Question

HBD HBB GFP br or + bnl On GFP end of class! • Assignment: Researchers created a CRISPR/Cas9 construct targeting HBB, which encodes a subunit of hemoglobin. To test for off-target effects, they looked at the closely-related HBD gene. They added increasing amounts of the construct and ran a PCR targeting HBB and HBD. The resulting gel is shown on the right. GFP was used as a control. 1. (1pt) Is this gel showing DNA or RNA? 2. (1pt) Interpret the experimental results. 3. (1pt) If the researchers forgot to include Cas9, how would the results in the HBB lanes change?

Answer 1

Answer 1:

Here we are studying the ability of CRISPR/Cas9 nucleases to cause off-target cleavage. That's why these are DNA sequences being run on PCR.  

Answer 2:

The results show that there were mismatch mutations in HBB gene and cleavage occurred in HBB genes that is why different bands are formed in each lane, i.e. 1 $\mu$ g, 2 $\mu$ g, 3 $\mu$ g and 4 $\mu$ g. Whereas, there is no cleavage seen in HBD gene, leading to one band formation in each lane.

Answer 3:

If Cas9 was not added, then single bands would have formed for HBB gene in all four lanes. The results for HBB and HBD would look similar.