Materials and Methods

A far-red light–inducible CRISPR-Cas12a platform for remote-controlled genome editing and gene activation

MATERIALS AND METHODSConstruction of the FICA systemThe constructs were assembled by standard molecular cloning techniques. Cas12a derived from Acidaminococcus sp. BV3L6 was amplified from the Addgene plasmid 69982 and cloned downstream of the FRL-responsive promoter PFRL using Eco RI and Hind III sites.Construction of the FIdCA systemTo generate the FIdCA system, the E993A mutation was introduced in Cas12a by overlap polymerase chain reaction (PCR). The 10 amino acid copies of the starvation-responsive transcription factor GCN4 fragment were amplified from the Addgene plasmid 78899 and ligated to the C terminus of dCas12a with a glycine-serine linker and an NLS. The scFv and p65-HSF1 fragments were amplified by standard PCR and then fused to the glycine-serine linker sequences using the MultiS One Step Cloning Kit (Vazym, Nanjing, China, catalog no. C113-01) according to the manufacturer’s instructions. All genetic components were confirmed by sequencing (Genewiz Inc., Suzhou, China). Detailed information on the plasmids constructed in this study is provided in table S1.Construction of crRNAs and sgRNAsThe crRNAs targeting DNMT1, FANCF, GRIN2B, CXCR, HBB, d2EYFP, SEAP, and EGFP were generated by the annealed oligo cloning strategy using the Bsm BI site of a constitutive PU6-driven crRNA vector (pZQ23) (19, 21). The PU6-crRNA cassette was amplified from the Addgene plasmid 78741 and cloned into pcDNA3.1 (+). For the construction of crRNAs bearing MS2 RNA aptamers, we generated crRNAs targeting HBB and SEAP with MS2 aptamers (Addgene no. 102560) by the annealed oligo cloning strategy using the Bsm BI site of the pZQ23 vector. The sgRNAs targeting IL12A-AS1 and PRKCH were constructed as previously described (37). The sgRNAs targeting HBB were constructed as previously described (36). For the construction of crRNA array, we designed oligonucleotides for one-directional annealing through their sticky ends. The oligonucleotides (final concentration, 10 μM) were mixed with 10× Primer start buffer (Takara, Dalian, China, catalog no. R010A) to 20 μl. The mixture was phosphorylated and annealed on a thermocycler (95°C, 5 min; 94°C, 2 s, −0.1°C per cycle, 200 times; 75°C, 1 s, −0.1°C per cycle, 600 times; 16°C, 5 min). The annealed pre-crRNA array was ligated to a pZQ23 using the Bsm BI site. The target sequences that were used for constructing the crRNA are provided in table S2.Cell culture and transfectionHEK cells (HEK-293T; American Type Culture Collection, CRL-11268) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, NY, USA, catalog no. 31600-083), supplemented with 10% (v/v) fetal bovine serum (Gibco, catalog no. 10270-106) and a 1% (v/v) penicillin and streptomycin solution (Beyotime Inc., Shanghai, China, catalog no. ST488-1/ST488-2). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. The cells were regularly tested for the absence of Mycoplasma and bacterial contamination. Transfections were performed using an optimized polyethyleneimine (PEI)–based protocol. Briefly, a total of 6 × 104 cells per well were seeded into a 24-well cell culture plate without exogenous supplement of biliverdin for 18 hours before transfection and then cotransfected with the corresponding plasmid mixtures for 6 hours with 50 μl of 3:1 PEI:DNA mixture (PEI; molecular weight, 40,000; stock solution, 1 mg/ml in ddH2O; catalog no. 24765, Polysciences). The concentration and viability of the cell lines were evaluated with a Countess II Automated cell counter (Life Technologies, USA).SEAP reporter assayThe production of human placental SEAP in the cell culture was quantified using a p-nitrophenylphosphate–based light absorbance time