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Examines the revolutionary CRISPR-Cas9 system, its bacterial origins, and its applications in targeted genome editing.
What if you could edit the 'source code' of a living organism as easily as fixing a typo in a text message? CRISPR-Cas9 has turned this science fiction into a laboratory reality.
Originally discovered as a bacterial immune system against viruses, CRISPR-Cas9 consists of two primary components. The Cas9 nuclease acts as 'molecular scissors,' capable of cutting double-stranded DNA. However, Cas9 is blind on its own; it requires guide RNA (gRNA) to find its target. The gRNA is a synthetic sequence designed to be complementary to the target gene. Once the gRNA binds to the DNA, Cas9 looks for a PAM (Protospacer Adjacent Motif) sequence—typically —which serves as a 'safety lock.' If the PAM is present and the gRNA matches the DNA, Cas9 performs a double-strand break (DSB).
Quick Check
What specific sequence must be present immediately following the target DNA for Cas9 to initiate a cut?
Answer
The PAM (Protospacer Adjacent Motif) sequence, specifically .
Once Cas9 cuts the DNA, the cell's natural repair machinery takes over. There are two main pathways. Non-Homologous End Joining (NHEJ) is the default 'quick fix.' It shoves the broken ends back together, but it is messy, often causing small insertions or deletions (indels). This usually breaks the gene, making NHEJ ideal for gene knockouts. Alternatively, if a scientist provides a 'donor template' of DNA, the cell may use Homology-Directed Repair (HDR). HDR uses the template to precisely rewrite or 'knock in' a new sequence, allowing for the correction of point mutations.
1. Scenario A: You want to stop a cancer cell from producing a growth protein. You would use NHEJ to create a frameshift mutation and disable the gene. 2. Scenario B: You want to fix a single nucleotide mutation in the hemoglobin gene to cure Sickle Cell Anemia. You would use HDR by providing a healthy DNA template along with the CRISPR components.
Quick Check
Which repair pathway is most likely to result in a 'gene knockout' due to random insertions or deletions?
Answer
Non-Homologous End Joining (NHEJ).
The application of CRISPR is divided into two categories. Somatic editing targets non-reproductive cells (like lung or blood cells). Changes made here affect only the patient and are not inherited. Germline editing targets embryos, sperm, or eggs. These changes are permanent and will be passed down to all future generations. While germline editing could theoretically eliminate hereditary diseases like Huntington's, it raises massive ethical concerns regarding 'designer babies' and permanent alterations to the human gene pool.
If a gRNA sequence is 20 nucleotides long, the theoretical number of unique sequences is . However, Cas9 can sometimes bind to sequences with 1-3 mismatches. If a genome has base pairs, scientists must use bioinformatics to ensure the chosen 20-bp sequence does not appear elsewhere in the genome to avoid 'off-target' mutations.
1. Identify target: 2. Check for PAM: 3. Run BLAST search: Ensure no similar sequences exist in non-target genes.
What is the primary role of the guide RNA (gRNA) in the CRISPR-Cas9 system?
Why is Homology-Directed Repair (HDR) preferred over NHEJ for correcting genetic diseases?
Genetic changes made via somatic cell CRISPR editing will be passed on to the patient's biological children.
Review Tomorrow
In 24 hours, try to explain the difference between NHEJ and HDR to someone else without looking at your notes.
Practice Activity
Research a real-world application of CRISPR currently in clinical trials (e.g., for Leber Congenital Amaurosis) and identify if it uses somatic or germline editing.