Learning Objective: At the end of this lesson, the learner will be able to explain the mechanism and applications of the CRISPR/Cas9 genome editing system and identify its clinical and research relevance, including therapeutic gene modification and disease correction.
CRISPR/Cas9 is a genome-editing tool derived from bacterial defense systems. It allows for precise modification of DNA sequences within living cells by using a guide RNA (gRNA) and a CRISPR-associated endonuclease (Cas9).
Mechanism
| Component | Function |
|---|---|
| Guide RNA (gRNA) | A short RNA molecule that is complementary to the target DNA sequence. It directs Cas9 to the specific genomic locus. |
| Cas9 Endonuclease | A DNA-cutting enzyme that introduces single- or double-strand breaks at the target site. |
After the break, the cell’s natural repair mechanisms modify the DNA:
- Non-homologous end joining (NHEJ) → results in frameshift mutations, leading to gene inactivation (“knock-out”).
- Homology-directed repair (HDR) → allows insertion of a corrected or new DNA sequence (“knock-in”) using a supplied template.
Applications
- Therapeutic gene editing: correction of disease-causing mutations (e.g., sickle cell disease).
- Cancer research: targeting and inactivating oncogenes or restoring tumor-suppressor genes.
- Microbial engineering: removal of virulence factors from pathogens.
- Functional genomics: studying gene function by creating knock-out or knock-in models.