course assay. Briefly, the cell supernatants were collected and heated for 30 min at 65°C to inactivate the cell culture supernatant. We added 120 μl of substrate solution 100 μl of 2× SEAP buffer containing 20 mM homoarginine, 1 mM MgCl2, and 21% (v/v) diethanolamine (pH 9.8), and 20 μl of p-nitrophenylphosphate substrate solution containing 120 mM p-nitrophenylphosphate to 80 μl of the heat-inactivated supernatants. The absorbance was measured at 405 nm using a Synergy H1 hybrid multimode microplate reader (BioTek Instruments Inc., USA) installed with the Gen5 software (version 2.04).FICA-mediated gene editing activity in HEK-293TFor FRL-controlled exogenous gene editing experiments, HEK-293T cells (6 × 104) were cotransfected with pXY137 (PhCMV-p65-VP64-BldD-pA-PhCMV-BphS-P2A-YhjH-pA), pDL192 (PFRL2-hCMVmin-Cas12a-pA), and pDL196 (PSV40-d2EYFP-pA), together with pDL194 (PU6-crRNA1d2EYFP-pA) and pDL197 (PU6-crRNA2d2EYFP-pA). The cells were then illuminated with FRL (730 nm, 2 mW/cm2) for 6 hours per day for 2 days. The expression of the d2EYFP reporter was visualized by fluorescence microscopy and quantified by flow cytometry 48 hours after the first illumination.For FRL-controlled endogenous gene editing experiments, HEK-293T cells (6 × 104) were cotransfected with pXY137, pDL192, and pZQ28 (PU6-crRNADNMT1-pA), pZQ206 (PU6-crRNAFANCF-pA), pZQ205 (PU6-crRNAGRIN2B-pA), or pZQ288 (PU6-crRNACXCR-pA) and illuminated with FRL (730 nm, 2 mW/cm2) for 6 hours per day for 2 days. The frequency of induced indel mutations was measured by a T7E1 assay 48 hours after the first illumination.Mismatch-sensitive T7E1 assayGenomic DNA was extracted from the cells using the TIANamp Genomic DNA Extraction Kit (TIANGEN Biotech Inc., Beijing, China, catalog no. DP304) according to the manufacturer’s instructions. The genomic region containing the target sequence was amplified by PCR with a 2× Taq Plus Master Mix II (Dye Plus) DNA polymerase (Vazyme, catalog no. P213). The PCR primers that were used for each target gene are shown in table S3. The amplified PCR products were purified using HiPure Gel Pure Micro Kits (Magen Inc., Shanghai, China, catalog no. D2111-03). To promote restriction enzyme, we mixed 500 ng of purified DNA products with 1.5 μl of 10× M Buffer (Takara, catalog no.1093A) and Milli-Q water to a final volume of 15 μl. The DNA was then reannealed to form heteroduplex DNA (95°C, 5 min; 94°C, 2 s, −0.1°C per cycle, 200 times; 75°C, 1 s, −0.1°C per cycle, 600 times; 16°C, 5 min), which was subjected to 5 U of T7E1 endonuclease I digestion (New England Biolabs, catalog no. M0302) for 30 min at 37°C, and, lastly, analyzed by 1.5% agarose gel electrophoresis. The gels were stained with GelRed (Vazyme, catalog no. GR501-01) and visualized using a Tanon 3500 gel imaging system (Tanon Science & Technology Inc.). The proportion of indel mutation promoted by Cas12a was calculated using the ImageJ software with the following formula: 100% × 1 − (1 − (b + c)/ (a + b + c))1/2, in which a is the integrated intensity of the undigested PCR product, and b and c are the integrated intensities of the T7E1-digested products.TIDE analysisTarget regions were amplified from genomic DNA by PCR using 2× Taq Plus Master Mix II (Dye Plus) DNA polymerase (Vazyme, catalog no. P213). Purified PCR products were analyzed by Sanger sequencing. The ab1 Sanger sequencing files were uploaded to the online TIDE analysis tool (https://tide.nki.nl/) (62) to quantify the nature and frequency of the generated indels.Targeted deep sequencingTargeted regions were amplified from genomic DNA using 2× Taq Plus Master Mix II (Dye Plus) DNA polymerase (Vazyme, catalog no. P213). The paired-end sequencing of PCR amplicons was performed on Illumina MiSeq (Sangon Biotech, Shanghai). The PCR primers