Module 6

CRISPR Therapeutics

CRISPR-Cas9 earned the 2020 Nobel Chemistry (Charpentier, Doudna) and delivered its first FDA approval in December 2023: Casgevy (exagamglogene autotemcel) for sickle-cell disease and transfusion-dependent beta-thalassemia. This module covers Cas9 biology, base editing, prime editing, delivery, and the current clinical landscape.

1. Cas9 Mechanism

CRISPR-associated protein 9 (Cas9) from Streptococcus pyogenes is guided by a 20-nt single guide RNA (sgRNA) to a matching DNA protospacer adjacent to a 5′-NGG PAM sequence. Cas9 generates a blunt double-strand break 3 bp upstream of the PAM. Repair by NHEJ (error-prone) produces indels; HDR (rare) enables precise template-guided edits.

\[ \text{sgRNA}_{20\text{nt}} + \text{DNA target} + \text{NGG PAM} \;\longrightarrow\; \text{Cas9 DSB 3 bp upstream of PAM} \]

2. Base & Prime Editing

Liu lab innovations eliminate the DSB:

Cytosine Base Editors (CBE) (Komor 2016): APOBEC1 cytidine deaminase fused to nickase Cas9 converts C→T (or G→A on the other strand).
Adenine Base Editors (ABE) (Gaudelli 2017): evolved TadA deaminase converts A→G (T→C).
Prime Editors (Anzalone 2019): Cas9 nickase + reverse transcriptase + pegRNA enables any single-nucleotide change plus small insertions/deletions, without DSB.

Simulation: Editing Methods & Specificity

Python

script.py46 lines

import numpy as np, matplotlib
matplotlib.use('Agg')
import matplotlib.pyplot as plt

# Off-target scoring by guide-target mismatch position
positions = np.arange(1, 21)
# Seed region (1-12 from PAM) more sensitive
weight = np.where(positions <= 12, 0.08, 0.02)

# Cumulative off-target probability
n_mismatches = np.array([0, 1, 2, 3, 4])
base_p = 1.0
off_tgt = base_p * np.prod([np.mean(weight)]*1) ** n_mismatches

# Editing outcomes: NHEJ indels vs base-edit vs prime-edit
methods = ['Cas9 + NHEJ', 'Cas9 + HDR', 'Cytosine BE', 'Adenine BE', 'Prime Edit']
efficiency = [0.75, 0.05, 0.60, 0.70, 0.30]
precision  = [0.20, 0.95, 0.90, 0.92, 0.98]

fig, (ax1, ax2) = plt.subplots(1, 2, figsize=(14, 5), facecolor='#0a0a1a')
for ax in (ax1, ax2):
    ax.set_facecolor('#111827'); ax.tick_params(colors='#cbd5e1')
    for s in ax.spines.values(): s.set_color('#334155')
    ax.grid(True, color='#334155', alpha=0.3)

ax1.bar(positions, weight, color='#2dd4bf', edgecolor='#5eead4')
ax1.axvline(12, color='#fbbf24', ls='--', label='Seed boundary')
ax1.set_xlabel('sgRNA position (PAM-proximal -> distal)', color='#cbd5e1')
ax1.set_ylabel('Off-target weight', color='#cbd5e1')
ax1.set_title('Seed region dominates specificity',
              color='#5eead4', fontweight='bold')
ax1.legend(facecolor='#1e293b', edgecolor='#334155', labelcolor='#cbd5e1')

x = np.arange(len(methods))
ax2.bar(x - 0.2, efficiency, 0.4, color='#2dd4bf', label='Efficiency')
ax2.bar(x + 0.2, precision, 0.4, color='#fbbf24', label='Precision')
ax2.set_xticks(x); ax2.set_xticklabels(methods, rotation=15, ha='right')
ax2.set_ylabel('Fraction', color='#cbd5e1')
ax2.set_title('Editing methods: efficiency vs precision',
              color='#5eead4', fontweight='bold')
ax2.legend(facecolor='#1e293b', edgecolor='#334155', labelcolor='#cbd5e1')

plt.tight_layout()
plt.savefig('output.png', dpi=120, bbox_inches='tight', facecolor='#0a0a1a')
print('Casgevy (exa-cel) 2023: first FDA CRISPR therapy, sickle-cell disease')
print('Base editing avoids double-strand breaks; prime editing enables precise insertions')

Click Run to execute the Python code

Code will be executed with Python 3 on the server

3. Delivery

Delivery is the central CRISPR translation challenge. Ex vivo (edit patient cells outside the body, then reinfuse) is the approved path for haematological diseases — Casgevy edits autologous haematopoietic stem cells. In vivo delivery is harder: AAV (size-limited for Cas9), LNP (mRNA-LNP for Cas9, like Verve Therapeutics’ VERVE-102 for hypercholesterolaemia), electroporation (for ex-vivo).

4. Clinical Approvals & Pipeline

Casgevy (exa-cel) approved 2023 (UK, FDA) for SCD and transfusion-dependent β-thalassemia: ex vivo Cas9 edit of BCL11A enhancer in autologous HSCs.
VERVE-102: in-vivo base-editor LNP targeting PCSK9 for hypercholesterolaemia.
Beam Therapeutics BEAM-101: base edit of HbS to HbG-Makassar in SCD.
Intellia NTLA-2001: in-vivo LNP Cas9 for hereditary ATTR amyloidosis (90% serum TTR reduction).
Prime Medicine: prime-edit programmes in chronic granulomatous disease and cystic fibrosis.

Key References

• Jinek, M. et al. (2012). “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Science, 337, 816–821.

• Komor, A. C. et al. (2016). “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature, 533, 420–424.

• Anzalone, A. V. et al. (2019). “Search-and-replace genome editing without double-strand breaks or donor DNA.” Nature, 576, 149–157.

• Frangoul, H. et al. (2021). “CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia.” N. Engl. J. Med., 384, 252–260.

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