8.) Genome Modification, Northern and Westem Blots To further investigate the fu
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8.) Genome Modification, Northern and Westem Blots To further investigate the function of CREB you decide to create a CREB-KO cell line. To this end you design a Cas9 system to create a doublestrand break in the middle of CREB exon 2 (indicate by lightning) that you expect will be fixed imperfectly by the cellular Non-Homologous End-Joining (NHEJ) DNA repair machinery. To check for the success of Cas9 treatment you collect RNA and Protein from your cell line before and after Cas9 treatment and run Northern and Western Blots using probes and antibodies binding CREB mRNA and protein as indicated below Cas9 Probe 1 Probe 2 GADNOOsGDAAVTE Antibody Antibody 2 A.) Northern Blots: What does the banding pattern produced by the two probes tell you about the properties of CREB mRNA before and after Cas9 treatment? (Word limit: 40) (6 points) Probe Before Ater Betore Afer Probe 2 10 B.) Western Blots: What does the banding pattem produced by the two antibodies tell you about the properties of CREB protein before and after Cas9 treatment (Word limit: 60 (12 points) C.) By combining the condusions drawn from Northern and Westem Blats, explain what kind of mutation the Cas9 mediated doublestrand break most likely created in CREB exon 2 and what the consequences of this mutation are . (Word limit: 40) (6 points)Explanation / Answer
A) Northern blot indicates that after Cas9 treatment the break site in CREB DNA has fixed by NHEJ and produced intact mRNA similar to before Cas9 treatment (control cells). So both the probes amplified complete mRNA
B) The banding pattern produced by antibody 1 indicates that NHEJ has not happened after Cas9 treatment and mRNA and protein produced is shorter than intact mRNA (before cas9 treatment). The antibody 1 detected protein fragment which is synthesized from 5' end of mRNA. The antibody 2 detected no protein because there is no mRNA from other part of CREB DNA.
C) As we can observe from northern blot. The mRNA produced after cas9 treatment is bigger than the mRNA before cas9 treament. This indicates that defective NHEJ repair has inserted extra sequence during repair in to CREB DNA. This results in longer mRNA product which will result in frame shift mutation and codes for a protein different aminoacid residues and inactive protein
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