76. CRISPR/Cas9 is an example of bacterial adaptive immunity. The transcription of CRISPR loci generates small
crispr-RNAs (crRNA) to specifically target viral DNA, but not CRISPR loci, by forming complex with guide RNA
and Cas9 nuclease. This prevention of autoimmunity is due to the:
(1) absence of protospacer adjacent motif sequence in CRISPR loci.
(2) absence of DNA sequence complementary to crRNA in CRISPR loci.
(3) absence of DNA sequence complementary to guide RNA in CRISPR loci.
(4) methylation of CRISPR loci.

Introduction:

CRISPR/Cas9 is a revolutionary tool in molecular biology, originally discovered as a part of the bacterial adaptive immune system. It helps bacteria recognize and defend against viral DNA by forming a complex between crRNA (CRISPR RNA), guide RNA, and the Cas9 nuclease. However, one major concern in immune systems is the prevention of autoimmunity—where the immune system mistakenly targets and damages its own cells. In this article, we explore how bacteria use CRISPR/Cas9 to avoid autoimmunity and protect their own genomic integrity while targeting viral invaders.

How CRISPR/Cas9 Works:

1. Bacterial Immunity:

   • Bacteria use CRISPR sequences to remember past viral infections. These sequences are stored in the form of CRISPR loci, which contain short segments of viral DNA known as spacers. When a virus infects a bacterium, it transcribes the CRISPR loci to generate small RNAs, known as crRNAs, that match viral DNA sequences. These crRNAs then guide the Cas9 nuclease to the target viral DNA to cut and neutralize the threat.

2. Role of Guide RNA:

   • The crRNA guides the Cas9 nuclease to the corresponding viral DNA sequence, facilitating a precise cut. However, it is crucial that the CRISPR system does not target the bacterial genome itself—this could lead to the destruction of the bacterium’s own DNA, causing autoimmunity.

How Autoimmunity Is Prevented in CRISPR/Cas9:

The prevention of autoimmunity in CRISPR/Cas9 systems involves a combination of mechanisms that ensure that the bacterial DNA is not mistakenly targeted. The key factor in preventing this self-targeting is the absence of protospacer adjacent motif (PAM) sequences or complementary sequences in the CRISPR loci.

• Protospacer Adjacent Motif (PAM):

  • The PAM is a short DNA sequence that is essential for the Cas9 nuclease to bind and cut the target DNA. For a crRNA to successfully guide Cas9 to the target site, the viral DNA must contain a PAM sequence adjacent to the target sequence. Importantly, CRISPR loci in the bacterial genome do not contain the PAM sequence, which ensures that the system does not target its own DNA.

• The Role of Complementary Sequences:

  • While CRISPR loci contain spacer sequences that match viral DNA, there are no complementary sequences to the crRNA or guide RNA within the CRISPR loci themselves. This ensures that the bacterial genome is not targeted.

Correct Answer:

(1) Absence of Protospacer Adjacent Motif Sequence in CRISPR Loci.

By lacking the PAM sequence, the CRISPR system can differentiate between foreign viral DNA and the bacterium’s own genome, preventing self-targeting and autoimmunity.

Conclusion:

The CRISPR/Cas9 system is an elegant bacterial immune mechanism that specifically targets viral DNA without harming the bacterium itself. The prevention of autoimmunity is achieved through the absence of the protospacer adjacent motif (PAM) sequence in the CRISPR loci. This careful selection ensures that bacteria can defend against viruses while maintaining the integrity of their own genomic material. The CRISPR/Cas9 system’s precise targeting mechanism is what makes it such a powerful tool in modern molecular biology and gene editing.