stimulus-Responsive CRISPR Cas9 Delivery Systems

Endogenous stimulus-responsive CRISPR Cas9 delivery systems generally utilize specific physiological or pathological factors within the body, such as pH, enzyme activity, redox environment, etc., to achieve temporal and spatial control of drug or gene editing tool release, thereby enhancing therapeutic efficacy and reducing side effects.

  • pH-responsive systems

These systems exploit differences in pH values across different parts of the body, designing carrier materials to respond to these differences, such as pH-sensitive polymers or compounds. These systems can release drugs within endosomes/lysosomes, avoiding issues of drug degradation and inactivation.

  • Redox-responsive systems

These systems leverage differences in the oxidative-reductive environment inside and outside cells, designing polymers or carrier materials that can be degraded by specific enzymes. Through this design, targeted delivery and release of gene editing tools can be achieved under specific pathological conditions.

  • Tissue-specific responsive systems

Targeting specific tissues or cells, these systems are designed with targeted delivery systems, such as carriers containing tissue-specific promoters or targeting ligands. These systems can reduce the impact on non-target tissues, improving the precision and safety of treatment.

  • Enzyme-responsive systems

By utilizing the activity of specific enzymes in the tumor microenvironment, these systems are designed with carriers or nanoparticles that can be degraded by these enzymes. These systems can achieve targeted release of drugs or gene editing tools in tumor tissues, enhancing therapeutic efficacy and minimizing effects on normal tissues.

Exogenous Stimuli-Responsive CRISPR Cas9 Delivery Systems

Exogenous stimuli-responsive CRISPR Cas9 delivery systems are gene editing tool delivery platforms that utilize external stimuli such as light, ultrasound, magnetic fields, etc., to precisely control the release and activity of the CRISPR Cas9 system. The design of these systems aims to achieve precise spatiotemporal control of gene editing tools, thereby enhancing therapeutic efficacy and reducing side effects.

  • Light-Responsive CRISPR Cas9 Delivery System

Light serves as a non-invasive, spatiotemporal control switch that enables precise control of gene editing system delivery. Light-induced photothermal conversion or ROS generation through photosensitive materials can achieve controlled delivery of the CRISPR Cas9 system. Utilizing the safety and excellent tissue penetration of near-infrared light (NIR), deeper tissue therapy can be realized. By connecting photosensitizers with CRISPR Cas9 and releasing RNP under specific conditions, light-controlled genome editing can be achieved.

  • Small Molecule-Responsive CRISPR Cas9 Delivery System

Spatial and temporal control of Cas9 activity can be achieved through the regulation of Cas9 function by organic small molecules. Precise control of genome editing is achieved by activating or inhibiting the nuclease activity of Cas9 with small molecules.

  • Ultrasound-Responsive CRISPR Cas9 Delivery System

Ultrasound can be used for intracellular delivery of gene editing tools, with deeper tissue penetration and biological effects. Thermal energy or ROS generated through sonosensitizers can achieve ultrasound-controlled genome editing.

  • Magnetic Field-Responsive CRISPR Cas9 Delivery System

Magnetic field-responsive delivery systems typically consist of magnetic nanomaterials and enable remote-controlled targeted drug delivery. External magnetic fields can guide carriers to target areas, reducing off-target editing and enhancing the precision of genome editing.

Prospects for CRISPR Cas9 Delivery System

Despite the significant achievements in recent years, intelligent stimuli-responsive materials still face numerous challenges in CRISPR Cas9 delivery. Firstly, although highly efficient and organ- or tissue-targeted CRISPR Cas9 stimuli-responsive delivery systems have been developed, their precision remains low, leading to decreased efficiency and rendering them unsuitable for clinical applications. Future directions may involve designing materials with multiple stimuli-responsive properties to enhance delivery precision and editing capabilities. Secondly, there is a shortage of co-delivery materials for gene editing tools and template DNA used in gene-directed repair, possibly due to the increased complexity associated with co-delivery. Additionally, material design should take into account the reproducibility of experimental results and the potential for industrial applications. Furthermore, although intelligent stimuli-responsive non-viral delivery systems have been applied in the research of other recently reported gene editing tools, such as base editors and prime editors, related studies are still limited. Lastly, the majority of reported intelligent stimuli-responsive CRISPR Cas9 delivery systems remain in the preclinical research stage. Overcoming challenges such as improving editing efficiency, enhancing specific targeting to target cells or tissues, reducing potential off-target effects, and addressing potential immune responses induced by CRISPR Cas9 tools are crucial steps for the clinical application of CRISPR Cas9-mediated genome editing. However, with the continuous development of CRISPR Cas9 technology and intelligent stimuli-responsive delivery techniques, it is believed that their combination will achieve more precise genome editing and promote their clinical application.

* Only for research. Not suitable for any diagnostic or therapeutic use.
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