Goal of this exercise: Analyze the first study that used the CRISPR/Cas9 system to provide a permanent cure for Sickle cell disease (Frangoul et al. 2021). Part 1 below provides background information on the disease, outlines the therapeutic protocol and summarizes the molecular mechanism of CRISPR/Cas 9 treatment. Note: This is a follow-up to the original sickle cell case, which involves genetic testing on family members in different case scenarios. The mobile version of the original case also uses NCBI tools to explore of the nature of the sickle cell mutation.
Part 1: Overview of CRISPR/Cas9 treatment for Sickle cell disease (this page).
Part 2: Using Case It v705 to locate and silence the BCL11 erythroid-specific enhancer (under construction)
Part 3: Using NCBI tools to examine BCL11A and the erythroid-specific enhancer (under construction)
Organization of Part 1
(1) Overview of Sickle cell disease
(2) Therapeutic concept of CRISPR/Cas9 treatment
(3) Molecular Mechanism of CRISPR/Cas9 treatment
(1) Overview of Sickle cell disease
Sickle cell disease (SCD) arises from a single point mutation in the β-gene of the HBB locus, encoding the β-globin subunit of adult hemoglobin (HbA). This mutation substitutes glutamic acid with valine, producing sickle hemoglobin (HbS). Fetal hemoglobin (HbF) does not contain beta-globin, and hence is free of the mutation.
- Click the video screen below for an overview of the disease including its evolutionary relationship with malaria.
- Click this link to see a another video animation showing how HbS polymerizes in deoxygenated conditions, deforming red blood cells into rigid, crescent shapes, causing anemia, vaso-occlusive crisis, and organ damage.
(2) Therapeutic concept
The mutation that causes sickle-cell disease occurs only in the beta-globin chains of adult hemoglobin. Enhancing the production of fetal hemoglobin via CRISPR treatment is one way to mitigate effects of sickle cell disease.
Fetal hemoglobin (HbF) consists of two alpha-globin chains and two gamma-globin chains (α₂γ₂). Fetal hemoglobin has a much higher affinity for oxygen than adult hemoglobin (HbA), which consists of two alpha-globin chains and two beta-globin chains (α₂β₂).
Before birth, most of the body’s hemoglobin is the (α₂γ₂) type. Shortly before or after birth, the body naturally transitions from producing gamma chains to beta chains, switching to adult hemoglobin. This structural difference allows the developing fetus to effectively obtain oxygen from the mother’s bloodstream across the placenta. This is critical because the fetus’s own lungs are not yet functional for gas exchange. By about 6 months of age, most of a baby’s hemoglobin is adult hemoglobin.
If a child receives the mutation from both parents, then all of the beta-globin chains will have the mutation, resulting in sickle-cell disease (homozygous recessive). If the child receives the mutation from one parent but the normal gene from the other parent, then they are heterozygous and would be asymptomatic for the disease. This is because a relatively small percentage of the hemoglobin has the mutation in both beta-globin chains, not enough to cause symptoms. Hemoglobin molecules with the mutation in only one of the two beta-globin chains function normally.
Fetal hemoglobin expression is normally repressed after birth by the transcription factor BCL11A, which binds to enhancer regions controlling γ-globin gene expression and silences them. CRISPR treatment targets the erythroid-specific enhancer for BCL11A and silences it, resulting in the continued production of fetal hemoglobin. Since it only targets this specific enhancer, CRISPR treatment does not adversely affect other critically important functions of BCL11A.
(3) Molecular Mechanism of CASGEVY (CRISPR/Cas9 Treatment)
Click here for a video overview of the therapeutic procedure outlined below, and here for a video interview with the first person to be treated with this therapy (Victoria Gray) along with the scientist (Jennifer Doudna) who received the Nobel Prize for her team’s work developing the CRISPR-Cas9 system as recreated in the CRISPR exercise. An overview of this system is provided in Part 1 of the ATTR exercise.
