CRISPR-Cas9 is a genome-editing tool known as "genetic scissors," and it earned Jennifer Doudna and Emmanuelle Charpentier the Nobel Prize in Chemistry in 2020. It is revolutionizing medicine, agriculture, and basic research.
Basic Knowledge
CRISPR is an abbreviation for the bacterial immune-memory mechanism "Clustered Regularly Interspaced Short Palindromic Repeats." Bacteria record DNA fragments from invading viruses in their genomes and, when reinvasion occurs, use Cas9 protein and guide RNA (gRNA) to cut the foreign DNA.
Principle of genome editing:
① Design a 20-base guide RNA complementary to the target sequence.
② The gRNA guides the Cas9 protein to the target sequence.
③ Cas9 cuts both DNA strands (DSB: double-strand break).
④ NHEJ (non-homologous end joining) repairs the break inaccurately → gene knockout, or HDR (homology-directed repair) uses template DNA for accurate replacement/insertion (knock-in).
Applications include gene-targeted therapy for cancer cells, treatment of genetic diseases (sickle-cell disease), crop improvement, and production of model animals.
Answer: (1) The guide RNA must have a sequence complementary to the target sequence (20 bases) of gene A, and a PAM sequence (NGG) must be present immediately downstream. (2) NHEJ directly joins cut ends through inaccurate repair, making frameshift mutations likely and being suitable for knockouts. HDR is accurate repair using homologous sequences as a template and is used for specific-sequence knock-ins. (3) Conventional genetic recombination randomly inserts a foreign gene, whereas genome editing can precisely modify a specific site in the endogenous genome.
- CRISPR-Cas9 = a genome-editing tool adapted from a bacterial immune-memory mechanism
- Guide RNA (20 bases) + Cas9 recognize and cut the target sequence
- NHEJ → inaccurate repair → knockout
- HDR → accurate repair → knock-in
- Doudna + Charpentier = 2020 Nobel Prize in Chemistry
- PAM sequence (NGG) is essential for Cas9 cutting
Practice
- Explain the role of guide RNA when editing a genome with CRISPR-Cas9.
- Compare the repair pathways NHEJ and HDR after a DSB, including their respective characteristics.
- State and discuss one ethical issue in treating genetic diseases using CRISPR-Cas9.