Hello again. My apologies for the delay in posting – I’ve been busy with other projects lately. As a follow up to my previous post, we wanted to further confirm our EZH1 antibody and test it in a knockout line. To do this, we generated a CRISPR knockout of EZH1 in an AML cell line that will be a great tool for future experiments. To generate the knockout line, we were helped by Fraser Soares, a postdoctoral fellow in Hansen He’s lab. He provided us with an AML cell line (OCI-AML-3) that expresses Cas9, along with guide RNAs that would allow us to knockout EZH1. Once the knockouts were generated, we tested the expression of EZH1 and EZH2. We determined that the guides knocked out EZH1, but had no effect on related EZH2 expression levels (Figure 1). We can now conclude we have a specific EZH1 antibody and EZH1 knockout lines that we can use in the future. For full experimental details, please see Zenodo post.
Figure 1: Westerns of Crispr knockouts in OCI-AML-3 Cas9 cells with either control guide RNA (sgLacZ) or guide RNA to EZH1. Levels of EZH1 were undetectable with knockout (A) and did not affect EZH2 levels (B).