Cell lines and cell culture conditions
hTert-immortalized retinal pigment epithelium (RPE-1) and derived cell lines were maintained in DMEM/F-12 + Glutamax (Gibco, Life Technology) supplemented with 1% penicillin/streptomycin and 6% fetal bovine serum (FBS, S-FBS-EU-015, Serana).
Taxol and Tariquidar treatment
Taxol and Tariquidar were dissolved in DMSO and prepared at stock concentrations before usage at varying final concentrations as indicated in each figure.
sgRNA designed and cloning
the gRNAs targeting ABCB1 were cloned into a lenti-guidePuro (Addgene plasmid # 52963) using the BsmBI restriction site. sgRNA sequences are summarized in Table 1.
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gRNA sequences targeting ABCB1
Colony Outgrowth Assays
1 million cells were treated with 8nM of taxol and allowed to grow out for 15 days. Plates were fixed in 80% Methanol and stained with 0.2% Crystal Violet solution. After fixation, the number of taxol resistant cells were counted.
For viability assays, 1000 cells were plated in a 96-well plate and treated for 7 days with indicated drug concentrations. Subsequently, plates were fixed in 80% Methanol and stained with 0.2% Crystal Violet solution.
RNA isolation and qRT-PCR analysis
RNA isolation was performed by using Qiagen RNeasy kit and quantified using NanoDrop (Thermo Fisher Scientific). cDNA was synthesized using Bioscript reverse transcriptase (Bioline), Random Primers (Thermo Fisher), and 1000 ng of total RNA according to the manufacturer’s protocol. Primers were designed with a melting temperature close to 60 degrees to generate 90–120-bp amplicons, mostly spanning introns. cDNA was amplified for 40 cycles on a cycler (model CFX96; Bio-Rad Laboratories) using SYBR Green PCR Master Mix (Applied Biosystems). Target cDNA levels were analyzed by the comparative cycle (Ct) method and values were normalized against GAPDH expression levels. qRT-PCR oligo sequences are summarized in Table 2.
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For western blot experiments, equal amounts of cells were lysed with Laemmli buffer and separated by SDS–polyacrylamide gel electrophoresis followed by transfer to a nitrocellulose membrane. Membranes were blocked in 5% milk in PBST for 1h at RT before overnight incubation with primary antibody in PBST with 5% BSA at 4°C. Membranes were washed three times with PBST followed by incubation with secondary antibody in PBST with 5% milk for 2h at RT. Antibodies were visualized using enhanced chemiluminescence (ECL) (GE Healthcare). The following antibodies were used for western blot experiments: α-Tubulin (Sigma t5168), MDR(PgP) (sc-8313). For secondary antibodies, peroxidase-conjugated goat anti-rabbit (P448 DAKO, 1:2000), goat anti-mouse (P447 DAKO, 1:2000) and rabbit anti-goat (P449) were used.
RPE-1 cells were plated on glass coverslips and washed twice with BS before fixation in 4% PFA in PBS for 10 minutes at room temperature. After two additional washes in 1x PBS coverslips were incubated in 70% ethanol at 4°C overnight. Coverslips were incubated for pre-hybridization in wash buffer (2x saline-sodium citrate (SSC) with deionized formamide (Sigma) 10%) for 2-5 minutes at room temperature. RNA FISH probe mix wash dissolved in hybridization buffer (wash buffer supplemented with 10% dextran sulfate). 38 probes labelled with Cy5 were targeted to the intronic regions of ABCB1 (Biosearch technologies). Coverslips were incubated in hybridization solution for at least 4h at 37°C. Then coverslips were washed twice for 30 minutes with wash buffer followed by a quick rinse with 2x SSC. Finally, coverslips were washed once for 5 minutes in 1x PBS before mounting on slides using Prolong gold DAPI mounting medium (Life Technologies). Images were acquired with the use of a DeltaVision Elite (Applied Precision) equipped with a 60x 1.45 numerical aperture (NA) lens (Olympus) and cooled CoolSnap CCD camera. ABCB1 transcription start site quantification was performed manually double blind.
TLA was performed as previously described with minor modifications. TLA libraries were sequenced on a MiSeq and were mapped to genome using bwa bwaswHeng Li ref: PMID: 20080505 to enable partial mapping of sequence reads. Reads were mapped to hg19 reference of the human genome.
The discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) and its development as a genome editing tool has revolutionized the field of molecular biology. In the DNA damage field, CRISPR has brought an alternative to induce endogenous double-strand breaks (DSB) at desired genomic locations and study the DNA damage response and its consequences. Many systems for sgRNA delivery have been reported in order to efficiency generate this DSB, including lentiviral vectors. However, some of the consequences of these systems are yet not well understood. Here we report that lentiviral-based sgRNA vectors can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. By generating a DSB in the regulatory region of the ABCB1 gene using a lentiviral sgRNA vector, we can induce the formation of taxol-resistant colonies. We show that these colonies upregulated ABCB1 via integration of the EEF1A1 and the U6 promoters from the sgRNA vector. We believe that this is an unreported CRISPR/Cas9 artefact that researchers need to be aware of when using lentiviral vectors for genome editing.