Mechanisms of action
The mechanism of action of CRISPR-Cas enzymes in human cells involves several steps:
- Recognition of target DNA: The Cas nuclease enzyme, guided by the sgRNA, recognizes and binds to a specific DNA sequence within the genome of human cells. The sgRNA is designed to be complementary to the target DNA sequence, leading the Cas nuclease to the desired site for genome editing.
- Cleavage of DNA: Once bound to the target DNA, the Cas nuclease cleaves both strands of the DNA, creating a double-stranded break (DSB) at the target site. This DSB triggers the cell's natural DNA repair mechanisms.
DNA repair mechanisms:
Human cells have two main DNA repair pathways that can be involved in the repair of the DSB created by Cas nuclease: non-homologous end joining (NHEJ) and homology-directed repair (HDR).
- NHEJ: These systems are the most well-known and widely used for genome editing. They are characterized by the Cas9 nuclease, which requires both tracrRNA and crRNA for target DNA recognition and cleavage. The tracrRNA and crRNA can be artificially linked to use as a single guide RNA (sgRNA)
- HDR: HDR is a more precise DNA repair pathway that can be used to introduce specific DNA sequences at the DSB site. HDR requires a template DNA molecule with homology to the sequences flanking the DSB site, which can be used as a template for repair. This allows for precise gene editing, insertion of desired sequences, or correction of specific mutations.
- Editing outcome The repair outcome depends on the type of DNA repair pathway that is predominantly used in the specific human cell type being targeted. NHEJ repairs often result in gene disruption or knockout, while HDR repairs can result in precise gene editing, insertion of desired sequences, or correction of specific mutations.
CRISPR-Cas is certainly a revolutionary tool in biotechnology due to its precision, simplicity, versatility, and potential applications in various fields. It has opened up new possibilities for genetic research, gene therapy, crop improvement, and other areas, while also raising ethical considerations that need to be carefully addressed.