used for the amplification of regions for deep sequencing are provided in table S4. The average depth of coverage for the sequencing runs was ~20,000 reads. The paired-end MiSeq raw data were analyzed using the SSH Secure Shell Client software from Sangon Biotech.Quantitative real-time PCR analysisTotal RNA was isolated using the RNAiso Plus Kit (Takara, catalog no.9109) according to the manufacturer’s instructions. A total of 500 ng of mRNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit with the genomic DNA Eraser (Takara, catalog no. RR047). Quantitative PCR (qPCR) analysis was performed on a real-time PCR instrument (Roche, LightCycler 96, Switzerland) using the SYBR Premix Ex Taq (Takara, catalog no. RR420) to detect each of the target genes. The list of qPCR primers used in this study is available in table S4. We used the following PCR cycling parameters: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s. Samples that were transfected with AsCas12a crRNA backbone plasmid (pZQ23) were used as negative controls. The relative cycle threshold (CT) of the housekeeping gene human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured as an endogenous control, and thus, the relative mRNA expression levels were normalized to match those of GAPDH using the standard ΔΔCt method.Flow cytometry analysisThe transfected HEK-293T cells were dissociated by 0.25% Trypsin-EDTA (Sangon Biotech, Shanghai, China, catalog no. AP10629-0050) for 3 min and then washed in phosphate-buffered saline for three consecutive times. The resuspended cells were analyzed using a Becton Dickinson LSRFortessa Flow Cytometer (BD Biosciences, San Jose, USA) equipped for EGFP/d2EYFP 488-nm laser, 505-nm long pass filter, and 530/30 emission filter (passband centered on 530 nm; passband width, 30 nm). Approximately 10,000 cells per sample were recorded, and the data were analyzed by the FlowJo software (version no. 7.6). The transfected HEK-293T cell populations were gated for cells with high EGFP/d2EYFP fluorescence beyond a threshold of 103 arbitrary fluorescence units. The expression values of weighted EGFP/d2EYFP were calculated as the percentage of gated cells multiplied by their median fluorescence, which is correlated with fluorescence intensity and cell number.Fluorescence microscopyFluorescence microscopy images of the cells expressing d2EYFP/EGFP were obtained using an inverted fluorescence microscope (Leica DMI8, Wetzlar, Germany) with a 20× objective and a 495/535-nm (B/G/R) excitation/emission filter set. Identical settings, including exposure times for d2EYFP/EGFP, were used for all fluorescence micrographs.FIdCA-mediated gene activation in mammalian cellsFor FRL-controlled exogenous SEAP reporter experiments, HEK-293T cells (6 × 104) were cotransfected with the pXY137, pZQ113 (PhCMV-scFv-p65-HSF1-pA), pZQ116 (PFRL3-dCas12a-NLS-GCN4-pA), pZQ28 (PU6-crRNASEAP-pA), and pZQ5 (crRNA targeting site-PhCMVmin-SEAP-pA). The cells were subsequently illuminated with FRL (730 nm, 2 mW/cm2) for 6 hours per day for 2 days. SEAP production levels in the culture medium were quantified 48 hours after the first illumination.For FRL-controlled endogenous gene experiments, HEK-293T cells (6 × 104) cotransfected with the pXY137, pZQ113, pZQ116, and pZQ34 (PU6-crRNAHBB-pA) were illuminated with FRL (730 nm, 2 mW/cm2) for 6 hours per day for 2 days, and the relative mRNA expression of HBB was quantified by qPCR 48 hours after the first illumination.Spatial control of FRL-dependent gene activation in mammalian cellsHEK-293T cells (3 × 106) were plated into a 10-cm dish, cultivated overnight to 60 to 70% confluency, and then transfected with 1000 μl of a 3:1 PEI:DNA mixture (w/w) containing 15 μg of plasmid DNA pXY137, pDL247, pZQ5, pZQ113, and pZQ116 at a ratio of 5:3:3:2:3 (w/w/w/w/w). Twenty-four hours after transfection, the cells were illuminated with FRL for 24 hours (730 nm, 0.5 mW/cm2, 1 min on, 5 min off) under a “BIO”-patterned photomask made from aluminum foil. The patterned photomask was placed between the upward-facing LED array and the bottom of the 10-cm culture dish. Fluorescence images were obtained 24 hours after illumination using the ChemiScope 4300 Pro imaging equipment (Clinx, Shanghai, China).In vivo gene activation using FIdCA systemWe randomly divided male wild-type C57BL/6 mice 6 weeks old; East China Normal University (ECNU) Laboratory Animal Center into two groups. Before injecting the mice, the plasmids encoding the FIdCA system pXY137, pZQ303, and pZQ315 at a 2:4:1(w/w/w) ratio were mixed into a Ringer’s solution (147 mM NaCl, 4 mM KCl, and 1.13 mM CaCl2) Each mouse was hydrodynamically injected with 350 μg of DNA without exogenous supplement of biliverdin within 8 s via tail vein. The injection volume of the plasmid solution was 2 ml per mouse weight (20 ± 2 g). Eight hours after injection, the mice were exposed to FRL (730 nm LED, 10 mW/cm2, 2 min on, 2 min off, alternating). Control mice were kept in the dark. Twelve hours after illumination, each mouse was intraperitoneally injected with 100 μl of Hanks’ balanced salt solution supplemented with 100 mM luciferin substrate solution (Synchem, CAS no. 115144-35-9) and anesthetized with ether before proceeding with imaging. Ten minutes after luciferin injection, we obtained bioluminescence images of the mice using the IVIS Lumina II in vivo imaging system (PerkinElmer, USA).EthicsThe animal experiments were approved by the ECNU Animal Care and Use Committee and in direct accordance with the Ministry of Science and Technology of the People’s Republic of China on Animal Care guidelines. The protocol (protocol ID: m20191212) was approved by the ECNU Animal Care and Use Committee. All mice were euthanized after the completion of the experiments.Statistical analysisAll in vitro data represent the means ± SD and are described separately in the figure legends. For the animal experiments, the results are expressed as the means ± SEM. Comparisons between the groups were performed using a Student’s t test. Differences were considered statistically significant at P < 0.05 (*), very significant at P < 0.01 (), and extremely significant at P < 0.001 (*). The Prism 5 software (version 6, GraphPad Software) was used for statistical analysis. n and P values are provided in the figure legends.

Article TitleA far-red light–inducible CRISPR-Cas12a platform for remote-controlled genome editing and gene activation

Abstract

The CRISPR-Cas12a has been harnessed as a powerful tool for manipulating targeted gene expression. The possibility to manipulate the activity of CRISPR-Cas12a with a more precise spatiotemporal resolution and deep tissue permeability will enable targeted genome engineering and deepen our understanding of the gene functions underlying complex cellular behaviors. However, currently available inducible CRISPR-Cas12a systems are limited by diffusion, cytotoxicity, and poor tissue permeability. Here, we developed a far-red light (FRL)–inducible CRISPR-Cas12a (FICA) system that can robustly induce gene editing in mammalian cells, and an FRL-inducible CRISPR-dCas12a (FIdCA) system based on the protein-tagging system SUperNova (SunTag) that can be used for gene activation under light-emitting diode–based FRL. Moreover, we show that the FIdCA system can be deployed to activate target genes in mouse livers. These results demonstrate that these systems developed here provide robust and efficient platforms for programmable genome manipulation in a noninvasive and spatiotemporal fashion.


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