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./18
Instructions
-
1. Cut out the gRNA models along the dotted lines,
so you have two pieces of paper, one for each
gRNA sequence
2. Compare the gRNA1 sequence on your cutouts
to the DNA sequence to locate the target DNA
sequence
• Starting at the beginning of the DNA
sequence, place the gRNA1 cutout over the
DNA in the space between the two strands
of DNA.
• The gray downward arrows on the gRNA1
cutout should be pointing from the gRNA1
bases to the bases on the bottom strand of
DNA.
• Move the gRNA1 cutout along the DNA
sequence, row by row, until you have located the complementary target DNA sequence.
• You will have located the complementary target DNA sequence when all 20 base pairs of
the spacer region are complementary to the bottom strand of the DNA. (e.g., a C in the
gRNA1 cutout should be pointing at a G on the bottom strand of DNA.)
Note: In the cell, this step would require a complexed Cas9 protein and gRNA. For
simplicity, in this activity, you are just using the gRNA. Also, the target sequence could
technically be on either strand, but in this activity, the target sequence is located on the
bottom strand of DNA.
3. Once you have located the target DNA sequence that is complementary to gRNA1, identify
where Cas9 will cut
• The Cas9 enzyme will typically cut the DNA
between the third and fourth base pairs from
the 3' end of the gRNA spacer region.
• There is a black arrowhead on the gRNA1
cutout that is three bases from the end of
the spacer region—this indicates where the Cas9 nuclease will cut.
• Make a mark on the DNA where the black arrowhead is pointing. The mark will be
between two bases on the top strand of DNA. Also make a mark on the bottom strand of
DNA in line with the mark on the top strand.
