Notes on Genome Editing and CRISPR-Cas9 Technology

Introduction to Genome Editing

• Genome editing involves targeted changes in an organism's genome, including insertions and deletions.
• CRISPR-Cas9 has become a popular method in the last decade due to its:
  • Speed
  • Cost-effectiveness
  • Precision
  • Ease of use

How CRISPR-Cas9 Works

1. Creation of RNA Guide
   • Researchers create a piece of RNA with a guide sequence complementary to the target DNA.
   • Example: For a target DNA sequence 5'GGCTAT 3', the RNA guide would be 3'CCGAUA 5' (with uracil in RNA instead of thymidine).
2. Binding Process
   • Cas9 protein attaches to the RNA guide.
   • The Cas9-RNA complex binds to the target DNA sequence.
3. DNA Modification
   • Cas9 makes a double strand cut in the genomic DNA.
   • An alternative piece of DNA can be inserted at the cut site.

Applications of CRISPR-Cas9

• Works in various cell types and organisms.
• Used to study diseases and generate tissues from stem cells (e.g., heart muscle and neuronal tissue).
• Potential to treat whole multicellular organisms.
  • Example: CRISPR-Cas9 treatment improved symptoms in a mouse with liver disease caused by genetic defects.

Germline vs. Somatic Cell Editing

• Somatic Cells
  • Changes are not passed to the next generation.
• Germline Cells
  • Genomic modifications can be transmitted across generations.
  • This has been performed in several organisms, including mice, monkeys, and humans.

Controversial CRISPR-Cas9 Case

• Chinese scientist He Zhangquai claimed to have edited the genes of twin girls to confer HIV resistance.
  • Altered the CCR5 gene in embryos which were carried to term.
  • Twins reportedly born healthy.

Ethical Concerns Raised

1. Secrecy
   • Zhangquai did not inform his institution about the experiments.
2. Informed Consent
   • Uncertainty regarding proper informed consent from parents.
3. Medical Necessity
   • Procedure done without known medical need as neither infant had HIV.
4. Risks of Technology
   • CRISPR-Cas9 technology is still in infancy; risks are hard to predict.
   • Not all cells in the embryos were edited, leading to potential outcomes:
     • Heterozygous: Cells may have normal and edited copies of CCR5, possibly not resistant to HIV.
     • Mosaic: Some cells have normal, others have edited genes.
5. Mutation Mismatch
   • The mutation did not match the known variation that confers HIV resistance, leading to uncertainty about its effects.

Future of Germline Genome Editing

• Despite the rushed nature of the research, there is support for ethical germline genome editing to prevent severe genetic diseases (e.g., cystic fibrosis, muscular dystrophy).
• General consensus: The scientific community has moved faster than ethical guidelines.