miniPCR bio
TM
Chopped! Using CRISPR/Cas9 to cut DNA - Student's Guide
Version: 1.0 - Release: May 2022 - © 2022 by miniPCR bio™
Student's Guide
P./17
Pre-lab activity
Predicting where the CRISPR/Cas9 system will cut
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Guide RNAs (gRNAs) can be designed to direct the Cas9 nuclease to specific DNA regions by
following the rules of base pairing. Today, you will test Cas9 alone or in combination with two
different gRNAs that are complementary to different target DNA sequences within the same DNA
molecule (see Figure 2 below).
Before you perform the lab, you will use the given gRNA and DNA sample sequences to identify
where Cas9 will cut the DNA sample and predict the sizes of the resulting DNA fragments. After the
DNA has been cut, you will experimentally check that your lab results match your predictions.
gRNAs:
Page 42 contains schematics of the two gRNAs that you will use in this lab.
• The spacer sequence of the gRNA is the 20-base region that can be designed to be
complementary to the target DNA sequence. The spacer sequences are shown as the
horizontal portion of the gRNAs with the bases labeled.
• The scaffold region of the gRNA is the 80-base region that binds to itself and forms a loop
that attaches to the Cas9 nuclease to form the Cas9/gRNA complex. The scaffold regions are
shown as the vertical loop of the gRNAs. Because the scaffold region plays a structural role
and is identical in both gRNAs, the individual bases have not been labeled.
• Remember that the gRNAs, like all RNA, use the base uracil (U) instead of thymine (T).
Target DNA:
The sequence of the DNA sample you will be cutting with CRISPR/Cas9 starts on page 40.
• The DNA used in this lab is a linear double-stranded fragment that is 3,142 base pairs long.
• Note that both strands of the DNA are shown in this activity.
• For simplicity, you have only been provided the middle 1,000 base pairs of the 3,142 base pair
DNA sequence (base pairs 1,101 to 2,100). For this reason, the numbering of the bases starts at
1,101.
Figure 2. Different gRNAs will target different DNA sequences in the same DNA sample.
