This mobile version links to a video of the Case It simulation running the procedure described in Part 4 of the CRISPR exercise, to recreate Figure S15D of Jinek et al. 2012. Research described in this paper resulted in the 2020 Nobel Prize in Chemistry.

Hypothesis to be tested: Cas9 can be used as a programmable restriction enzyme, able to cut any DNA sequence close to a 3-bp PAM site.

Summary of the experimental procedure

• Create plasmid DNA with 5 unique recognition sites, three on the non-complementary strand and two on the complementary strand, and also include a recognition site for the restriction enzyme SalI (see Fig S15 A of the supplement to Jinek et al. 2012)
• Create five different guide RNA/Cas9 enzyme complexes programmed to recognize the five unique sites via complementary base pairing (Fig S15 B and C)
• Digest the plasmid DNA with the five enzyme complexes along with the enzyme SalI.
• Digest the non-complementary strand with SalI.
• Run gel electrophoresis on the products of the above reactions, including a 100-bp DNA ladder (Fig S15 D).
  

Video of Case It v705 simulating above procedure

Note: When using the actual simulation, sequence files are accessed via the ‘DNA’ button. This process is not shown in the video, as the relevant files have already been opened.

Questions:

A. Why was SalI included in the experimental procedure? Why was it necessary to include a DNA ladder?

B. Do the results support the hypothesized function of Cas9 as a programmable DNA-cutting enzyme? Why or why not?

C. One could argue that the virtual gel and Fig S15 summarize one of the more important experimental results in biological history. Do you agree or disagree with this? Justify your answer.

D. Revisit your answer to B above after watching this video, starting at the 6:37 mark. Why does Jennifer Doudna refer to results of the experiment in Fig S15 as their “aha moment”?