RPE1 cells were grown in Dulbecco’s Modified Eagle’s Medium F-12 (DMEM/F-12) with 10% FBS and 1% penicillin/streptomycin. All cell lines were cultured at 37°C in a humidified 5% CO2 incubator.
sgRNAs were transcribed in vitro from PCR-generated DNA templates according to a previously published method (DeWitt et al., 2016) with slight modifications. Briefly, template DNA was assembled by PCR from five different primers: 1) a variable forward primer containing T7 promoter and desired guide sequence, 2) a variable reverse primer containing the reverse complement of the guide sequence and the first 15 nt of the non-variable region of the sgRNA, 3) a forward primer containing the entire invariant region of the sgRNA, and 4), 5) two amplification primers. The assembled template was purified and subjected to in vitro transcription by T7 RNA polymerase using the Hiscribe T7 High Yield RNA Synthesis Kit (New England Biolabs). The reaction product was treated with DNAse I, and the synthesized sgRNA was purified using the RNA Clean & Concentrator Kit (ZYMO RESEARCH). All sgRNA and primer sequences used in this study are listed in Supplementary Table1 and 2, respectively.
CRISPR-del-mediated gene knockout
For large chromosomal deletions, the CRISPR-del method was performed with Cas9 protein and two synthesized sgRNAs. Cas9/sgRNA RNPs were electroporated into RPE1 cells using the Neon Transfection System (Thermo Fischer Scientific) according to the manufacturer’s protocol. Briefly, HiFi Cas9 protein (1.55 μM) from Integrated DNA Technologies (IDT) and two sgRNAs (0.92 μM each) were pre-incubated in Resuspension buffer R and mixed with cells (0.125 x105/μl) and Cas9 electroporation enhancer (1.8 μM, IDT). After resuspension, electroporation was immediately conducted using a 10 μl Neon tip at a voltage of 1300 V with two 20 ms pulses. The transfected cells were seeded into a 24-well plate. After recovery from the electroporation, single cells were isolated into 96-well plates using cellenONE (cellenion) according to the manufacturer’s protocol. After cell expansion, each 96-well plate was duplicated for genotyping and preparation of a frozen stock. Briefly, cells in 96-well plates were washed with PBS and treated with 25 μl of Trypsin/EDTA solution (nacalai tesque). After brief incubation at 37°C, the detached cells were resuspended with 75 μl of Cell Reservoir One (nacalai tesque), a DMSO-free cryopreservation medium. 25 μl of the cell mixture were transferred into a well of another 96-well plate filled with 175 μl of growth medium for cell expansion followed by genotyping analysis. The 96-well plate with the remaining cell suspension was placed in a deep freezer at −80°C.
For high-throughput genotyping, genomic DNA was directly extracted from single cell clones in 96-well plates using DNAzol Direct (Molecular Research Center) and subjected to PCR for the detection of both WT and the deleted alleles using appropriate primers. Briefly, after removal of culture medium, 20 μl of DNAzol Direct was added to each well and the 96-well plate was shaken at 800 rpm for 20 min at room temperature. 1μl of the lysate containing genomic DNA was used for 10 μl of PCR reaction using KOD One PCR Master Mix (TOYOBO). The PCR products were analyzed by the automated microchip electrophoresis system MCE-202 MultiNa (Shimadzu). To confirm the genotype of homozygous KO clones, their genomic DNA was purified using NucleoSpin DNA RapidLyse kit (Macherey-Nagel) and subjected again to genotyping PCR. The PCR products were analyzed by agarose gel electrophoresis.
