1. Therapeutic Genome Editing
A. Genetic Disease Correction
- In Vivo Base Editing: Liver-directed LNP delivery of ABE8.8 mRNA corrects SERPINA1 E342K mutation in α1-antitrypsin deficiency, achieving 30% editing efficiency and restoring protease inhibitor function .
- Prime Editing: Dual AAV vectors deliver pegRNA-nCas9-M-MLV to correct CFTR ΔF508 in cystic fibrosis organoids, restoring chloride channel activity .
B. Cancer Immunotherapy
- CAR-T Engineering: Ex vivo CRISPR knockout of PDCD1 and TCR genes enhances anti-tumor activity .
- Tumor-Specific Targeting: gRNAs targeting BCL11A enhancer induce fetal hemoglobin in sickle cell disease .
Suggested Figure 1: In Vivo Base Editing Workflow
LNP encapsulation → Hepatocyte delivery → A→G conversion → Functional protein restoration.
2. Functional Genomics & Disease Modeling
A. High-Throughput Screening
- CRISPRko/i/a Libraries: Genome-scale gRNA pools identify synthetic lethal interactions (e.g., PARP1 knockout sensitizing BRCA1-mutant tumors) .
- Single-Cell CRISPR Screens: Paired scRNA-seq and gRNA barcoding reveal cell-type-specific gene networks .
B. Organoid Engineering
- Multi-Gene Editing: Simultaneous targeting of APC, KRAS, and TP53 generates colorectal cancer organoids for drug testing .
Suggested Figure 2: CRISPR Screening Pipeline
gRNA library → Lentiviral delivery → Phenotypic selection → NGS deconvolution → Hit identification.
3. Diagnostic Applications
A. Infectious Disease Detection
- SHERLOCKv2: Cas13a collateral cleavage of fluorescent RNA reporters detects Zika virus at 2 aM sensitivity .
- DETECTR: Cas12a-based lateral flow assay identifies HPV16 in cervical swabs with 95% concordance .
B. Cancer Liquid Biopsies
- CRISPR-Cas9-NSG: Enrichment of EGFR T790M mutations in ctDNA improves detection limit to 0.01% allele frequency .
Suggested Figure 3: CRISPR Diagnostics Workflow
Sample → Cas13/Cas12 activation → Collateral cleavage → Fluorescent/lateral flow readout.
4. Agricultural & Environmental Engineering
A. Crop Improvement
- Multiplex Editing: Cas12a-mediated knockout of OsSWEET14 confers bacterial blight resistance in rice .
- Climate Resilience: Cas9-edited ZmCCT gene reduces photoperiod sensitivity in maize .
B. Bioremediation
- Microbial Pathway Engineering: Pseudomonas putida edited for enhanced polyethylene degradation .
5. Advanced Delivery Strategies
| Delivery System | Application | Editing Efficiency |
|---|---|---|
| AAV Vectors | Inherited retinal diseases | 15–40% in photoreceptors |
| Gold Nanoparticles | Skin regeneration | 25–60% in fibroblasts |
| Magnetofection | Brain tumor editing | 18% in glioma models |
Suggested Figure 4: Nanoparticle Delivery
AuNP-CRISPR complex → Cellular uptake → Endosomal escape → Genome editing.
6. Emerging Frontiers
A. Epigenome Editing
- dCas9-DNMT3A: Methylates FMR1 promoter to silence fragile X syndrome alleles .
- CRISPRa/i: Activates VEGF for ischemic heart repair .
B. Chromosome Engineering
- CRISPR-C: Programmable telomere fusion induces synthetic lethality in cancer .
C. In Utero Editing
- Base Editing: Corrects COL1A1 mutation in osteogenesis imperfecta murine model .
Conclusion
CRISPR-targeted editing transforms biomedicine through:
- Precision Therapeutics: Single-base correction for monogenic diseases.
- Functional Discovery: Genome-scale screens mapping disease pathways.
- Point-of-Care Diagnostics: Field-deployable pathogen detection.
- Sustainable Agriculture: Climate-resilient crop engineering.
With advances in delivery (LNPs, AAVs) and editing fidelity (base/prime editing), CRISPR therapeutics will enter routine clinical practice within this decade.
Data Source: Publicly available references.
Contact: chuanchuan810@gmail.com
