Question

Which is an endonuclease in CRISPR Cas9 Technique?

• A. gRNA
• B. Cas 9
• C. PAM
• D. Cr RNA

Hint:

• CRISPR-Cas9 System Components
  • Cas9 Protein An RNA-guided DNA endonuclease (enzyme that cuts DNA).
  • Guide RNA (gRNA/sgRNA) Directs Cas9 to the target DNA sequence. Contains a crRNA-like spacer for target recognition and a tracrRNA-like scaffold for Cas9 binding.
• PAM Sequence A short sequence in the target DNA, adjacent to the target site, required for Cas9 recognition and cleavage. Not part of the Cas9 enzyme or gRNA.

Answer 1

The Correct Option is B

The CRISPR-Cas9 system is a powerful gene editing tool derived from a bacterial adaptive immune system. It involves two key components for targeted DNA cleavage: 1. Cas9 (CRISPR-associated protein 9): This is an RNA-guided DNA endonuclease. It's an enzyme that uses a guide RNA (gRNA) molecule to find a specific target DNA sequence and then makes a double-strand break (DSB) in the DNA at that site. Cas9 has two nuclease domains (HNH and RuvC) that cut the two DNA strands. 2. Guide RNA (gRNA): In engineered CRISPR-Cas9 systems, this is often a synthetic single guide RNA (sgRNA), which combines the features of two natural bacterial RNAs:

• crRNA (CRISPR RNA): Contains a ~20 nucleotide sequence (spacer) that is complementary to the target DNA sequence, providing specificity. (Option d)
• tracrRNA (trans-activating crRNA): Binds to the crRNA and also to the Cas9 protein, forming a scaffold for the complex.

The gRNA directs the Cas9 endonuclease to the correct target site in the genome. (Option a refers to the guide RNA). Other terms:

• PAM (Protospacer Adjacent Motif) (Option c): A short DNA sequence (e.g., NGG for Streptococcus pyogenes Cas9) that is located immediately downstream of the target DNA sequence complementary to the gRNA spacer. The Cas9 enzyme requires the presence of a PAM site near the target sequence for efficient DNA binding and cleavage. The PAM itself is part of the target DNA, not the endonuclease.

The question asks which component is an endonuclease. The Cas9 protein is the endonuclease enzyme responsible for cutting the DNA. \[ \boxed{\text{Cas 9}} \]