Materials and methods

Easy multiple sequential CRISPR/Cas9 knockouts in cell lines using a Cre/LoxP re-cyclable vector

Cell culture and reagents

HEK 293T cells were cultured in DMEM (Thermo Fisher Scientific, Cat# 11885-084) supplemented with 8% fetal bovine serum (FBS, Sigma Cat# F9423). WEHI7 cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with 8% fetal bovine serum. Cells were cultured at 37 °C and 10% CO2. All cell lines used were routinely tested for mycoplasma contamination.

Doxycycline were purchased from Sigma-Aldrich.

Plasmid construction and inducible expression

LentiCRISPRv2-mCherry was a gift from Agata Smogorzewska (Addgene plasmid # 99154;; RRID: Addgene99154). In order to insert the two loxP sites, we designed two separate fragments, F1 and F2. F1 contained a loxP site and NotI and KpnI restriction sites. F2 contained a loxP site and BsrGI and SacII restriction sites. The F1 and F2 fragments were synthesised by Integrated DNA Technology (IDT). The fragments were amplified and sub-cloned into a TA vector. The F1 and F2 fragments were digested out of TA vectors using their respective restriction enzymes and then ligated to LentiCRISPRv2-mCherry in two steps. The target sequences of sgRNAs for _Bcl2 and Bak1 were described previously 15. Complementary oligonucleotides were annealed and cloned into the BsmBI sites of the LentiCRISPRv2-loxP-sgRNA-Cas9 plasmid.

For inducible Cre expression, the doxycycline inducible lentiviral vector pFTRE 3G rtTA EGFP/Cre, kindly provided by Prof. John Silke (WEHI), was used to infect cells, and clonal lines were obtained by sorting single EGFP positive cells.

Preparation of lentivirus

HEK293FT cells were transfected with a lentiviral vector with pVSV-G and pCMV8.2 using Effectene (QIAGEN, Hilden, Germany). Forty-eight hours after transfection, supernatant containing lentivirus was collected for spin-infection as described previously18.

CRISPR/Cas9 gene mutation

For CRISPR/Cas9 gene deletion, parental cells were infected with lentiviral constructs encoding Cas9 and mCherry, and a single guide RNA targeting the desired gene. Successful transfected cells were isolated by sorting mCherry positive cells on a flow cytometer (FACS, Becton Dickinson). Independent single cell clones lacking the targeted protein were confirmed by immunoblotting.

Flow Cytometry and cell Sorting

For flow cytometric analysis of mCherry, cells were harvested and then resuspended in phosphate-buffered saline (PBS) before being analyzed by a LSRFortessa X-20 flow cytometer (BD Biosciences San Jose, CA). The threshold for detection was set at 10,000 counting events. Data were analysed using FlowJo software (BD Biosciences, San Jose, CA). Wild-type (WT) WEHI7 cells were used to design gate for analysis with FACS.

For FACS sorting, cell suspensions were resuspended in PBS containing 2% FBS and then separated on a FACSAria Fusion flow cytometer (BD Biosciences, San Jose, CA). For single cell sorting, one cell was seeded per well into flat-bottomed 96 well plates (BD Biosciences) containing 100 μL RPMI 1640 medium supplemented with 8% FCS. At day 14, the clones were moved to 24 well plates (BD Biosciences) for further expansion. Confluent clones were split and replicated plates were harvested for western blot analysis.

Western blot

Total cell lysate was prepared in RIPA buffer (50 mM Tris-HCl, 0.1% SDS, 1% Nonidet P-40, 0.5% deoxycholate and 150 mM NaCl, pH 8.0) complemented with protease inhibitors. Proteins at the same amount were separated by Tris-glycine gels (BioRad) and transferred onto polyvinylidene difluoride membranes. Membranes were then incubated with blocking buffer for 1 h at room temperature, followed by indicated antibodies for 16 h at 4 °C. After probing with primary antibody, membranes were probed with HRP conjugated anti-IgG secondary antibodies and ECL (GE Healthcare Life Sciences). The antibodies used were: ACTIN (AC-15, Sigma #A1978), BAK1 (aa23-38, #B5897 Sigma), BCL2 (#610539, BD Biosciences).

Article TitleEasy 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.

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