All reagents were ordered from Sigma-Aldrich (St. Louis, MO, USA) unless specified otherwise. Linear PEI (molecular weight, 40 kDa) was obtained from Polysciences Inc. (Warrington, PA, USA). U2OS and 3T3-L1 were obtained from the American Type Culture Collection (ATCC). U2OS.EGFP reporter cell line was obtained from J. K. Joung at Massachusetts General Hospital (36).
Expression and purification of Cas12a
The ORF of humanized Cas12a was amplified from plasmid pY016 (Addgene #69988) (13) using primers Cas12a-f/Cas12a-r (table S1), and the purified amplicon was then cloned into the Bam HI and Hind III sites of a bacterial expression vector (pET-28a). An NLS was appended on the C terminus of Cas12a. E. coli Rosetta (DE3) pLysS was used as the host for Cas12a expression as described before (37). Briefly, the E. coli strain with pET-28a-Cas12a was seeded in LB medium (5-ml medium with kanamycin and chloromycetin supplemented at 10 and 34 μg/ml). The cells were cultured at 220 rpm overnight and then transferred into a 500-ml LB medium supplemented with the same concentrations of kanamycin and chloromycetin. When the OD600 (optical density at 600 nm) of the E. coli reached 0.6 to 0.8 after 2 to 3 hours of continued culture, isopropyl β-d-1-thiogalactopyranoside (IPTG) was added (0.5 M). Induction of Cas12a expression was done at 20°C for 8 hours before harvest. Cas12a was subsequently purified through the Ni-NTA agarose beads (Qiagen)–based chromatography. The cells were centrifuged at 4000g for 15 min, washed once with H2O, and resuspended in suspension buffer containing tris-HCl buffer (20 mM pH 8.0), imidazole (10 mM), NaCl (0.5 M), and phenylmethylsulfonyl fluoride (1 mM). The cells were then lysed using a probe sonicator. After removing cell debris by high-speed centrifugation (20,000g) for 20 min, supernatant containing Cas12a was loaded into Ni-NTA resin column and equilibrated for 1 hour. Unbound proteins were removed with wash buffer containing tris-HCl buffer (20 mM pH 8.0), imidazole (60 mM), and NaCl (0.5 M). Cas12a was then eluted with a buffer that contains tris-HCl buffer (20 mM pH 8.0), imidazole (500 mM), and NaCl (0.5 M). The obtained Cas12a was dialyzed overnight in a buffer that was composed of Hepes (20 mM, pH 7.0), KCl (150 mM), dithiothreitol (1 mM), and glycerol (10%). The purified Cas12a was then analyzed by 12% SDS-PAGE (polyacrylamide gel electrophoresis) for purity and quantified by Bradford assay (Bio-Rad).
In vitro transcription and purification of crRNA
The crRNAs were designed with the assistance of Benchling (http://benchling.com). The transcription templates containing a T7 promoter and the crRNA were synthesized by Integrated DNA Technologies (IDT). Sequences of the template for in vitro transcription of crRNA were listed in table S1. The crRNAs were transcribed in vitro using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB). The transcribed crRNA products were purified using the MEGAclear Transcription Clean-Up Kit (Ambion). Purity and yield of the obtained crRNA were examined using NanoDrop 2000c (Thermo Fisher Scientific).
In vitro cleavage assay of Cas12a/crRNA RNP
To test the DNA cleavage activity of Cas12a/crRNA RNP in vitro, plasmid DNA pCAG-EGFP acquired from Addgene (#11150) (54) was used as the substrate. Plasmid DNA was amplified in E. coli DH5α and purified with the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific). The plasmid was linearized with Pvu I (NEB) and purified again before use. Cas12a was complexed with crRNA-EGFP, and its cleavage led to cleavage of the plasmid DNA. The cleavage reaction was performed in 20-μl NEBuffer 3 containing 300 ng of pCAG-EGFP plasmid, 160 ng of Cas12a, and 14 ng of crRNA. The reaction was kept under 37°C for 1 hour, and the cleavage bands were analyzed by 0.8% agarose gel electrophoresis and stained by GelRed (Biotium) for visualization.
Preparation of the DNA NCs
DNA NCs for loading the Cas12a/crRNA RNP were synthesized by RCA. Sequences of the primer and templates were shown in table S1, where sequences with complete or partial complementation to the target crRNA were incorporated to adjust the interaction between the DNA NCs and Cas12a/crRNA RNP. A palindromic sequence was incorporated to induce the self-assembly of the DNA NC. The templates were 5′-phosphorylated linear single-stranded DNA ordered from IDT. It was cyclized with CircLigase II ssDNA ligase (Epicenter), and the remaining ssDNA was removed by treating the template DNA with Exonuclease I (NEB). After heat inactivation of Exonuclease I, the template (10 pmol) was hybridized with the primer (0.5 μM) in a 1-ml isothermal amplification buffer (NEB) under 95°C for 5 min. After cooling the mixture to room temperature, Bst 2.0 DNA polymerase was added (0.2 U/μl) and the RCA reaction was conducted under 60°C for 17 hours. Precipitates from the reaction were removed by centrifugation at 14,000g for 2 min, and the obtained DNA NC was dialyzed against deionized water in a Slide-A-Lyzer dialysis unit (20-kDa molecular weight cutoff) for 48 hours. Purity and concentration of the synthesized DNA NCs were analyzed using NanoDrop 2000c. Agarose gel electrophoresis (0.8%) was also performed to analyze the DNA NCs.
