DNA Technology Part 5: Gene Editing with CRISPR Cas9

Introduction to CRISPR Cas9

• CRISPR stands for Clustered Regulatory Interspace Short Palindromic Repeats.

• It is a revolutionary gene editing technology used to modify DNA sequences within an organism.

• Development and Recognition:

  • The technology was developed by two female scientists: Duna and Sharpender.

  • They were awarded the Nobel Prize in Chemistry in 2020 for this discovery.

Case Study: Sickle Cell Disease

• Sickle cell disease, also referred to as sickle cell anemia, is a primary application for CRISPR Cas9 gene editing.

• Genetic Basis of the Disease:

  • It is caused by a specific mutation at the DNA level in the beta globin, specifically the $HBV$ gene.

  • It is an autosomal recessive condition, meaning an individual must inherit two copies of the mutated gene to manifest the disease.

  • The mutation causes red blood cells to lose their round shape and form a sickle shape.

• Clinical Presentation:

  • Patients experience significant symptoms including anemia and a high susceptibility to infections.

  • These symptoms result in a poor quality of life for the individual.

Research and Therapeutic Applications

• A key paper in this field is Frangoule et al., published in 2021 (the transcript also refers to this as Fangoule et al.).

• This research focuses on using CRISPR Cas9 to treat two single-gene disorders involving the $HBV$ gene:

  • Sickle cell disease.

  • Beta thalassemia.

• Regulatory Milestone: This technology was approved by the US Food and Drug Administration (FDA) in December $2023$ for the treatment of sickle cell disease.

Mechanism of the CRISPR Cas9 System

• Cas9 Protein: Cas9 is a nuclease that acts as "molecular scissors." Its primary function is to cut double-stranded DNA molecules.

• Guide RNA (gRNA):

  • The Cas9 protein is directed to a specific target sequence by a guide RNA molecule.

  • The Cas9 and the guide RNA form a complex.

  • The guide RNA serves as a "homing device," ensuring the complex only cuts DNA sequences that are complementary to the guide RNA.

• Functional Utility:

  • Knock out: Disrupting a gene to study its function (e.g., determining what happens during development if a gene is missing).

  • Repair: Introducing a wild-type copy of a gene or repairing a specific mutation.

Clinical Procedure for Treating Sickle Cell Disease

• Step 1: Collection: Hematopoietic stem cells and progenitor cells ($HSPC$) are collected from a patient with sickle cell disease.

• Step 2: Editing: The collected $HSPC$ are edited outside the body using CRISPR Cas9 technology. The goal of this editing is to reactivate the production of fetal hemoglobin.

• Step 3: Transplantation: The modified cells are transplanted back into the patient.

• Step 4: Engraftment: The modified cells engraft within the bone marrow.

• Step 5: Symptom Reduction: The cells increase the production of fetal hemoglobin, which alleviates the symptoms of sickle cell disease.

Targeting the BCL11A Transcription Factor

• Hemoglobin Transition:

  • At birth, the body produces fetal hemoglobin.

  • By roughly $3$ months of age, fetal hemoglobin levels decrease as adult hemoglobin production takes over.

  • This decline in fetal hemoglobin is when sickle cell symptoms typically begin for affected individuals.

• The Role of $BCL11A$:

  • $BCL11A$ is a transcription factor that is responsible for the repression of fetal hemoglobin expression.

• Therapeutic Strategy:

  • Scientists use CRISPR Cas9 to target and "switch off" the $BCL11A$ transcription factor.

  • By disabling this repressor, the expression of fetal hemoglobin can continue throughout adulthood, reducing the impact of the mutated adult hemoglobin.

Limitations and Risks

• Off-target mutations: A significant challenge in CRISPR technology is the potential for the system to generate mutations in regions of the genome other than the intended target.

• Safety Protocols: To mitigate this risk, scientists must perform further steps, such as sequencing other regions of the genome, to confirm that the editing was site-specific and did not cause unintended damage.

Questions & Discussion

• Multiple Choice Question: Which of the following characteristics allows the CRISPR Cas9 enzyme to be an effective way to generate mutations that block the function of a specific protein (a knockout)?

  • 1. It makes double stranded breaks in DNA throughout the genome.

  • 2. Its function is regulated by complementary guide RNA.

  • 3. It forms a complex with DNA.

  • 4. It removes random DNA bases.

  • 5. It forms a complex with protein.

• Solution and Reasoning: Option 2 is the correct answer. The CRISPR Cas9 enzyme's function is regulated by a complementary guide RNA, which acts as a homing device that allows the enzyme to find and edit a specific, targeted region of DNA rather than cutting randomly or throughout the entire genome.