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./19
• Underline the entire complementary sequence and label it "gRNA1 target DNA
sequence."
• Remove the gRNA1 cutout.
4. Repeat steps 2 and 3 for gRNA2
Now that you have predicted where the gRNAs will cut, you can predict the sizes of the resulting
DNA fragments. Remember that the sequence you are using spans nucleotides 1,101 to 2,100 of
the complete DNA molecule. Use the numbering on the DNA strands to predict the size of the
DNA fragments produced by Cas9.
5. For each gRNA, determine at which
numbered base pair Cas9 will cut
• Circle the base pair to the left
of where Cas9 will cut the
DNA.
• Use the labeled numbers at the beginning of each row to determine the position of the
circled base pair (i.e., count from the number at the beginning of the row to your circled
base pair).
• Note the position of that circled base pair for each of the gRNAs below:
gRNA1: ________
gRNA2: ________
6. Calculate the size of the resulting DNA fragments
Remember, the total size of the uncut DNA is 3,142 base pairs:
gRNA1: Cas9 cuts at base pair (from 5, above) ______
The entire length of the DNA is 3,142 bp. Based on where you predict the Cas9 nuclease to cut the
DNA, predict the length of the two DNA fragments that will be produced.
Fragment 1: ___________ bp (same number as question 5 above)
Fragment 2: __________ bp (subtract Fragment 1 from 3,142 bp)
