Question 1

How does CRISPR function as an immune system in bacteria and archaea?

Accepted Answer

CRISPR, combined with Cas proteins, acts as an adaptive immune system that identifies, remembers, and neutralizes viral infections. When foreign DNA enters the cell, Cas proteins recognize specific sequences called Protospacer Adjacent Motifs (PAMs) and extract viral DNA segments called protospacers. These fragments are stored in the CRISPR array as a genetic memory, allowing the cell to recognize and destroy the same virus during future infections.

Question 2

What role does the Protospacer Adjacent Motif play in CRISPR defense?

Accepted Answer

The Protospacer Adjacent Motif (PAM) is a specific DNA sequence that Cas proteins recognize to identify foreign genetic material. By requiring a PAM adjacent to the target sequence, the CRISPR system ensures specificity and prevents accidental cleavage of the host's own genome. This requirement protects bacterial and archaeal cells from self-targeting while maintaining effective defense against viral invaders.

Question 3

How are CRISPR RNA molecules processed and used during viral reinfection?

Accepted Answer

The CRISPR array is transcribed into pre-crRNA, which pairs with tracrRNA. RNase III and Cas9 then process this duplex into individual crRNAs, each carrying one spacer and repeat fragment. During reinfection, these crRNAs guide Cas proteins to scan invading viral DNA for matching sequences. Upon finding a match adjacent to a PAM, Cas proteins cleave the viral DNA, neutralizing the infection.

Question 4

What information does the CRISPR array store about past viral infections?

Accepted Answer

The CRISPR array functions as a molecular record by storing viral DNA fragments called spacers between palindromic repeats. Each spacer represents a segment from a previous invader's genome, creating a genetic memory of past infections. This stored information enables the cell to recognize and respond rapidly to subsequent infections by the same virus, providing long-term adaptive immunity.

Question 5

How do Cas proteins recognize and extract viral DNA during initial infection?

Accepted Answer

When viral DNA enters the cell, Cas proteins scan for the Protospacer Adjacent Motif (PAM), a specific sequence that signals foreign DNA. Once a PAM is identified, Cas proteins excise the adjacent protospacer segment from the viral genome. This extracted DNA fragment is then inserted into the CRISPR array, where it becomes a spacer that serves as a template for future immune responses against that particular virus.

Question 6

Why is the PAM requirement essential for preventing self-targeting in CRISPR systems?

Accepted Answer

The PAM requirement enhances specificity by ensuring Cas proteins target only foreign DNA and not the bacterium's own CRISPR array. Since the host's CRISPR sequences lack PAMs in the same configuration as invading viral DNA, the system distinguishes between self and non-self genetic material. This protective mechanism preserves cellular integrity while maintaining effective defense against viral infections.

Question 7

What happens when a virus reinfects a cell with an active CRISPR immune system?

Accepted Answer

Upon reinfection, the crRNA-Cas complex scans the invading viral DNA for sequences complementary to stored spacers. When a match is found adjacent to a PAM, the Cas protein cleaves the viral DNA at the target site, halting the infection. This rapid recognition and destruction mechanism demonstrates how CRISPR provides adaptive immunity by leveraging the genetic memory stored during previous infections.

The Antiviral System of Bacteria and Archaea: CRISPR

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