CRISPR/Cpf1-mediated gene knock-in
Endogenous mNG tagging of HNRNPA1 by CRISPR-Cpf1 system was performed with the electroporation of Cpf1/crRNA RNP and dsDNA repair template. crRNA was designed to target the site immediately downstream of the stop codon of HNRNPA1 and transcribed in vitro as described above. The DNA template for crRNA synthesis was assembled by PCR using a forward primer containing T7 promoter and the target sequence, and a reverse primer containing the reverse complement of the target sequence and the non-variable region of crRNA. The dsDNA repair template was amplified by PCR from a plasmid encoding the mNG sequence using two primers containing 90 bp left and right homology arm sequence, respectively. Electroporation of Cpf1/crRNA and the repair template was conducted similarly to the Cas9/sgRNA condition described above, with a modification in the electroporation solution. A.s.Cpf1 Ultra (1 μM, IDT) and crRNA (1 μM) were pre-incubated in buffer R and mixed with cells (0.125 ×105 /μL), Cpf1 electroporation enhancer (1.8 μM, IDT) and the repair template (33 nM). mNG-positive cells were sorted using FACS Aria III (BD Biosciences), equipped with 355/405/488/561/633 nm lasers. After cell expansion, cell sorting was repeated to re-enrich mNG-positive cells. The knock-in cell pool was subjected to CRISPR-del experiments. The crRNA sequence is listed in Supplementary Table1.
CRISPR-del efficiency assessed by Flow cytometry
HNRNPA1-mNG knock-in cells were electroporated with Cas9 and the indicated sgRNA pairs for CRISPR-del application. After 10 days in culture, the cells were harvested with trypsin/EDTA solution, washed in cold PBS, and analyzed by FACS Aria III for mNG expression. Data for approximately 5,000 gated events were collected. Due to the presence of a very small portion of mNG-negative cells in control samples, the mean percentage (1.47 %) was then subtracted from that of each CRISPR-del sample before comparison.
Cells were lysed on ice in lysis buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1% Triton X-100, 5 mM EGTA, 1 mM DTT, 2 mM MgCl2, and 1:1,000 protease inhibitor cocktail Nakarai Tesque). After centrifugation, the supernatant was added to Laemmli sample buffer, boiled and subjected to SDS-PAGE. Separated proteins were transferred onto Immobilon-P membrane (Merck) using Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories). The membranes were probed with the primary antibodies, followed by incubation with their respective HRP-conjugated secondary antibodies (Promega). The membrane was soaked with Chemi-Lumi Super (Nakarai Tesque) for the signal detection using ChemiDoc XRS+ (Bio-Rad Laboratories).
For the immunofluorescence analyses, cells cultured on coverslips (Matsunami) were fixed with −20 °C methanol for 7 min, or 4 % PFA at room temperature for 30min. Fixed cells were incubated with blocking buffer (1 % bovine serum albumin in PBS containing 0.05 % Triton X-100) for 30 min at room temperature. The cells were then incubated with primary antibodies in the blocking buffer for 1 hour in a humid chamber. After washing with PBS, the cells were incubated with secondary antibodies and Hoechst 33258 (DOJINDO, 1:3000–1:5000) in the blocking buffer for 30 min, followed by final wash with PBS. The coverslips were mounted onto glass slides (Matsunami) using ProLong Gold (Molecular Probes), with the cell side down.
The following primary antibodies were used in this study: anti-CEP128 (abcam, ab118797; IF 1:500, WB 1:1000), anti-γ-tubulin (Merck, GTU88; IF 1:1,000), anti-p15 (Santa Cruz Biotechnology, sc-271791; WB 1:1000), anti-HSP90 (BD Biosciences, 610419; WB 1:1000) and anti-β-actin (Santa Cruz Biotechnology, sc-47778; WB 1:1000). The following secondary antibodies were used: anti-mouse IgG Alexa Fluor 488 (Molecular Probes, 1:1000), anti-rabbit IgG Alexa Fluor 555 (Molecular Probes, 1:1000), anti-mouse IgG HRP (Promega, WB 1:10000) and anti-rabbit IgG HRP (Promega, WB 1:10000).
Statistical comparison between the data from different groups was performed in PRISM v.8 software (GraphPad) using either a Mann–Whitney U test or a Tukey–Kramer test as indicated in the legend. P values <0.05 were considered statistically significant. All data shown are mean ± S.D. The sample size is indicated in the figure legends.
The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion/deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked-out by the indel-based gene disruption. In this study, we optimized CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform “complete” gene knockout in non-transformed human diploid RPE1 cells. By improving several procedures, the optimized CRISPR-del allowed us to generate knockout cell lines harboring bi-allelic large chromosomal deletions in a high-throughput manner. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the ability of this method to introduce a large chromosomal deletion enables the generation of a model cell line having a bi-allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the optimized CRISPR-del is a high-throughput method for performing “complete” gene knockout in RPE1 cells.