How CRISPR works at the molecular level

– CRISPR works at the molecular level by using a guide RNA molecule and a Cas9 enzyme to target and edit specific DNA sequences.

– The guide RNA is a short piece of RNA that is complementary to the DNA sequence that needs to be modified.

– The Cas9 enzyme is a protein that can cut DNA at specific sites.

– Together, they form a complex that can recognize and bind to the target DNA sequence.

– Once bound, the Cas9 enzyme can cut both strands of the DNA, creating a double-strand break. This break can then be repaired by the cell's own DNA repair mechanisms, which can either join the ends back together (non-homologous end joining) or use a template DNA molecule to fill in the gap (homology-directed repair).

– Depending on the type of repair and the presence of a template, CRISPR can be used to delete, insert, or replace DNA sequences in the genome.

🔅 The role of guide RNA in CRISPR-Cas9?

– Guide RNA (gRNA) is a key component of the CRISPR-Cas9 system that directs the Cas9 enzyme to the specific DNA sequence that needs to be edited.

– The gRNA is a short piece of RNA that is complementary to the target DNA sequence and binds to it by base pairing. The gRNA also contains a scaffold region that interacts with the Cas9 enzyme and forms a ribonucleoprotein (RNP) complex.

– The gRNA-RNP complex can recognize and bind to the target DNA sequence only if it is followed by a specific protospacer adjacent motif (PAM), which is a short DNA sequence that varies depending on the type of Cas9 enzyme used.

– The PAM serves as a signal for the Cas9 enzyme to cut both strands of the DNA, creating a double-strand break. The double-strand break can then be repaired by the cell's own DNA repair mechanisms, which can result in gene knockout, knock-in, or correction, depending on the presence and design of a repair template.