Questions: CRISPR-Cas Systems and Adaptive Bacterial Immunity

Question 1

A bacterium survives a phage attack. Researchers examine its genome the following day and find a new 30-bp sequence inserted into its CRISPR array that perfectly matches a segment of the attacking phage's genome. What does this represent?

• A. A random mutation that happened to match the phage sequence by coincidence
• B. The acquisition step of CRISPR adaptive immunity — Cas1/Cas2 captured a fragment of the phage DNA and inserted it as a heritable molecular record of that infection
• C. Transcriptional activation of an anti-viral gene that was previously silenced in the genome
• D. Horizontal gene transfer from a nearby phage-resistant bacterium that already had immunity

Answer: (B)

This is the acquisition step of CRISPR immunity. Cas1 and Cas2 proteins recognize foreign DNA during an infection, cleave out a short fragment called a protospacer, and integrate it into the CRISPR array as a new spacer. The resulting spacer is a permanent, heritable record of that specific phage — molecular memory that will be passed to all daughter cells. This is what makes CRISPR adaptive (memory-based) rather than innate (generic) immunity.

Question 2

A phage that previously infected strain A no longer does — strain A has a CRISPR spacer matching the phage. The phage then mutates two nucleotides in its protospacer region. Which prediction follows?

• A. Strain A remains fully protected because Cas9 tolerates a few mismatches anywhere in the protospacer
• B. Strain A is now vulnerable to the mutant phage because guide RNA binding requires near-perfect complementarity; the mutations disrupt recognition, allowing the phage to escape immunity
• C. Neither strain is vulnerable because the mutations affect only surface proteins, not the DNA that Cas9 targets
• D. Both strains become equally vulnerable because any phage mutation resets CRISPR immunity in all bacteria

Answer: (B)

CRISPR guide RNA (crRNA) binds to target DNA by Watson-Crick base pairing. Mismatches in the spacer-protospacer match — especially near the PAM-proximal 'seed region' — disrupt recognition and prevent Cas9 from cutting the phage DNA. This is one of the primary mechanisms by which phages evolve to escape CRISPR immunity: mutation in the protospacer or PAM disrupts the complementarity that the guide RNA depends on. This coevolutionary arms race drives rapid diversification of both phage genomes and bacterial CRISPR arrays.

Question 3

The CRISPR spacer array functions as a chronological record of past phage infections: newer spacers are added at one end of the array, so older spacers are farther from the leader sequence — allowing researchers to infer a bacterium's infection history.

• T. True
• F. False

Answer: True

New spacers are integrated at the leader-proximal end of the CRISPR array during each acquisition event. This means the array accumulates a temporal archive of phage encounters: the most recent infection is represented by the spacer closest to the leader, and older spacers are progressively farther away. Researchers can read this molecular timeline to reconstruct which phages a bacterial lineage has encountered, in roughly what order — a form of microbial paleovirology.

Question 4

CRISPR-Cas systems provide bacteria with essentially complete, fail-safe immunity against any phage whose sequence matches a stored spacer, since the guide RNA will typically find and destroy the invader.

• T. True
• F. False

Answer: False

CRISPR immunity is powerful but not infallible. Phages counter it through multiple mechanisms: mutating protospacer or PAM sequences to disrupt guide RNA binding; encoding anti-CRISPR (Acr) proteins that directly inhibit Cas enzymes; or evolving genomic regions that lack any stored spacer match. Some phages even evolve phage-encoded CRISPR systems to target bacterial defense genes. The ongoing arms race between bacterial CRISPR acquisition and phage escape mechanisms drives enormous genetic diversity in both parties.

Question 5

Explain why the PAM sequence requirement is essential for CRISPR-Cas function. What problem would arise if the Cas9 system cut DNA without checking for a PAM?

Model answer: The PAM (protospacer adjacent motif) is a short sequence (e.g., NGG for SpCas9) that must be present on the target DNA adjacent to the protospacer for Cas9 to cleave it. Foreign DNA (phage genomes) has PAM sequences flanking protospacers. Crucially, the bacterium's own CRISPR array also contains spacer sequences that match past invaders — but the stored spacers lack PAM sequences flanking them. Without PAM recognition, Cas9 could not distinguish between a phage's protospacer (target) and the bacterium's own stored spacer (self). It would cut the CRISPR array itself, destroying the immune memory it relies on. PAM recognition is the self/non-self discrimination mechanism that makes the system safe to operate inside the bacterium's own genome.

This is why PAM sequences are critical for the biotechnology applications too: guide RNAs for gene editing must be designed to target sequences adjacent to the correct PAM in the genome of interest, and the PAM requirement limits where in any given genome Cas9 can cut.