Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)
Type
Blog Post
...the cells will still appear green despite having active Cas9 (see tip 2).
Pro-Tips
1. To quantify Cas9 activity...I got clear bands using the conditions above.
2. Design your primers so that the digestion site isn’t ...transductions and use a range of reasonable MOIs.
2. Create a monoclonal Cas9-expression cell line. Especially...doing a screen. I made a few monoclonal lines (Figure 2) and I was surprised at how variable Cas9-expression...across the different cells (Figure 3).
Figure 2: Cas9 expression in monoclonal cell lines generated... expression
Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn...Western blotting against GFP expression, but I didn’t.
2. In the original publication they see GFP expression...