Easy multiple sequential CRISPR/Cas9 knockouts in cell lines using a Cre/LoxP re-cyclable vector
To easily generate cell lines lacking multiple proteins, we inserted Cre/Lox sites flanking the guide RNA, Cas9, and mCherry fluorescent protein coding regions of a CRISPR/Cas9 lentiviral vector. Cells bearing an inducible Cre recombinase construct can be transfected with the CRISPR/Cas9 lentiviral vector, and mCherry positive cells sorted by flow cytometry. Induction of Cre causes deletion of the guide RNA, Cas9, and mCherry genes, so that mCherry negative cells can be isolated. After confirming successful targeting of the gene, the cells can be re-infected with the same vector bearing a different guide RNA, and mCherry-positive cells sorted once more. In this way, multiple genes can be mutated sequentially using the same vector and selection marker, without persistent expression of the guide RNA or Cas9. We used this system to sequentially mutate two candidate genes, Bak1 and Bcl2, and generated lines that lacked expression of both proteins.