For Feb 3: Discussion paper
Sternberg, S.H., Redding, S., Jinek, M., Greene, E.C., and Doudna, J.A. (2014) DNA
interrogation by the CRISPR RNA-guided endonuclease Cas9, Nature, in press.
1) What are PAM sites and why are they significant?
2) Explain the single-tethered DNA curtains assay. What technical issues
contribute to its limited resolution, as evidenced by the broad distributions
in Figure 1d?
3) What factors might the authors have considered when choosing the six target
sites shown in Figure 1b?
4) What method did the authors use to compare DNA-binding affinities of apoCas9 to the RNA-bound form (Extended Data Figure 3)? What other methods
might they have used?
5) Why is heparin used as a competitor in Figure 1g? What features does it
share with the crRNA–tracrRNA competitor?
6) What would Figure 2e have looked like if Cas9–RNA associated with target
sites by one-dimensional sliding? What about one-dimensional hopping?
7) Why were the authors surprised to find that the lifetimes of “nonspecific
binding events” were insensitive to salt concentration in Figure 2f?
8) Why does the data in Figure 3e not perfectly match the predictions of the
sequential unwinding model? Can you propose a model that better fits the
data?
9) What is the evidence that PAM sites are needed not only for DNA recognition
but also for Cas9 catalysis?
10)What are the implications of the results for achieving high DNA-binding
specificity and minimizing off-target effects when using Cas9 for genome
engineering?
Assignments:
Odd number questions – Crick
Even number questions - Franklin