I. Core Challenges of CRISPR-Cas System Delivery and Advantages of Amino Acid Carriers
The key bottlenecks in CRISPR-Cas gene editing lie in the efficiency and safety of delivery systems: Macromolecules such as Cas nucleases (e.g., Cas9), sgRNA, or mRNA are prone to degradation by nucleases and struggle to penetrate cell membranes and nuclei. Fmoc-Arg(Pbf)-OH, as a protected arginine derivative, offers distinct advantages:
Natural transmembrane property: The multi-positive-charged guanidyl group of arginine (Arg) mediates endocytosis via the cell-penetrating peptide (CPP) mechanism, enhancing cellular uptake efficiency of nucleic acids/proteins.
Modifiability: Fmoc (amino-protecting group) and Pbf (guanidyl-protecting group) serve as chemical handles to conjugate CRISPR components or carrier materials, while protecting Arg's active groups from enzymatic degradation.
Responsive release: By regulating protective group dissociation conditions (e.g., light, pH, enzymes), precise intracellular release of Cas components can be achieved, reducing off-target effects.
II. Structural Modification of Fmoc-Arg(Pbf)-OH and Construction of Delivery Systems
Covalent Conjugation Strategies for CRISPR Components
sgRNA/DNA conjugation: The carboxyl group of Fmoc-Arg(Pbf)-OH links to the 3'-terminal amino group of sgRNA via an amide bond or stable linkage through click chemistry (e.g., azide-alkyne cycloaddition). For instance, modifying Fmoc-Arg(Pbf)-OH as an azide and conjugating it with alkynylated sgRNA forms an "Arg carrier-sgRNA" complex, where Arg's positive charge neutralizes RNA's negative charge to enhance complex stability.
Cas protein conjugation: The carboxyl group of Fmoc-Arg(Pbf)-OH reacts with lysine amino groups on the Cas9 protein surface to form peptide bonds. For example, site-specific conjugation of multiple Arg derivatives to non-active regions of Cas9 enhances protein cell penetration without affecting nuclease activity.
Assembly Design of Nano-Delivery Systems
Polymeric nanoparticles: Using Fmoc-Arg(Pbf)-OH as a monomer, copolymerizing with polylactic acid (PLA), polyethyleneimine (PEI), etc., to form amphiphilic polymers. For example, Fmoc-Arg(Pbf)-OH-PEI copolymer can encapsulate sgRNA via electrostatic interaction to form nanoparticles. After cellular entry, Pbf protective groups dissociate in the acidic lysosome, restoring Arg's positive charge to promote lysosomal escape and sgRNA release.
Liposome/lipid-like nano-carriers: Modifying Fmoc-Arg(Pbf)-OH on the liposome membrane surface enhances endocytosis via Arg's targeting (e.g., binding to cell surface glycoproteins), while light or pH triggers Pbf dissociation to disrupt the liposome membrane and release Cas9/sgRNA complexes into the cytoplasm.
III. Protective Group Regulation and Intracellular Release Mechanisms
Responsive Dissociation Design of Protective Groups
pH-sensitive dissociation: Pbf protective groups exhibit reduced stability in acidic environments (pH 5.0–6.5, lysosomal conditions). Modification with carboxylic ester groups can induce hydrolysis under acidic conditions to release free Arg. For example, replacing Pbf with pH-sensitive tert-butoxycarbonyl (tBoc) or introducing ortho-carboxyl groups in the Fmoc structure accelerates protective group detachment via intramolecular catalysis under acidic conditions.
Enzyme-triggered dissociation: For matrix metalloproteinases (MMPs) highly expressed in the tumor microenvironment, inserting an MMPs recognition peptide (e.g., PLGVR) into the linker of Fmoc-Arg(Pbf)-OH exposes Arg's transmembrane activity upon enzymatic cleavage, while releasing Cas components.
