miniPCR bio
TM
Chopped! Using CRISPR/Cas9 to cut DNA - Instructor's and Student's Guide
Version: 1.0 - Release: May 2022 - © 2022 by miniPCR bio™
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Student's Guide
• Tube D should show that the DNA sample was cut by Cas9.
° Tube D contains Cas9 nuclease and gRNA2. This forms a Cas9/gRNA complex that cuts at
the sequence targeted by gRNA2.
° gRNA2 targets the DNA sample at around base pair 1,977, resulting in a band of 1,165 bp and
a band of 1,977 bp when cut by Cas9.
° You may also continue to see a 3,142 bp band. This represents residual uncut DNA. See
below for more discussion of partial cutting.
Partial cutting
• You may observe a top band at 3,142 bp in Tubes C and D that represents uncut DNA.
• This happens if the CRISPR/Cas9 reaction has not completely cut all of the DNA molecules in
the tubes.
• The brightness of this band roughly indicates how much of the DNA sample was cut—a faint
uncut band means more of the DNA was cut into the two smaller bands, while a brighter uncut
3,142 band means less of the DNA was cut.
• The presence of some uncut DNA is expected, since there are billions of copies of the DNA
sample in each tube.
• You may see variability between groups in how much uncut DNA is present, which may occur
due to variability in micropipetting, sample handling, and experimental timing and conditions.
• Having some uncut DNA is more likely in Tube D than in Tube C. Our testing indicates that
gRNA1 is a more efficient guide RNA than gRNA2. It is not completely known why certain
gRNA sequences are more effective at directing Cas9 to cut DNA than others, but this
experimental observation demonstrates why screening and testing multiple gRNAs for a given
target molecule is important when using CRISPR/Cas9.
