Building a Paper Model of CRISPR-Cas9
Cas9 will keep binding to other PAM sequences until it finds the matching target DNA.
You will now model how the guide RNA finds its matching target DNA using target DNA 1. The target DNA 1
sequence is from a real gene called MC1R. This gene codes for a protein that affects the color of skin and hair.
5. Slide target DNA 1 under guide RNA 1, through the rightmost tab
on the Cas9 enzyme. Line up the red targeting sequence on the
guide RNA with the complementary sequence on the target DNA,
as shown in Figure 5.
Question 1a. Write d
own the guide RNA 1 sequence that binds to
the DNA, and the complementary DNA 1 sequence that it binds to.
Label the 5' and 3' ends of both strands.
Once the guide RNA binds to the DNA, it activates the nuclease activity
(DNA-cutting ability) of the Cas9 enzyme. Cutting DNA is also called
“cleaving.” Cas9 always cleaves both strands of the DNA three
nucleotides upstream (toward the 5’ end) of the PAM sequence.
6. To model how Cas9 cleaves the DNA, use scissors to cut target DNA
1 along the dotted line, as shown in Figure 6. Do not cut the guide
odeling DNA Repair
After Cas9 cleaves the DNA, cellular enzymes will attempt to repair the break. CRISPR-Cas9 takes advantage of
these repair mechanisms to alter the target gene sequence. You will now explore two applications of CRISPR-
Cas9 that use different repair mechanisms.
First, you will model how CRISPR-Cas9 can be used to inactivate a target gene (“gene knockout”). In this case,
the cell uses nonhomologous end joining (NHEJ), a repair mechanism that is sometimes error-prone, to repair
the DNA break.
7. To model a possible outcome of NHEJ when CRISPR-Cas9 is used, tape the cut pieces of target DNA 1
together with the “Random Nucleotides” piece between them. These random nucleotides represent a
mutation that is likely to inactivate the target gene.
Question 1b. Compare the sequences in the model you just made to your answer to Question 1a. How did
the sequence of the gene change due to CRISPR-Cas9? Where was this change made (to the RNA, to one
DNA strand, or to both DNA strands)?
Question 1c. How might this change inactivate, or “knock out,” a gene?
Figure 5. Model of the Cas9-guide RNA
complex bound to target DNA 1.
Figure 6. Model of Cas9 cleaving target