What is the design of the vector backbones used in your library?
What is the design of the vector backbones used in your library?
Both our sgRNA library and the Cas9 sequence are cloned into a lentiviral vector backbone consisting of a 3' self-inactivating LTR and sequence elements (e.g., cPPT/CTS and WPRE) to enhance titer and transduction efficiency.
Cas9 (from S.pyogenes) is expressed from the human EF1-alpha promoter, and stable integrants can be selected using puromycin.
The sgRNA library is expressed from a human U6 promoter, and hygromycin B can be used to select for stable integration of the lentivirus. The red fluorescent protein mCherry is also expressed, which significantly simplifies the process to determine which MOI gives a transduction efficiency of 30–40% in your target cells. It also allows visualization of a fully selected population (i.e., one in which 100% of the cells are red).