Molecular Machines

Cas9

The 28 review prompts from the notes on Cas9. Answer each in your head, then reveal it. Your grade decides when the card comes back.

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Summary 9

Read this section: Summary, in the Cas9 notes

  1. What does Cas9 do for the bacterium that carries it?

  2. In Cas9, what part of the machine holds the address of the target?

  3. Cas9 spends no ATP on targeting; cleavage needs blank and the free energy of RNA-DNA base pairing.

  4. Which DNA strand does the HNH domain of Cas9 cut?

  5. Which DNA strand does the RuvC domain of Cas9 cut?

  6. The two catalytic residues of SpCas9 are blank.

  7. Which two Cas9 residues read the two guanines of the NGG PAM?

  8. What share of known disease-associated genetic variants do the prime editing authors estimate it could in principle correct?

  9. Which goal on the Cas9 frontier list is still only proposed rather than demonstrated?

How it works 6

Read this section: How it works, in the Cas9 notes

  1. What has to happen before Cas9 can accept DNA at all?

  2. Why does Cas9 pass over a perfect sequence match that has no nearby PAM?

  3. Strand separation in Cas9 starts at the blank, and the RNA-DNA heteroduplex then grows away from there toward the far end of the target.

  4. What is the first checkpoint in the Cas9 cycle, the one that allows catalysis?

  5. Which step is the final specificity checkpoint of Cas9?

  6. What makes the two nuclease domains of Cas9 fire together instead of one at a time?

Big picture 6

Read this section: Big picture, in the Cas9 notes

  1. How many virus infections start in the ocean every second, the pressure that CRISPR immunity answers?

  2. How long does one Cas9 need to find its target in E. coli?

  3. In random DNA an NGG PAM turns up about once every blank base pairs, counting both strands.

  4. What does the approved Cas9 therapy cut in a patient's own blood stem cells?

  5. Calling Cas9 search-and-cut scissors gets one tool and one target right. What does it get wrong?

  6. How is the bacterial CRISPR memory unlike our own immune memory?

Evidence 7

Read this section: Evidence, in the Cas9 notes

  1. The ternary complex structure crystallised a full-length SpCas9 chain of blank residues.

  2. What does a Cas9 carrying only one of the two active-site mutations do to DNA?

  3. Gel binding assays gave dCas9 a Kd of 0.80 nM on target. What was the Kd with 8 PAM-distal mismatches?

  4. How was the link between HNH conformation and cleavage rate in Cas9 measured?

  5. Cryo-EM of the active Cas9 complex with magnesium shows the HNH domain travelling about blank A to reach the target-strand scissile bond.

  6. Where does Cas9 cut the target strand, relative to the PAM?

  7. Single-molecule imaging on DNA curtains found Cas9 reaching targets only by three-dimensional collisions. How did later FRET work qualify that?

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