Optimization of Nuclear Targeted Release
Nuclear localization signal (NLS) integration: Introducing an NLS sequence (e.g., PKKKRKV from SV40 large T antigen) into the Fmoc-Arg(Pbf)-OH structure synergizes Arg's transmembrane ability with NLS's nuclear targeting to enhance Cas9/sgRNA nuclear entry efficiency. For instance, linking Fmoc-Arg(Pbf)-OH to an NLS peptide via a cleavable disulfide bond allows glutathione-mediated reduction after cellular entry, releasing the NLS-Arg complex to guide Cas9 to the nucleus.
Light-controlled nuclear release enhancement: For light-responsive systems, introducing light-sensitive groups like o-nitrobenzyl into Fmoc or Pbf triggers protective group dissociation upon UV/NIR irradiation, restoring Arg's positive charge to interact with negatively charged nuclear pore proteins and facilitate Cas component nuclear entry.
IV. Verification of Delivery Efficiency and Gene Editing Performance
Characterization of Cellular Uptake and Subcellular Distribution
Fluorescence labeling tracking: Labeling sgRNA with Cy5 and conjugating it to Fmoc-Arg(Pbf)-OH, then detecting cellular uptake efficiency via flow cytometry and observing lysosomal escape (LysoTracker staining) via confocal microscopy. For example, unmodified sgRNA shows <10% cellular uptake, while Arg modification boosts it to >60% with significantly improved lysosomal escape.
Protective group dissociation verification: Analyzing intracellular Fmoc-Arg(Pbf)-OH protective group dissociation rate via mass spectrometry (MS) or nuclear magnetic resonance (NMR) to confirm responsiveness in the intracellular microenvironment. For instance, incubation at pH 5.5 for 2 hours achieves 75% Pbf dissociation, releasing free Arg.
Evaluation of Gene Editing Efficiency
Mutation rate detection: Using HEK293T cells as a model to target GFP gene editing, detecting the proportion of GFP-negative cells via flow cytometry or calculating insertion-deletion (Indel) frequency via deep sequencing. For example, Cas9/sgRNA complexes delivered by Fmoc-Arg(Pbf)-OH achieve an Indel rate of 45%, significantly higher than liposome transfection (25%).
Off-target effect analysis: Detecting off-target mutations via whole-genome sequencing (WGS) or GUIDE-seq to verify whether Arg modification affects Cas9 specificity. Results show Arg modification does not significantly increase off-target rates, as Arg's targeting primarily enhances cellular uptake rather than altering Cas9's nucleic acid binding properties after protective group dissociation.
V. Technical Challenges and Future Optimization Directions
Current Key Issues
Toxicity from residual protective groups: Incompletely dissociated Fmoc or Pbf may affect Arg's biocompatibility, requiring optimized protective group structures for higher dissociation efficiency.
Large-scale synthesis challenges: The complex process and high cost of modifying CRISPR components with Fmoc-Arg(Pbf)-OH limit clinical translation.
Improvement Strategies
Dual-responsive protective group design: Developing pH/enzyme dual-responsive Pbf analogs, such as introducing enzyme cleavage sites and pH-sensitive groups into Pbf structures to trigger dissociation dual-dimensionally and improve intracellular release efficiency.
Simplification with protective group-free carriers: Leveraging Arg's natural transmembrane property to directly encapsulate Cas9/sgRNA via electrostatic interaction, avoiding protective groups while reducing immunogenicity through PEGylation. For example, Arg-PEG copolymer nanoparticles enable covalent-free CRISPR delivery with editing efficiency reaching 30%–40%.
VI. Clinical Translation Prospects and Application Scenarios
Fmoc-Arg(Pbf)-OH-mediated delivery systems hold potential in treating genetic diseases, such as:
Sickle cell anemia: Delivering Cas9/sgRNA via Arg-modified nanoparticles to target-edit the HBB gene in hematopoietic stem cells and repair mutation sites.
Ocular diseases: Utilizing Arg's corneal penetrability to locally deliver CRISPR components to the retina for treating monogenic eye diseases like retinitis pigmentosa.
Future research should further optimize in vivo metabolic pathways, reduce immunogenicity, and validate editing efficiency and safety in organ targeting (e.g., liver, muscle) via animal models to advance clinical application of gene editing therapies.
