Cellecta’s Tetracycline-Inducible CRISPR Cas9 Lentiviral Vector enables researchers to make cells capable of high Cas9 expression for a limited time during which CRISPR-mediated targeted rearrangements can occur, and then shut off Cas9 expression for downstream assays with the modified cells.
Over the past seven years, the CRISPR Cas9 system has become a critical tool for cell engineering, gene-function cell screens, and gene target validation. CRISPR-based targeted gene knockout occurs relatively quickly with Cas9 and the appropriate sgRNA. However, for studies requiring editing in the majority of cells in a population, for example CRISPR sgRNA screens, several days may be needed to maximize DNA repair failure.
As a result, for some applications, it is important to maintain significant levels of Cas9 in cells over longer periods to maximize the occurrence of the knockout or genomic editing in a sufficient number of cells. For this purpose, lentiviral-based constructs expressing sgRNA and Cas9 have proven to be a highly effective means to introduce and carry out CRISPR-mediated genome editing in virtually any mammalian cell. Once the genomic editing event occurs, however, the changes to the DNA are permanent and the CRISPR elements mediating the knockout are no longer necessary.
While most cells tolerate Cas9 expression reasonably well, high-level expression of the protein will often slow down cell doubling rates. Also, the rate of CRISPR knockout has been shown to be dependent Cas9 expression. The higher the Cas9 expression, the faster knockout occurs.
However, high expression of the Cas9 nuclease can strain cell biology and elevate levels of DNA damage.
Given this situation, it is desirable to select cells expressing high levels of the Cas9 nuclease for genomic editing purposes, but then remove Cas9 for use of the engineered cells in downstream experiments. The tet-inducible Cas9 vector provides researchers with the flexibility to address both of these situations. Simply removing doxycycline from the medium turns off expression of the Cas9 gene after editing.
Left: Dox treatment time course GFP knockout in HEK293 cells with three different GFP-targeting sgRNA after induction of Cas9. Within 3 days of induction, maximal levels of knockout occurred.
Right: Tet-inducible Cas9, pool and clones Cells were transduced with GFP-targeting sgRNA and the tet-responsive Cas9 construct, then incubated 7 days with 1ug/ml dox. After this induction, individual cells were isolated and expanded to make clonal cultures. Results show that ~50% of the cell pool show tet-responsive knockout of the GFP gene. However, clones with significantly better responsiveness can be easily isolated.