The Lenti-X Tet-On 3G CRISPR/Cas9 System is a complete system for lentiviral-mediated CRISPR/Cas9 genome editing under the control of the Tet-On 3G expression system. The Tet-On 3G system enables doxycycline (dox)-inducible Cas9 expression with an extremely low background. Even residual levels of Cas9 expression from a leaky tet system can cause unwanted gene editing in the absence of doxycycline, which makes our tightly controlled Tet-On 3G system advantageous for developing an inducible Cas9 gene editing system. Here we present detailed data demonstrating a tight control of Cas9 expression and doxycycline-induced gene knockouts in HEK293 and Jurkat cells.
The advantage of using the Lenti-X Tet-On CRISPR-Cas9 System is its ability to generate parental cell lines with inducible Cas9 expression that can be used for editing many different gene targets or for sgRNA library screening techniques. Because the Cas9 is only expressed when needed for editing, it is possible to avoid the toxicity associated with persistent expression of Cas9. This can be especially important in more sensitive cell lines such as human induced pluripotent stem cells (hiPSC).
There are several steps involved in using a Tet-On 3G system with dox-inducible Cas9 for gene editing experiments. The schematic below represents a standard approach to using this system for gene editing. It is important to note that we have seen successful gene editing even when Cas9 expression was too low to be detected on a Western blot. For this reason, we suggest transduction with the pLVX-EF1a-Tet-3G construct and screening clones with the provided luciferase reporter to identify clones with a low background and a high inducible expression. This is then followed by a similar screen using qRT-PCR after transduction with the TRE3G-Cas9 vector.
Figure 1. Protocol overview for the Lenti-X Tet-On 3G CRISPR/Cas9 System. We suggest screening for a low background and high inducibility with a luciferase reporter after the first transduction, followed by qRT-PCR for a similar screen after transduction with the TRE3G-Cas9 vector.
Results
Screening for highly inducible clones
As previously mentioned, it is important to choose the right clones to ensure low background and highly inducible expression of Cas9. Two clones are shown below with their associated Western blot and qRT-PCR data for inducible Cas9 expression in the presence and absence of dox. Editing performance was assayed using our Guide-it Mutation Detection Kit followed by functional confirmation using FACS with a fluorescent anti-CD81 antibody.
Figure 2. Choosing an optimal clone for inducible genome editing in HEK293 cells. Clones may be selected by assaying Cas9 expression or genome editing in both uninduced and induced cell populations. However, while Western blot detection of Cas9 is helpful for prescreening clones, CRISPR/Cas9-mediated editing is so efficient that even when Cas9 protein is undetectable by Western blot, genome editing can still occur in the corresponding clones. We recommend qRT-PCR for identifying clones in which the residual expression of Cas9 is low enough for editing to be avoided in the absence of doxycycline. Data in the upper row is from a desirable clone (C-1), which exhibits robust expression of Cas9 protein in induced cells (Western blot, +), and the lowest residual levels of Cas9 mRNA (qRT-PCR). Genome editing occurs at much higher frequencies upon induction of Cas9 expression in cells derived from this clone (+Dox) relative to uninduced cells (–Dox), as evidenced by the increase in smaller-sized bands in the Resolvase assay (indicated by blue arrows) and the greater proportion of cells in the "knockout" category as determined by FACS, with the lowest residual editing in un-induced cells. In contrast, data in the lower row is from an undesirable clone (C-3), which exhibits residual transcription of Cas9 (qRT-PCR) and detectable frequencies of genome editing in the absence of doxycycline, even though Cas9 protein expression in uninduced cells (–) is undetectable by Western blot. This demonstrates the importance of screening clones using qRT-PCR for low background expression of Cas9.
Inducible genome editing
After selecting clones with a high inducible expression and a low background expression of Cas9, we demonstrated inducible genome editing by transducing cells with a lentivirus encoding for an anti-CD81 sgRNA constitutively expressed from the U6 promoter. These cells expressed sgRNA but did not express Cas9 in the absence of doxycycline, which enabled tight control over genome editing, as seen in the figure below.
Figure 3. Inducible knockout of CD81 in Jurkat cells. Tet-On 3G-Cas9-positive Jurkat cells with low residual Cas9 expression were generated using the established protocol and transduced twice with CD81-sgRNA lentivirus at an MOI of 5 on successive days. Cells were then divided between two wells of a 12-well plate and cultured in the absence (–Dox) or presence (+Dox) of doxycycline at a concentration of 0.5 µg/ml for seven days. Cells were then treated with FITC anti-human CD81 antibody and analyzed by FACS. Only a small proportion of cells (0.4%) cultured in the absence of doxycycline (–Dox; top) exhibit residual editing. In contrast, a significant proportion of cells (32.0%) cultured in the presence of doxycycline (+Dox; bottom) exhibit knockout of CD81.
Conclusions
The Lenti-X Tet-On 3G CRISPR Cas9 System gives you control over Cas9 expression and lets you generate Cas9-expressing cell lines with the confidence that toxic effects or drifts in cell phenotype will be limited over time. Our Tet-On 3G system provides the extremely low background expression necessary to avoid unwanted gene editing.
Methods
The methods used here can be found in the Lenti-X Tet-On 3G CRISPR/Cas9 System User Manual. For the clone selection, cells were transduced with pLVX-EF1a-Tet-3G, selected with G418, and screened using a luciferase reporter. After selecting clones with a low background and high inducible expression, clones with acceptable levels of expression were further transduced with pLVX-TRE3G-Cas9-puro, selected using puromycin, and then screened using qPCR. Clones with sufficiently low background and inducible Cas9 expression were then transduced with pLVX-hyg-sgRNA1 with a sequence targeting CD81 and selected for sgRNA expression using hygromycin. These final cell populations were tested for gene editing using either our Guide-it Mutation Detection Kit or FACS combined with a fluorescent anti-CD81 antibody. Similar methods were used to generate Jurkat clones with inducible Cas9 expression.