Preparation of the assembly
The purified Cas12a and crRNA were mixed at equimolar ratio in PBS, and the mixture was incubated at room temperature for 5 min for complexing Cas12a with crRNA. The Cas12a/crRNA RNP was then mixed with DNA NC at a weight ratio of 4:1 and incubated at room temperature for another 5 min to obtain Cas12a/crRNA/NC. PEI was then coated on the assembly with different weight ratios of PEI:DNA NC (0:1 to 3:1), and the Cas12a/crRNA/NC/PEI assembly was equilibrated at room temperature for 5 min. To find the optimal concentration of the Gal-PEI-DM layer for the assembly, Gal-PEI-DM was added to the solution at different weight ratios to PEI (0:1 to 3:1). Hydrodynamic size and zeta potential of the assembly were measured using a Zetasizer (Malvern). Transmission electron microscopy (TEM) imaging was performed with 2% uranyl acetate as the negative stain on a JEM-2000FX TEM. The assembly was also imaged by CLSM to confirm the colocalization of the layers (SP5, Leica), where Gal-PEI-DM was modified with AF647, PEI was stained with FITC, and the DNA NC was visualized by Hoechst 33342.
Zeta potential measurement for the charge reversal process
The charge reversal behavior of the assembly was analyzed by measuring the zeta potentials of the assembly under different pH. The assembly was dissolved in 0.1 M PBS of different pH (7.4, 6.5, and 5.5). The samples were incubated at 37°C with constant stirring. Aliquots were taken from the incubation at planned time intervals, and the zeta potentials were measured using a Zetasizer (Malvern).
In vitro hemolysis assay
Human red blood cells (RBCs) suspended in PBS were obtained from Thermo Fisher Scientific. The human RBC was washed with PBS at 500g for 5 min and then diluted in PBS at pH 7.4 for the assay. The assemblies were prehydrolyzed in PBS with different pH (7.4, 6.5, and 5.5) for 8 hours before incubating with human RBC (100 μg/ml in terms of Gal-PEI-DM) for another 2 hours in PBS at pH 7.4. Human RBC treated with 0.5% Triton X-100 was used as the positive control, while human RBC treated with PBS at pH 7.4 was used as the negative control. After the incubation, intact human RBC was precipitated by centrifugation at 500g for 5 min. The absorbance of the supernatant was recorded on a plate reader at 420 nm (Tecan).
Intracellular distribution of the assembly
For convenient imaging of the intracellular distribution of the assemblies, U2OS (ATCC HTB-96) cells were seeded in glass-bottomed confocal dishes (MatTek) and incubated for 24 hours before the treatment with assemblies. Assemblies containing Cas12a-AF647 was then incubated with the cells for 2, 4, and 6 hours. The cells were then washed with cold PBS twice and stained for 30 min with LysoTracker Green at 50 nM (Life Technologies) and 10 min with Hoechst 33342 (1 μg/ml). The stained cells were washed with cold PBS twice again and imaged with CLSM immediately.
In vitro EGFP disruption and cytotoxicity assay
U2OS.EGFP cells were seeded in 24-well plates and cultured for 24 hours before the treatment with assemblies. When the cells are approximately 70% confluent, 0.5 ml of Opti-MEM medium containing Cas12a/crRNA-loaded assemblies was used to replace existing culture medium. After 4 hours of incubation, the Opti-MEM was replaced with full serum medium and the cells were allowed to grow for another 48 hours. The cells were then imaged using a fluorescence microscope (IX71, Olympus). Percentage of GFP disruption was quantified by flow cytometry (CytoFLEX, Beckman Coulter; LSRII, Becton Dickinson). To evaluate the cytotoxicity during gene editing, a TO-PRO-3 live/dead stain–based assay was adopted after the EGFP disruption assay. The cells were then washed with PBS and stained with TO-PRO-3 live/dead stain (1 μM) for 15 min. Signal of TO-PRO-3 was also analyzed by flow cytometry.
In vitro Pcsk9 gene disruption
For in vitro disruption of Pcsk9, mouse cell line 3T3-L1 was used. Specific crRNAs were designed to target exons 2 and 3 of Pcsk9 gene. 3T3-L1 cells were seeded in six-well plates and cultured for 24 hours before the treatment with assemblies. When the cells grow to 70% confluency, the culture medium was replaced with 1 ml of Opti-MEM containing assemblies loaded with Pcsk9 targeting Cas12a/crRNA RNP (Cas12a at 200 nM). Four hours after the treatment, the medium was replaced with full serum medium. Two days after incubating 3T3-L1 cells with the formulations, genetic alterations were analyzed by T7EI assay.
