HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

Abstract CRISPR/Cas9 technology enables targeted gene editing; yet, the efficiency and specificity remain unsatisfactory, particularly for the nonvirally delivered, plasmid‐based CRISPR/Cas9 system. To tackle this, a self‐assembled micelle is developed and evaluated for human papillomavirus (HPV) E7 oncogene disruption. The optimized micelle enables effective delivery of Cas9 plasmid with a transient transgene expression profile, benefiting the specificity of Cas9 recognition. Furthermore, the feasibility of using the micelle is explored for another nucleic acid‐guided nuclease system, Natronobacterium gregoryi Argonaute (NgAgo). Both systems are tested in vitro and in vivo to evaluate their therapeutic potential. Cas9‐mediated E7 knockout leads to significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo, while NgAgo does not show significant E7 inhibition on the xenograft mouse model. Collectively, this micelle represents an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine‐based therapeutics.


This file includes:
Supplementary Materials and Methods Figure S1. Optimization of micelle formulation. Figure S2.
Characterization of the optimized micelle. Figure S3.
Sequence verification of the pCas9 constructs used in this study. Figure S6.
Potential off-target sites of the gRNAs used in this study. Figure S9.
Sequences of the primers and gDNAs used in this study.

DNase I digestion assay
The naked pCas9 plasmid or pCas9-encapsulated micelle (equivalent plasmid concentration = 10 ng/L) was incubated with DNase I (Thermo Fisher) under physiological DNase I condition (DNase I activity = 1.1×10 -3 mg min -1 mL -1 ) [1] at 37 o C for 0, 2, 4, 8 or 24 h. After the incubation, DNase I was inactivated by heating the samples at 95 o C for 10 min. All the samples were subsequently incubated with heparin (20 mg/mL) at 37 o C for 30 min and run on a 1.5% ethidium bromide-prestained TAE-agarose gel directly.

Lentiviral FLAG-NgAgo-NLS plasmid construction
NgAgo expression in the lentiviral plasmid is controlled by a tetracycline-inducible promoter. To construct the FLAG-NgAgo-NLS lentiviral plasmid, both NLS-NgAgo-pCDNA3.1 (Miaoling Bio, China) and Tet-O-FUW-Myt1l (Addgene #27152) were first digested with NheI-HF and EcoRI-HF (NEB) restriction enzymes to produce compatible sticky ends between the NLS-NgAgo fragment and the Tet-O-FUW backbone, both of which were extracted from an 1% agarose gel using QIAquick gel extraction kit. The Tet-O-FUW backbone was further dephosphorylated with Antarctic phosphatase (NEB). The two fragments were subsequently ligated using T4 ligase (NEB). The ligated product was transformed to the Stbl3 competent cells (Thermo Fisher), and the plasmid was extracted using Macherey-Nagel NucleoSpin® plasmid purification kit (Germany).
Lentivirus production was performed in HEK 293T cells cultured in a T75 flask by introducing pFLAG-NgAgo-NLS, psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) at the 3:2:1 mass ratio, to the cells in the presence of CalFectin (SignaGen Laboratories, Rockville, MD), based on the manufacturer's recommendation. Cells were replenished with fresh medium 16 hours post-transfection. Media containing viruses were collected at 40 h and 64 h post-transfection and concentrated using centrifugal filter units with 100 kD membrane (EMD Millipore).

FLAG-NgAgo-NLS-expressing HeLa stable cell line generation
HeLa cells were first seeded in 6-well plate with a density of 2×10 5 cells per well in 2.5 mL of DMEM and incubated for 24 h. For transduction, the cells were incubated with the reverse tetracyclinecontrolled transactivator-carrying and FLAG-NgAgo-NLS-carrying lentiviruses with a total MOI of 50, and polybrene (8 µg/mL) was used to enhance the efficiency. To further improve the efficiency, the transduction was repeated 3 times in a 3-day schedule. The transduction efficiency was evaluated by immunostaining. Briefly, the transduced HeLa cells were first seeded in a 24-well plate, and FLAG-NgAgo-NLS expression was induced using doxycycline (3 µg/mL). After 2-day doxycycline induction, the transduced cells were fixed and permeabilized. The cells were subsequently stained with anti-FLAG tag antibody (Thermo Fisher) and the secondary antibody, Alexa Fluor® 488 donkey anti-mouse IgG (Thermo Fisher), for immunofluorescent visualization. The cells were also stained with DAPI to visualize the nucleus. Fluorescent images were taken using Nikon Eclipse TE2000-U inverted microscope.

