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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
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different types of Cas nucleases. The different Cas nucleases are derived from different species
of bacteria and each works in slightly different ways. Cas9 is the enzyme most commonly used in
genome editing and is the specific enzyme we will focus on today. Cas9 is a powerful tool because it
can be programmed to specifically cut nearly any DNA sequence.
How CRISPR/Cas9 works
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The CRISPR/Cas9 system uses two main components: a Cas9 enzyme and a guide RNA (Figure 1).
Each component has an essential role in the function of the CRISPR/Cas9 system.
The Cas9 nuclease cuts DNA
The basic function of the Cas9 nuclease is simple: it cuts both strands of a DNA molecule. What
sets Cas9 apart from other nucleases is that it can be directed to cut virtually any DNA sequence.
To which specific DNA sequence the Cas9 nuclease will be directed is determined by the other key
component in the CRISPR/Cas9 system: the guide RNA (gRNA).
Guide RNAs control where Cas9 cuts
One end of the gRNA is a region referred to as the scaffold. The scaffold region is purely structural.
The nucleotides of the scaffold region fold to create a double-stranded RNA structure. This structure
binds to the Cas9 nuclease, creating a Cas9/gRNA complex.
On other end of the gRNA are twenty nucleotides called the spacer. The spacer region is what
directs the Cas9 enzyme to cut a specific DNA sequence.
To do this, Cas9 will unwind a small section of DNA and the single-stranded RNA bases in the spacer
will align with the bases in the DNA. If, according to the rules of base pairing, all 20 bases of the
gRNA spacer region are complementary to the bases of the DNA to which they are aligned, the Cas9
nuclease will cut the DNA. If the bases are not complementary, the Cas9 nuclease will not cut the
DNA and will move to a new region of the DNA.