In vivo biodistribution
All animal studies were performed following the animal protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina (UNC) at Chapel Hill and North Carolina State University. C57BL/6 male mice (5 to 6 weeks) were purchased from the Jackson Laboratory and used throughout this study. Cas12a was labeled with Cy5.5 for in vivo imaging. The mice were intravenously injected with assembly containing Cas12a-Cy5.5 via the tail vein. The mice were imaged on the IVIS Lumina imaging system (Caliper) 1 hour after administration of the assembly. The mice were euthanized, and major organs were harvested for ex vivo imaging, including heart, liver, spleen, lung, and kidney. Quantification of fluorescence intensities of the regions of interest was done with the Living Image Software.
In vivo Pcsk9 disruption
C57BL/6 male mice (5 to 6 weeks) were intravenously injected with the assemblies. The mice were divided into three groups (n = 5): PBS, Cas12a/crRNA-control/NC-control/PEI/Gal-PEI-DM, and Cas12a/crRNA-exon3/NC-exon3/PEI/Gal-PEI-DM. Each mouse was injected with assemblies containing 50 μg of Cas12a (~2.5 mg/kg mouse) every other day (days 1, 3, and 5) for three times. The mice were fed under normal conditions for another week until the extraction of tissue and blood samples (day 12). After overnight fasting (day 13), the mice were euthanized for liver and peripheral blood collection. From the serum samples, PCSK9 levels were determined using the Mouse PCSK9 ELISA Kit (R&D Systems). Serum cholesterol, ALT, and albumin levels were tested at the UNC Histopathology and Laboratory Medicine. Genomic DNA of the collected livers was extracted for T7EI assay and deep sequencing. Other major organs of the mice were also harvested for H&E staining to assess potential pathological abnormalities.
Genomic DNA of collected tissues or cells was extracted using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific) following the manufacturer’s manual. Polymerase chain reaction (PCR) amplicons of the targeted region were amplified with Phusion Hot Start II High Fidelity DNA polymerase (NEB). Potential off-target sites similar to Pcsk9 exon 3 were identified by Benchling (55). Primers for the amplicons were shown in table S2. A touchdown PCR program was used to generate the amplicons with reduced nonspecific bands: 10 cycles of (98°C, 10 s; 66 to 56°C, −1°C/cycle for 15 s; 72°C, 60 s) followed by 25 cycles of (98°C, 10 s; 56°C, 15 s; 72°C, 60 s). The obtained amplicons were gel-purified for the T7EI assay. In a reaction mixture containing 1 × NEBuffer 2 (20 μl), 200 ng of purified amplicons was added and annealed by heating at 95°C for 5 min. After cooling to room temperature, 1 μl of T7EI nuclease (10 U/μl, NEB) was added and the cleavage reaction was performed under 37°C for 15 min. The cleaved amplicons were analyzed by 2% agarose gel electrophoresis with GelRed to visualize the DNA. Indel formations were quantified by ImageJ.
The next-generation sequencing was applied to quantify on-target disruption of Pcsk9 of the treated liver. Liver DNA was extracted as mentioned above, and amplicons centered on the targeted region were amplified using primers in table S2. A 150-bp region was amplified by touchdown PCR and gel-purified. Deep sequencing was performed in UNC Genomics Core. Sequencing library was prepared with a KAPA Hyper Prep kit (Kapa Biosytems), and the library was subjected to MiSeq (Illumina) using 150-bp runs. Cas-analyzer was used to analyze the indels (56).
All data were presented as mean value ± SD from independent tests. Statistical analysis between different treatment groups was done by Student’s t test. The difference was considered significant when P < 0.05.
Article TitleCRISPR-Cas12a delivery by DNA-mediated bioresponsive editing for cholesterol regulation
CRISPR-Cas12a represents an efficient tool for genome editing in addition to the extensively investigated CRISPR-Cas9. However, development of efficient nonviral delivery system for CRISPR-Cas12a remains challenging. Here, we demonstrate a DNA nanoclew (NC)–based carrier for delivery of Cas12a/CRISPR RNA (crRNA) ribonucleoprotein (RNP) toward regulating serum cholesterol levels. The DNA NC could efficiently load the Cas12a/crRNA RNP through complementation between the DNA NC and the crRNA. Addition of a cationic polymer layer condensed the DNA-templated core and allowed further coating of a charge reversal polymer layer, which makes the assembly negatively charged under a physiological pH but reverts to positive charge under an acidic environment. WhenPcsk9was selected as the target gene because of its important role in regulating the level of serum cholesterol, efficientPcsk9disruption was observed in vivo (~48%), significantly reducing the expression of PCSK9 and gaining the therapeutic benefit of cholesterol control (~45% of cholesterol reduction).