Non-viral NgAgo-EGFP plasmid construction
The mammalian codon-optimized NgAgo coding sequence (CDS) was obtained from the NLS-NgAgo-pCDNA3.1 construct by PCR using NEB heat-activated Q5 DNA polymerase, and the EGFP-FLAG vector was purchased from Addgene (#46956). For pNgAgo-EGFP construction, the vector was first digested with the NEB EcoRI-HF restriction enzyme at 37 o C for 1h and then purified by Thermo Fisher PureLink DNA purification kit. Subsequently, the linear vector was blunted and de-phosphorylated using NEB's recommended protocols with Klenow polymerase and Antarctic phosphatase, respectively. The blunted, de-phosphorylated vector was ligated with the purified NgAgo CDS PCR product (insert/vector molar ratio = 3) using NEB T4 ligase at 16 o C for overnight. Followed by the ligation, the plasmid was transformed to the Clontech Stellar competent cells by following manufacturer's protocol. Singe clone was picked and further amplified in a large-scale culture for overnight at 37 o C. The plasmid was purified using S2 Macherey-Nagel NucleoBond® Xtra Midi Plus EF plasmid purification kit, and the sequences were verified by both PCR and Eton Bioscience (Union, NJ).

Transmission electron microscopy (TEM) measurement
The pCas9-and pNgAgo-EGFP-encapsulated micelles were negatively stained with 5% phosphotungstic acid (Electron Microscopy Sciences, Hatfield, PA). TEM measurement to visualize the micelles was then performed using FEI Talos F200X transmission electron microscope under an accelerating voltage of 200 KV.     For the cellular uptake study, cells were seeded in a 24-well plate one day prior to transfection. The Cas9 plasmid (Addgene #62934) was first stained with TOTO3 under a base-pair/dye molar ratio of 10/1 and complexed with PPO-NMe3 and FITC-F127. Cells were treated with the micelle for 4 h in Opti-MEM and then either fixed for microscope measurement or harvested for FACS analysis. Cellular uptake rate (TOTO3 + % and FITC + TOTO3 + %) are presented as average ± SEM (n=3). Figure S5. Sequence verification of the pCas9 constructs used in this study. The gRNA sequences are colored red, and the 20mer targeting region of each gRNA is underlined. Three plasmids were purified using Macherey-Nagel NucleoBond® Xtra Midi Plus EF plasmid purification kit (Germany), and the sequences were identified by Eton Bioscience (Union, NJ).  Mismatched bases and the PAM motif were colored red and underlined, respectively. The numbers represent the locations of each locus and its corresponding primer pair; these correspond to the locations in the chromosomes of GRCh38.p7 Primary Assembly. Primers were designed using NCBI Primer BLAST and listed in Table S1.     For the cellular uptake study, cells were seeded in a 24-well plate one day prior to transfection. The NgAgo plasmid (Addgene #78253) was first stained with YOYO3 and complexed with PPO-NMe3 and FITC-F127. Cells were treated with the micelle for 4 h in Opti-MEM and then either fixed for microscope measurement or harvested for FACS analysis. Cellular uptake rate (YOYO3 + % and FITC + YOYO3 + %) are presented as average ± SEM (n=3).

Figure S14.
Validation on real-time PCR result variation. The result variation was defined as the ratio of the mRNA expression level determined using the previously reported primers (qPCR_E7_F2/R2) to that using the primers used in this study (qPCR_E7_F1/R1). For each group, the variation was calculated from at least 10 individual results. Data are presented as average ± SEM.   Data are presented as average ± SEM (n = 5). One-way ANOVA with Dunnett's multiple comparison test was used for p-value calculation. The significant level is represented as n.s. (no significance). (C) Representative images of the H&E stained organs extracted from the pNgAgo-micelle-treated mice. Scale bar represents 100 m.