How might you use CRISPR–Cas9 to create a large deletion?

Key Concepts

CRISPR-Cas9 Mechanism

CRISPR-Cas9 is a revolutionary gene-editing technology that allows for precise modifications in DNA. It utilizes a guide RNA to direct the Cas9 enzyme to a specific location in the genome, where it creates a double-strand break. This break can then be repaired by the cell's natural repair mechanisms, which can lead to insertions, deletions, or modifications of the genetic material.

Large Deletions

Large deletions refer to the removal of significant segments of DNA from the genome. In the context of CRISPR-Cas9, creating a large deletion typically involves designing two guide RNAs that target sequences flanking the region to be deleted. When both guides direct Cas9 to cut at their respective sites, the intervening DNA can be excised during the repair process, resulting in a deletion.

Homology-Directed Repair (HDR) and Non-Homologous End Joining (NHEJ)

After CRISPR-Cas9 induces a double-strand break, the cell can repair the break through two main pathways: Homology-Directed Repair (HDR) and Non-Homologous End Joining (NHEJ). HDR can be used for precise edits if a template is provided, while NHEJ often leads to insertions or deletions (indels) at the break site. For large deletions, NHEJ is typically the pathway utilized, as it can result in the removal of the DNA between the two cut sites.