CRISPR-FAST: Ultrafast Light-Activated Gene Editing Technology and Its Core Applications
CRISPR-FAST (Far-red light-Activated Split-Cas9) is a groundbreaking gene-editing technology that uses far-red light to activate split Cas9 enzymes (N-Cas9 and C-Cas9), enabling spatiotemporal control of DNA modifications. Its advantages— deep tissue penetration (>5 mm), low phototoxicity, and programmable dynamic regulation—position it as a revolutionary tool in biomedical research, precision medicine, and synthetic biology. Below are its key applications and technological breakthroughs:

1. High-Precision Gene Editing in Deep Tissues

CRISPR-FAST overcomes the limitations of traditional CRISPR systems, enabling non-invasive editing of internal organs (e.g., liver, tumors) in mammals:

• Targeted Cancer Therapy:
  In mouse xenograft models, LED-activated CRISPR-FAST edits oncogenes (e.g., PLK1), suppressing tumor growth while minimizing chemotherapy side effects. A single light pulse achieves >50% editing efficiency in tumors with no off-target effects on healthy tissues.
• Organ-Specific Editing:
  In tdTomato reporter mice, non-invasive far-red light activates CRISPR-FAST to edit genes in deep tissues (e.g., liver, kidney), offering potential treatments for inherited metabolic disorders like phenylketonuria.

Key Innovations:

• Light-Activation Module: Bacterial photoreceptor BphS generates c-di-GMP under far-red light (730 nm), driving BldD-p65/VP64 transcriptional activation of N-Cas9, which binds C-Cas9 via Coh2-DocS to reconstitute active Cas8.
• Nanodelivery Optimization: Minicircle DNA vectors and lipid nanoparticles (LNPs) enhance delivery efficiency and reduce immunogenicity.

2. Spatiotemporal Control and Functional Genomics

CRISPR-FAST’s subcellular resolution and millisecond activation enable real-time studies of gene dynamics:

• DNA Repair Kinetics:
  Light-controlled Cas9 activation (within 30 seconds of illumination) captures the recruitment of repair proteins (e.g., BRCA1, Rad51) to DNA break sites, revealing repair initiation within 2 minutes and completion within 15 minutes.
• Cell Cycle-Dependent Editing:
  Synchronized cell populations allow study of gene function in specific cell cycle phases (e.g., MYC’s role in G1-phase proliferation).

Key Innovations:

• Synchronized Double-Strand Breaks: Photocaged sgRNA ensures Cas9 binds and cuts only post-illumination, minimizing off-target effects.
• Single-Allele Editing: Focused light beams edit individual alleles in diploid genomes, modeling dominant diseases like Huntington’s.

3. Synthetic Biology and Gene Circuit Design

CRISPR-FAST’s programmable logic gates drive dynamic metabolic engineering:

• Conditional Gene Circuits:
  AND-gate plasmids (e.g., pAND-gate) activate Cas9 under hypoxia or high lactate levels, enabling tumor microenvironment-responsive suicide gene therapy.
• Metabolic Pathway Optimization:
  Intermittent light pulses regulate acs and acc genes in cyanobacteria, boosting ethylene production from 2 g/L/h to 10 g/L/h without toxic byproduct accumulation.

Key Innovations:

• Dose-Response Linearity: Editing efficiency correlates linearly with light intensity (R²>0.95).
• Multiplex Editing: CRISPR array plasmids (e.g., pCRISPR-MultiAg) enable single-light activation of 10 targets for genome-wide screens or metastatic cancer studies.

4. Clinical Translation and Precision Medicine

CRISPR-FAST’s safety and on-demand activation support clinical applications:

• Genetic Disease Therapy:
  In hemophilia A mice, liver-targeted CRISPR-FAST restores Factor VIII to 70% of normal levels without hepatotoxicity.
• Enhanced Immunotherapy:
  Light-triggered PD-1 knockout in CAR-T cells activates tumor-specific cytotoxicity while reducing systemic immune storms.

Key Innovations:

• Dual-Factor Authentication: Blockchain tracks plasmid usage, requiring two-factor authorization for antibiotic resistance genes.
• Reversible Editing: Temperature-sensitive vectors (e.g., pX335-TS) degrade Cas9 post-editing (37°C→42°C) to prevent genomic instability.

5. Challenges and Future Directions

• Tissue Penetration Limits: Upconversion nanoparticles (UCNPs) or fiber optics may extend editing to deeper organs (e.g., brain).
• Delivery Efficiency: Organ-specific promoters (e.g., lung-targeted SFTPC) could improve non-liver targeting.
• Clinical Standardization: Global protocols for light parameters (wavelength, intensity, duration) and large-animal safety studies are needed.

Conclusion
CRISPR-FAST transforms gene editing from a static tool into a dynamically programmable system, advancing cancer therapy, genetic disease correction, and industrial biomanufacturing. With emerging technologies like quantum dot lighting and AI-driven parameter optimization, it promises single-cell precision and multi-organ coordination, heralding a new era of light-controlled life systems.

Data sources: Publicly available references. For collaborations or domain inquiries, contact: chuanchuan810@gmail.com.