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Chopped! Using CRISPR/Cas9 to cut DNA - Student's Guide
Version: 1.0 - Release: May 2022 - © 2022 by miniPCR bio™
Student's Guide
P./16
Today's lab
CRISPR/Cas9 has revolutionized biology by making it possible for scientists to manipulate DNA in
the living cells of virtually any organism. To understand how CRISPR/Cas9 can be such a versatile
tool, it helps to first break down exactly how this system works. Today, you will use CRISPR/Cas9 in
a system that you can easily manipulate and that will allow you to directly see the changes that you
make to DNA.
Your goal is to determine how each component of the CRISPR/Cas9 system contributes to cutting
DNA in a way that is both specific and programmable. To do this, you will use CRISPR/Cas9 in vitro
(in a test tube). This will allow you to experiment with the Cas9 enzyme and different guide RNAs
without the difficulty and time needed to rear and manipulate live organisms. But make no mistake,
today you are using real Cas9 enzymes, guide RNAs, and DNA.
You will be provided with Cas9 enzyme, two different gRNAs, and a sample of DNA. You will also be
provided with the sequences of both guide RNAs and the DNA.
Can you predict where the DNA will be chopped? Can you then prove that you are correct?
Lab activities
-
DNA
DNA
gRNA
gRNA
Cas9
1. Pre-lab analysis: You will analyze the sequences of each gRNA and your DNA sample to predict
where Cas9 will be directed to cut the DNA.
2. Set up the CRISPR/Cas9 reaction: You will create two different Cas9/gRNA complexes using
the Cas9 enzyme and your two gRNAs. Then, you will use these complexes to target and cut
your DNA.
3. Analyze results by gel electrophoresis: You will use gel electrophoresis to separate and
visualize the DNA fragments produced in your CRISPR/Cas9 reactions. Finally, you will
compare your experimental results to your predictions.
Step 1: Make DNA cutting
predictions based on
gRNA sequences
Step 2: Form Cas9/gRNA
molecular complexes
Step 3: Analyze cut DNA
using gel electrophoresis
