CRISPR in Medicine

What is CRISPR?

How the CRISPR-Cas9 System Works

Fig. 39-2: CRISPR-Cas9 overview. The guide RNA (green) base-pairs with the target DNA strand; Cas9's RNA-binding domain locks in; the nuclease domain introduces a double-strand break (DSB) 3 bp upstream of the PAM sequence (NGG). - Harper's Illustrated Biochemistry, 32nd Ed.

1. Adaptation - invader DNA is integrated as a new spacer into the CRISPR array
2. crRNA synthesis - the array is transcribed and processed by Cas proteins into mature CRISPR RNA
3. Interference - crRNA directs Cas to cleave matching foreign sequences

• NHEJ (Non-Homologous End Joining) - error-prone; causes insertions/deletions (indels) → gene knockout
• HDR (Homology-Directed Repair) - precise; uses a donor template → gene correction or insertion
• Schwartz's Principles of Surgery, 11th Ed.

CRISPR Variants Beyond Cas9

• Zhang, Ma & Liu, Genomics Proteomics Bioinformatics, 2025 [PMID: 40268745]

Medical Applications

1. Hemoglobinopathies (FDA-Approved)

• Edits autologous CD34+ hematopoietic stem cells ex vivo
• Reactivates fetal hemoglobin (HbF) by disrupting the BCL11A enhancer (which silences the γ-globin gene)
• Approved for sickle cell disease (SCD) and transfusion-dependent beta-thalassemia (TDT)

2. Cancer (CAR-T and Beyond)

• CRISPR is used to enhance CAR-T cell therapy - editing T cells to knock out immune checkpoint genes (e.g., PD-1) or HLA genes for allogeneic ("off-the-shelf") CAR-T products
• CRISPR screening (genome-wide loss-of-function screens) is identifying new cancer drug targets

3. Infectious Diseases

• SHERLOCK (CRISPR-Cas13): Specific High Sensitivity Enzymatic Reporter Unlocking - a SARS-CoV-2 SHERLOCK assay was the first CRISPR-based diagnostic to receive FDA Emergency Use Authorization
• DETECTR (CRISPR-Cas12): Used to detect HPV16/18 in clinical specimens
• CARVER (Cas13): Identifies viral infection AND directs RNA viral destruction
• FLASH (CRISPR-Cas9 + NGS): Detects antimicrobial resistance genes

• HIV: gRNA-Cas9 constructs silence proviral HIV DNA in cell culture
• Herpes viruses, HPV, HBV: targeted elimination in research models
• "Resensitization to antibiotics": CRISPR targets ESBLs and restores β-lactam susceptibility in resistant bacteria
• Tietz Textbook of Laboratory Medicine, 7th Ed.

4. Genetic / Rare Diseases (Pipeline)

• Duchenne Muscular Dystrophy - exon skipping to restore dystrophin reading frame
• Transthyretin amyloidosis - in vivo CRISPR (NTLA-2001) targets the TTR gene in the liver via lipid nanoparticles
• Retinal dystrophies - AAV-delivered CRISPR for CEP290 mutation (Leber congenital amaurosis)
• Chronic pain - CRISPR-based epigenome editing to silence pain genes without permanent DNA changes

5. Research Tools

• Large-scale genetic screening (CRISPR libraries)
• Lineage tracing in developmental biology
• Generating disease models (cell lines, animal models)

Challenges and Limitations

Recent Literature

• CRISPR Technology and Its Emerging Applications (Zhang et al., Genomics Proteomics Bioinformatics, 2025) - broad review of Cas9, Cas12a, Cas13, base editing, prime editing, and clinical applications [PMID: 40268745]
• Advancing CRISPR genome editing into gene therapy clinical trials (Cetin et al., Expert Rev Mol Med, 2025) - covers Casgevy approval, SCD/TDT outcomes, ongoing cancer and HIV trials, and challenges including off-target effects and delivery systems [PMID: 40160040]

⚡ CRISPR - High Yield MD Exam Notes

🧠 FIRST, THE MNEMONIC

"CRISPR CUTS DNA SMARTLY"

📌 ORIGIN - 1 LINE ANSWER

CRISPR = Part of bacterial adaptive immunity against bacteriophages First described in E. coli in 1987

🔧 MECHANISM (Most Asked)

Guide RNA (gRNA)
     ↓
Binds to TARGET DNA (Watson-Crick base pairing)
     ↓
Cas9 recognizes PAM sequence (5'-NGG-3')
     ↓
Cas9 cuts BOTH strands → Double Strand Break (DSB)
     ↓
Cell repairs the break via:
   ├── NHEJ → Indels → Gene KNOCKOUT ❌
   └── HDR  → Precise edit using template → Gene CORRECTION ✅

🔑 KEY COMPONENTS

🧬 CAS PROTEIN VARIANTS (High Yield for Step 1 / NEET PG)

Exam tip: Cas13 targets RNA - useful for viral RNA destruction (CARVER system)

💊 FDA-APPROVED CRISPR DRUG (Highest Yield Clinical Fact)

Casgevy (exa-cel / CTX001) - Approved December 2023

Memory trick: "CASgevy uses CAS9 to CASt away sickle cell disease"

🔬 CRISPR DIAGNOSTICS (Exam Favorite)

Trick to remember: SHERLOCK = SARS-CoV-2. First EUA. Cas13 (RNA).

🦠 THERAPEUTIC APPLICATIONS BY DISEASE

⚠️ CHALLENGES / LIMITATIONS (MCQ trap answers)

⚡ REPAIR PATHWAY COMPARISON (High Yield)

🏆 ONE-LINERS FOR VIVA / SHORT ANSWER

1. First CRISPR drug = Casgevy (sickle cell, Dec 2023)
2. PAM sequence = NGG (required for Cas9)
3. SHERLOCK = first CRISPR diagnostic with FDA EUA (for SARS-CoV-2)
4. Cas13 = only targets RNA (all others target DNA)
5. NHEJ = messy = knockout; HDR = precise = correction
6. BCL11A = the target in Casgevy (suppressor of fetal Hb)
7. Ex vivo = cells taken out for editing (like Casgevy); in vivo = editing inside the body
8. He Jiankui scandal = first (illegal) human germline editing (CCR5 gene, HIV resistance, 2018)
9. Base editing = single nucleotide change WITHOUT cutting both strands
10. Prime editing = most precise; no DSB, no donor template needed ("the next generation")

🗂️ COMPARE WITH OTHER GENE EDITING TOOLS

Why CRISPR won: RNA-guided (easy to reprogram) vs. protein-guided (ZFN/TALEN = must engineer new protein each time)

🎯 PREDICTED MCQ STEMS

• "A patient with sickle cell disease receives autologous stem cell therapy that reactivates fetal hemoglobin. What gene was targeted?" → BCL11A
• "Which CRISPR diagnostic received the first FDA EUA?" → SHERLOCK (COVID)
• "Which Cas protein targets RNA instead of DNA?" → Cas13
• "What repair pathway results in precise gene correction?" → HDR
• "The PAM sequence required by Cas9 is?" → 5'-NGG-3'
• "First FDA-approved CRISPR therapy?" → Casgevy (exa-cel)
• "CRISPR was adapted from which organism's immune system?" → Bacteria (originally E. coli)

📚 Sources: Tietz Textbook of Laboratory Medicine 7th Ed; Harper's Illustrated Biochemistry 32nd Ed; Schwartz's Principles of Surgery 11th Ed; Cetin et al., Expert Rev Mol Med 2025 [PMID 40160040]; Zhang et al., Genomics Proteomics Bioinformatics 2025 [PMID 40268745]