- Hematopoietic Stem Cell (HSC) Extraction:
The patient’s bone marrow stem cells are harvested via apheresis, after inducing movement of the cells from the bone marrow to the blood. - Targeted Gene Editing:
- A guide RNA (gRNA) directs Cas9 nuclease to the erythroid-specific enhancer of BCL11A (see this image from Frangoul et al. 2021). This enhancer is a crucial regulatory DNA sequence located within the second intron of the BCL11A gene. It controls the expression of BCL11A specifically in red blood cell precursors (erythroid cells).
- Cas9 introduces a double-stranded break at this locus.
- A guide RNA (gRNA) directs Cas9 nuclease to the erythroid-specific enhancer of BCL11A (see this image from Frangoul et al. 2021). This enhancer is a crucial regulatory DNA sequence located within the second intron of the BCL11A gene. It controls the expression of BCL11A specifically in red blood cell precursors (erythroid cells).
- Disruption of the erythroid-specific enhancer of BCL11A:
- DNA repair occurs primarily via Non-Homologous End Joining (NHEJ), producing insertions or deletions that disable the enhancer.
- Functionally, this reduces BCL11A expression in red blood cell precursors, stopping repression of γ-globin genes.
- DNA repair occurs primarily via Non-Homologous End Joining (NHEJ), producing insertions or deletions that disable the enhancer.
- Reactivation of Fetal Hemoglobin (HbF):
- The Hematopoietic Stem Cells (HSCs), now engineered to reduce BCL11A activity, preferentially express γ-globin thus increasing HbF production.
- HbF compensates for defective the β-globin in adult hemoglobin, preventing red blood cell sickling.
- The Hematopoietic Stem Cells (HSCs), now engineered to reduce BCL11A activity, preferentially express γ-globin thus increasing HbF production.
- Reinfusion and Engraftment:
- Edited HSCs are transplanted back into the patient after myeloablative conditioning, which clears space in the bone marrow. Note: This procedure has serious side effects but its use is considered justified due to the severe nature of sickle-cell disease.
- The cells engraft and generate a lifelong supply of red blood cells expressing non-sickling HbF.
- Edited HSCs are transplanted back into the patient after myeloablative conditioning, which clears space in the bone marrow. Note: This procedure has serious side effects but its use is considered justified due to the severe nature of sickle-cell disease.
Molecular Outcomes
- HbF competes with HbS, inhibiting polymerization of defective β-globin
- Red blood cells regain flexibility and normal morphology, reducing anemia and preventing vaso-occlusion.
- Patients experience long-term symptomatic relief with a functional cure.
Key Molecular Insights
- CRISPR exploits the cell’s native DNA repair machinery to disrupt a regulatory gene rather than directly correcting the structural mutation.
- Targeting the erythroid-specific enhancer allows selective modulation of BCL11A without compromising its roles in other tissues.
- The treatment demonstrates precision gene regulation, effectively “reprogramming” the patient’s hematopoietic system.
References from Current Research
- CASGEVY (exagamglogene autotemcel) was approved by the FDA in December 2023 using this strategy.
- Clinical trials show high efficacy: most patients maintain HbF levels sufficient to prevent vaso-occlusive crises for extended periods (≥12 months).
- Long-term monitoring is ongoing for potential off-target effects, efficiency of engraftment, and durability of HbF expression.
- The search for an alternative to the current form of myeloablative conditioning continues.
Summary
At a molecular level, CRISPR treatment for sickle cell disease reactivates fetal hemoglobin by selectively disabling the BCL11A enhancer, using patient-derived stem cells edited ex vivo. This precision-editing restores functional hemoglobin, prevents sickling, and represents a paradigm shift from symptomatic management to curative, gene-level treatment.
In the context of human biology, (α₂γ₂) represents the chemical structure of fetal hemoglobin (HbF), which is the primary type of oxygen-carrying protein in a developing fetus (in utero).