The following two figures were taken from: http://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/

Figure 1 ~ The steps of CRISPR-mediated immunity. CRISPRs are regions in the bacterial genome that help defend against invading viruses. These regions are composed of short DNA repeats (black diamonds) and spacers (colored boxes). When a previously unseen virus infects a bacterium, a new spacer derived from the virus is incorporated amongst existing spacers. The CRISPR sequence is transcribed and processed to generate short CRISPR RNA molecules. The CRISPR RNA associates with and guides bacterial molecular machinery to a matching target sequence in the invading virus. The molecular machinery cuts up and destroys the invading viral genome. Figure adapted from Molecular Cell 54, April 24, 2014 [5].

Questions:1. Why would it be advantageous for bacteria to be able to cut up viral DNA?

2. Based on what we learned in class, how could the CRISPR RNA “guide” the molecular machinery to specific viral DNA sequences? What biochemical process is happening?

3. What part of the DNA molecule do you think is damaged when a CRISPR cuts a DNA strand?

4. How is CRISPR similar to and different from a restriction enzyme?

Figure 2 ~ Gene silencing and editing with CRISPR. Guide RNA designed to match the DNA region of interest directs molecular machinery to cut both strands of the targeted DNA. During gene silencing, the cell attempts to repair the broken DNA, but often does so with errors that disrupt the gene—effectively silencing it. For gene editing, a repair template with a specified change in sequence is added to the cell and incorporated into the DNA during the repair process. The targeted DNA is now altered to carry this new sequence.

5. Name and define three types of mutations that could be introduced during DNA repair that could inactivate a gene’s function

6. Can you think of at least two reasons why researchers would want to break or inactivate a gene?

7. Why might researchers want to put a new sequence into a gene?

8. In what kind of cell would you need to do this sort of editing for the new information to be transmitted to the next generation?

Now go to : https://media.hhmi.org/biointeractive/click/CRISPR/ and click on “How it works”Scroll through the animation and answer the following questions:

9. What kind of enzyme is Cas9? Google this term and confirm your answer to question 3 on the first page.

10.Based on your answers to the first page questions, where did the guide RNA come from and how was it made? What sequence does it match if we are looking at CRISPRs in a bacterial cell? What sequence does it match if we are looking at human cells studied by researchers?

11.What are the three DNA bases in yellow, and why are they important?

12.Based on your knowledge of DNA binding proteins, describe how Cas9 binds to a PAM.

13.What happens immediately after Cas9 binds to the PAM? Why is step necessary? What other enzyme have we seen that has a similar ability, and what cellular process does this enzyme participate in?

14.How does binding of the guide RNA to a target DNA molecule affect the Cas9 enzyme?

15.Explain how CRISPRs illustrate an example of an emergent property.

STOP HERE AND WAIT FOR DISCUSSION

One promising use of CRISPR is to fix disease-causing mutations. Muscular dystrophy is a hereditary disease characterized by progressively weakening muscle function due to mutations in a protein called Dystrophin. Researchers used mice with a recessive mutation in the Dystrophin gene (on the X chromosome) that causes an early stop codon. They used CRISPRs targeting this stop codon and a repair template that would repair Dystrophin to a functional sequence without a stop codon. mdx is the name of the mutated allele of Dystrophin.

16.The authors used the following experimental design. Based on the genotypes of the parents, what fraction of offspring do you expect to be males with muscular dystrophy (in the absence of CRISPRs)? Females with muscular dystrophy?

17.Challenge question: why might not all cells in the embryo carry a change induced by the CRISPR? (hint: think about what happens to cells in embryos)

18.Dystrophin is a protein that is found near the plasma membrane of muscle cells. Below are cross sections of the mouse’s muscle tissue with Dystrophin labeled fluorescently. HDR-17% means that 17% of cells had a replacement of the mutated sequence with a corrected copy; HDR-41% means that 41% of cells had a replacement of mutated sequence with a corrected copy. Interpret these results; make sure to mention the importance of the controls.

One way that CRISPR has been modified is for base editing: a targeted replacement of one DNA base with another specific base. Instead of creating a random mutation or DNA replacement, a specific DNA letter can be converted to another DNA letter.

19.Based on the figure below, why is this useful? What is a “pathogenic human SNP”? Which type of conversion is MOST useful?

20.No enzymes that can change As to Gs in DNA exist in nature, so researchers used experimental evolution to make one. They used an enzyme that can perform the following reaction on RNA:

a) Inosine is not part of the standard genetic code. Why is it still useful?

b) Why might this enzyme only work on RNA and not DNA? Propose a specific mechanism of how the enzyme interacts with DNA vs RNA

21.The researchers used evolution/selection to find mutations that would allow this enzyme to work on DNA instead of RNA. Essentially they gave bacteria a copy of an antibiotic resistance gene with a nonsense point mutation in it. If this gene was successfully base-edited, then the nonsense mutation would be corrected and the resistance gene would work. They then created lots of mutations in the gene encoding the base editing protein. They grew the bacteria containing the mutant base editing gene and the mutant antibiotic resistance gene in the presence of the antibiotic. Explain why this scheme would help identify mutations that improve the function of the based editor to work on DNA.

22.Below are results for the optimized base editor (ABE7.10) compared to CRISPR-Cas9. 48h and 120h refer to the amount of time that passed after cells were treated. Which one works better for changing A-T to G-C? How much better is it? Is it perfect?

23.More recent data has shown that these tools can cause off-target effects by editing bases in RNA and also unintended DNA sequences. Why would this be problematic?