Takara Lenti-X Tet-On 3G CRISPR/Cas9 System \1 System

The Lenti-X Tet-On 3G CRISPR/Cas9 System is designed for Tet-inducible Cas9 expression along with expression of a custom sgRNA. Because Cas9 is only expressed in the presence of doxycycline, genome editing is achieved on demand and without the side effects of persistent Cas9 expression in cell culture. This kit contains all of the components necessary for generating three types of lentivirus: one for constitutive expression of the Tet-On 3G transactivator protein, a second for Tet-inducible Cas9 expression under the PTRE3GV promoter, and a third for constitutive sgRNA expression under the U6 promoter

Inducible knockout ofCD81in HEK293 cells.Target cells were first transduced with Tet-On 3G lentivirus and selected with G418.24 independent Tet-On 3G-positive clones were assayed for induction of the luciferase reporter, and one clone exhibiting the lowest residual expression was selected and transduced with Cas9 lentivirus at an MOI of 5. Target cells were selected with 1 µg/mL puromycin for two weeks, and six stable clones were randomly selected for analysis of Cas9 induction. Cells from each clone were divided between two wells of a 12-well plate and incubated either in the presence or absence of doxycycline (0.5 µg/mL) for two days. Cas9 induction was then assayed by Western blot using polyclonal anti-Cas9 antibody (Cat. No. 632607), diluted 1:1,000, and ECL reagent. Cells derived from three of the Tet-On 3G-Cas9-positive clones (C-1, C-2, and C-3) were then divided between two wells of a 12-well plate and transduced with CD81-sgRNA lentivirus at an MOI of 5. Eight hours after transduction, doxycycline was added to one well for each clone at a concentration of 0.5 µg/mL, and cells were cultured for six days. Cells were then harvested and stained with a FITC-tagged anti-human CD81 antibody and scanned for CD81 expression by FACS.Panel A.Western blot results for six independent Tet-On 3G-Cas9-positive cell populations cultured in the absence (–) or presence (+) of 0.5 µg/mL doxycycline, assayed with anti-Cas9 antibody. Cas9 expression is either undetectable or highly elevated in the absence or presence of doxycycline, respectively.Panel B.FACS results for three independent Tet-On 3G-Cas9-positive 293T cell populations transduced with CD81-sgRNA lentivirus and cultured in the absence (–Dox) or presence (+Dox) of 0.5 µg/mL doxycycline. Two of the populations (C-1 and C-2) exhibited no residual editing in the absence of doxycycline, and knockout ofCD81in either 58.6% or 30.7% of cells, respectively, while the third population (C-3) exhibited residual editing (27.9% of cells) in the absence of doxycycline.

Inducible editing ofAAVS1in HEK293 cells.Induction of Cas9 expression in six stable Tet-On 3G Cas9-positive HEK293 cell lines was assayed by qRT-PCR. Cells from each line were then divided between two wells of a 12-well plate and transduced with AAVS1-sgRNA lentivirus at an MOI of 5. Eight hours after infection, doxycycline was added to one well for each cell line at a concentration of 0.5 µg/mL, and the cells were cultured for six days. The Guide-it Mutation Detection Kit (Cat. # 631443) was then used to screen for editing ofAAVS1.Panel A.qRT-PCR results for Cas9 expression in uninduced (–Dox) and induced (+Dox) cells for each clone. Ct and ΔCt values for uninduced and induced clones are indicated in the corresponding columns, and the calculated fold-difference in expression for each clone is indicated in the column on the right. Of the various clones tested, Clone 1 (numbers in green) exhibited the highest-fold inducibility, while only Clone 3 (numbers in red) exhibited residual expression of Cas9 in the absence of doxycycline and low inducibility.Panel B.Results of Guide-it Resolvase assay for detection ofAAVS1gene editing in uninduced (–) and induced (+) clones. In contrast with the assay of the parental line, which yielded bands of equivalent size for uninduced and induced cells, assays of the induced clones yielded additional bands of smaller sizes, indicating the occurrence of gene editing atAAVS1for all clones tested. Cleavage products are also visible for the uninduced cells from Clone 3, consistent with the residual expression of Cas9 observed in the qRT-PCR assay.

Vector components for Lenti-X Tet-On 3G CRISPR/Cas9 System.pLVX-EF1a-Tet3Gencodes the Tet-On 3G transactivator protein under the control of the constitutive EF-1 alpha promoter. In contrast with the commonly used CMV promoter, EF-1 alpha is less susceptible to silencing by certain cell types (including various types of stem cells), enabling the establishment of inducible cell lines that are suitable for long-term use. Tet-On 3G is a modified form of the Tet-On Advanced transactivator protein, which was evolved to have a far higher sensitivity to doxycycline.pLVX-TRE3G-Cas9-puroencodes a modified version of the Cas9 endonuclease under the control of the induciblePTRE3GVpromoter. The Cas9 sequence in pLVX-TRE3G-Cas9-puro was originally derived from the bacteriumS. pyogenesand has been codon optimized for expression in mammalian cells. Additional Cas9 modifications include the incorporation of a C-terminal nuclear localization signal (NLS).pLVX-hyg-sgRNA1encodes an sgRNA (designed and inserted by the user) under the constitutive human U6 promoter.

Choosing an optimal clone for inducible genome editing.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 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 (+ or +Dox) relative to uninduced cells (– or –Dox), as evidenced by the increase in smaller-sized bands in the resolvase assay (blue arrows) and the greater proportion of cells in the “knockout” category as determined by FACS, with the lowest residual editing in uninduced 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 (Resolvase, FACS) in the absence of doxycycline, even though Cas9 protein expression in uninduced cells (–) is undetectable by Western blot