Materials and Methods

Improving transgene expression and CRISPR‐Cas9 efficiency with molecular engineering‐based molecules

4.1. Cell culture and cell transfection

HEK293T, 5637, SW80, and Hela cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). HEK293T, SW780, and Hela cells were maintained in Dulbecco's Modified Essential Medium, while 5637 cells were maintained in 1640 medium. All media were supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and maintained in 5% CO2 at 37°C. For transient transfection assays, the cells were seeded in six‐well plates the day before transfection using Lipofectamine‐3000 (Invitrogen). At 48 h post‐transfection, cells were harvested for follow‐up assays.

The human iPS cell line was purchased from ATCC and cultured in mTeSR1 (STEMCELL Technologies) in Geltrex (Gibco)‐coated six‐well plates. Cells were transfected with plasmids using Lipofectamine Stem Reagent (Invitrogen) and were harvested for follow‐up assays at 48 h post‐transfection.

4.2. Plasmid construction

All ANAMs were constructed by chemical synthesis based on the designed sequence. The ANAM sequences are listed in Table S1. The U6 promoter was used to drive the expression of ANAMs, and the U6‐ANAMs were incorporated into a pcDNA3.1 plasmid vector. The original plasmids (Addgene) of CRISPR‐Cas9, CRISPR‐dCas9‐vp64, and CRISPR‐dCas9‐Krab used in this study were previously reported. 39 The bpcDNA3.1 sequence was designed based on the sequence of pcDNA3.1 after deleting the resistance gene and marker protein‐coding gene from its backbone and was then chemically synthesized. β‐Catenin‐pcDNA3.1, NF‐κB‐pcDNA3.1, and CD23‐pcDNA3.1 plasmids were purchased from Beijing SyngenTech Co., Ltd.

4.3. Generation of the stable transgenic cell lines

The 293t and 5637 cell lines were transfected with pcDNA3.1/GFP/Neo plasmids using Lipofectamine 3000 (Invitrogen) and treated with G418 to select the 293t‐GFP and 5637‐GFP stable transgenic cell lines. Also, the 293t and 5637 cell lines were transfected with the AON promoter‐GFP expression pcDNA3.1 plasmid to generate the 293t‐AON and 5637‐AON stable transgenic cell lines. The stable transgenic cell lines were obtained by G418 resistance screening.

4.4. RNA extraction and real‐time qPCR

The total RNAs of transfected cells were extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The RevertAid First Strand cDNA Synthesis Kit (Fermentas, Hanover, MD, USA) was used to synthesize cDNAs from total RNAs. The All‐in‐One qPCR Mix (GeneCopoiea, Rockville, MD, USA) was used to perform the real‐time qPCR reactions on an ABI PRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA, USA). The PCR cycling parameters were as follows: 95°C for 15 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 45 s. The primer sequences are listed in Table S2.

4.5. Dual‐luciferase reporter assay

To measure the transcriptional activity of β‐catenin and NF‐κB, a dual‐luciferase reporter was constructed using β‐catenin or NF‐κB responsive elements. The β‐catenin reporter contained Tcf‐binding elements and the minimal promoter, which were inserted upstream of the firefly luciferase gene on the dual‐reporter vector. Similarly, the NF‐κB reporter contained multiple binding sites for NF‐κB and the minimal promoter. Cells were seeded in six‐well plates (5 × 10 5 per well) and co‐transfected with the β‐catenin or NF‐κB dual‐luciferase reporter vector and the ANAM expression vector. Luciferase activity was measured using the dual‐luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer's instructions at 48 h after transfection. The firefly luciferase activity was normalized to the Renilla luciferase activity.

4.6. Surface plasmon resonance assay

The affinity of the constructed ANAMs for β‐catenin or NF‐κB proteins was measured by surface plasmon resonance using a Biacore X100 instrument (GE Healthcare, Uppsala, Sweden). Briefly, the CM5 sensor chip (GE Healthcare) was preequilibrated with running buffer prior to the immobilization of ligands onto the chip surface and activated with 0.05 M N‐hydroxysuccinimide (NHS; GE Healthcare) and 0.2 M 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC; GE Healthcare). Each protein was then injected into the flow cells of the sensor chip, and amine coupling chemistry was used for covalently attaching ligands to the sensor chip surface. After immobilization of the ligand, the chip surface was deactivated with 1 M ethanolamine hydrochloride to block the remaining unreacted groups. DNA template oligonucleotides for ANAMs were transcribed into RNA using the MEGA‐shortscript T7 kit (Life Technologies). Then various concentrations of the ANAMs were injected over the sensor surface for 1.5 min at 5 μL/min, and subsequently analyzed for equilibrium binding properties.

