How does CRISPR-Cas9 generate targeted genome edits?

How does CRISPR-Cas9 generate targeted genome edits?

• A. RNA polymerase transcribes target gene without breaks
• B. Cas9 nuclease with guide RNA introduces double-strand break at target sequence; cellular repair introduces mutations or allows insertion
• C. Cas9 binds but does not cut DNA
• D. Cas9 integrates plasmid into genome

Answer: (B)

CRISPR-Cas9 edits genomes by using a guide RNA to bring the Cas9 nuclease to a specific DNA sequence and create a double-strand break there. Once the break is made, the cell’s repair machinery kicks in. If the break is repaired by non-homologous end joining, the process often inserts or deletes a few bases, disrupting the gene. If a donor DNA template with matching sequences (homology arms) is provided, the cell can use it to repair the break via homology-directed repair and incorporate a precise sequence change or insertion. This combination of targeted cutting and cellular repair is what enables targeted genome edits. The other options don’t fit because transcription can happen without breaks, Cas9’s role is to cut DNA rather than merely bind, and plasmid integration isn’t the standard mechanism for creating the intended edits (it’s the repair process, not random integration, that mediates the edit when a donor template is used).

CRISPR-Cas9 edits genomes by using a guide RNA to bring the Cas9 nuclease to a specific DNA sequence and create a double-strand break there. Once the break is made, the cell’s repair machinery kicks in. If the break is repaired by non-homologous end joining, the process often inserts or deletes a few bases, disrupting the gene. If a donor DNA template with matching sequences (homology arms) is provided, the cell can use it to repair the break via homology-directed repair and incorporate a precise sequence change or insertion. This combination of targeted cutting and cellular repair is what enables targeted genome edits. The other options don’t fit because transcription can happen without breaks, Cas9’s role is to cut DNA rather than merely bind, and plasmid integration isn’t the standard mechanism for creating the intended edits (it’s the repair process, not random integration, that mediates the edit when a donor template is used).