4.7. ELISA assay

The cells were seeded in 6‐well plates (5 × 105 cells/well) and transfected using the firefly luciferase reporter system. After 36 h, the medium was removed, and the cells were lysed with lysis buffer (Analytical Luminescence Laboratories, Mansfield, MA, USA). The level of firefly luciferase activity was measured by the Firefly Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's protocol.

4.8. Western blotting

Cells were washed with phosphate‐buffered saline (PBS) and lysed in the RIPA buffer (Beijing Solarbio Science and Technology Co.). The protein concentration was determined using a BCA kit (Beijing Solarbio Science and Technology Co., Ltd.). Equal amounts of whole protein extracts were electrophoresed onto SDS–polyacrylamide gels and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). The samples were blocked in 5% dry milk and incubated overnight with the primary antibodies against BCL2 (ab32124; 1:1000) and BAX (ab32503; 1:10 000). The next day, samples were incubated with the horseradish peroxidase‐conjugated secondary antibody (Amersham, Piscataway, NJ) and immunoblots were developed with Super Signal chemiluminescence reagents (Pierce Chemical Co.).

4.9. Flow cytometry

To determine fluorescent protein expression, the cells were suspended in PBS and analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA). We also used flow cytometry to determine the level of apoptosis. The cells were resuspended and treated with fluorescein isothiocyanate (FITC) and propidium iodide (PI) dye (Transgene, Beijing, China) according to the manufacturer's instructions. Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

4.10. Detection of GFP expression

The transfected cells were cultured in the normal growth medium, and the GFP expression was visualized using fluorescent microscopy (MicroPublisher 3.3 RTV; Olympus, Tokyo, Japan). The images were captured using the auto‐exposure mode.

4.11. Determination of NHEJ‐mediated indel mutations

Human iPS cells were harvested at 48 h post‐transfection, and the genomic DNA was extracted using the QuickExtract DNA Extraction system (Epicentre). Subsequently, PCR was performed to amplify the target regions using the genomic DNA as a template. The PCR products were purified using the ISOLATE II PCR and Gel Kit (Bioline) and subjected to Sanger sequencing. Total NHEJ frequencies were determined by decomposition of the sequencing chromatogram using the TIDE software program (https://tide-calculator.nki.nl/). Depicted values were generated from TIDER analyses with R 2 values > 0.9 and P < .001.

4.12. Statistical analyses

Data were summarized as the mean ± SEM. Significance tests were performed using SPSS statistical software for windows, version 21.0 (SPSS, Chicago, IL, USA). Statistical significance was determined using Student's t‐test or analysis of variance and a value of P < .05 was considered to be statistically significant.

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Article TitleImproving transgene expression and CRISPR‐Cas9 efficiency with molecular engineering‐based molecules

Abstract

As a novel and robust gene‐editing tool, the Clustered Regularly Interspaced Short Palindromic Repeats CRISPR‐associated protein 9 (CRISPR‐Cas9) system has revolutionized gene therapy. Plasmid vector delivery is the most commonly used method for integrating the CRISPR‐Cas9 system into cells. However, such foreign cytosolic DNAs trigger an innate immune response (IIR) within cells, which can hinder gene editing by inhibiting transgene expression. Although some small molecules have been shown to avoid the action of IIR on plasmids, they only work on a single target and may also affect cell viability. A genetic approach that works at a comprehensive level for manipulating IIR is still lacking. Here, we designed and constructed several artificial nucleic acid molecules (ANAMs), which are combinations of aptamers binding to two key players of IIR (β‐catenin and NF‐κB). ANAMs strongly inhibited the IIR in cells, thus improving transgene expression. We also used ANAMs to improve the gene‐editing efficiency of the CRISPR‐Cas9 system and its derivatives, thus enhancing the apoptosis of cancer cells induced by CRISPR‐Cas9. ANAMs can be valuable tools for improving transgene expression and gene editing in mammalian cells.


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