All reagents used in the experiments were purchased from commercial sources without further purification. Anhydrous dichloromethane (DCM), anhydrous N,N-dimethylformamide (DMF), HBr 48% (w/w) in H2O, DCC, 4-dimethylaminopyridine (DMAP), rhodamine B, dimethyl sulfoxide (DMSO), and TMP were purchased from Energy Chemical Co. Ltd. (Shanghai, China). Branched polyethyleneimine PEI25K; weight-average molecular weight (_M_w): 25 kDa was supplied by Meilunbio Co. Ltd. (Dalian, China). Dulbecco’s modified Eagle’s medium, RPMI 1640, and FBS were purchased from Sigma-Aldrich (USA). Lipofectamine 2000 and Lipofectamine 3000 transfection reagents were purchased from Thermo Fisher Scientific (Germany). The transfection reagent jetPEI was supplied by PolyPlus. The CCK-8 and LDH release assay kit were obtained from Beyotime Co. Ltd. (Shanghai, China). The dialysis bag molecular weight cutoff (MWCO): 1000 Da was obtained from Yuanye Bio-Technology Co. Ltd. (Shanghai, China). T7E1 enzyme was purchased from New England Biolabs (USA). Ultrapure water was obtained from a Milli-Q system. The plasmid encoding CMV-DHFR-Cas9-DHFR-U6-sgRNA (no. 85447) was supplied by Addgene.org, which was examined by Sanger sequencing and enzyme digestion. CMVIE94-3XFLAG-NLS-Cas9-NLS-P2A-EGFP (CMV-Cas9-P2A-EGFP) and CMV-3XFLAG-NLS-Cas9-NLS (wild-type) were constructed in our laboratory. Target sgRNA (sgPHD2) was designed by online tools (http://crispr.mit.edu/ and http://chopchop.cbu.uib.no/). All primers used in this work were listed in tables S1 to S5. The plasmids used in this work and corresponding nanocomplex information were listed in table S6 and in the “Plasmid information” section in Supplementary Materials. Primary antibodies used in this project included the following: Anti–TNF-α (1:1000) antibody was obtained from Beyotime (Shanghai, China). Anti-PHD2 (1:1000) antibody and cell/tissue lysis buffer radioimmunoprecipitation assay (RIPA) buffer were obtained from Solarbio Science and Technology (Beijing, China). Anti-FLAG antibody was obtained from HuaAn Biotechnology (HUABIO, Hangzhou, China). Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody was obtained from Abcam (Shanghai, China). All enzyme-linked immunosorbent assay (ELISA) kits used in this project were supplied by Multisciences (Hangzhou, China). Cationic PBAE was synthesized according to the previous literature (scheme S1 and fig. S1) (16, 17).
The 1H NMR spectra and heteronuclear singular quantum correlation spectrum of PBAE were carried out on a Bruker 500-MHz NMR spectrometer and were reported as chemical shifts (δ) in ppm relative to tetramethylsilane (δ = 0). Proton spin multiplicities are reported as a singlet (s), doublet (d), triplet (t), quartet (q), and quintet (quint), with coupling constants (J) given in hertz, or multiplet (m). Mass spectra were recorded on a Shimadzu liquid chromatography–mass spectrometry (LC-MS) mass spectrometer (LC-MS 2020). TEM was carried out on a TEM (HT7700, Hitachi, Japan). Sizes and zeta potentials of the nanoparticles were characterized by Malvern Nano ZS90. Mass analysis of OE-PEG, BAM-TK-COOH, and BAM-TK-TMP was performed on MALDI-TOF (Ultraflextreme, Bruker, Germany). The molecular weight analysis of PBAE was performed on a Waters 1525/2414 GPC system. Confocal microscopy was performed on LSM-880 (Carl Zeiss, Germany). The DiI intensity was evaluated with an in vivo imaging system (IVIS Spectrum, PerkinElmer).
Synthesis of 4-((2,4-diaminopyrimidin- 5-yl) methyl)-2,6-dimethoxyphenol (TMP-OH)
TMP-OH was synthesized according to the previous report (33). Briefly, TMP (1.00 g, 3.4 mmol) was dissolved in HBr (12.5 ml, 48% in H2O) and stirred at 95°C. After 20 min, the mixture was cooled down to room temperature and 2.97 ml of 50% (w/w) NaOH aqueous was added dropwise during stirring. After that, the solution was kept at 4°C overnight. The white precipitate was filtered and washed with double-distilled water (ddH2O). Residues were redissolved in boiling water, and 1 N NaOH was added to adjust pH to ~7.0 for recrystallization. After being cooled down at 4°C for a while, a pink solid, TMP-OH, was precipitated and washed by water (630 mg, 66%). 1H NMR (DMSO-d6) δ 8.06 (s, -OH), 7.45 (s, 1H), 6.48 (s, 2H), 5.99 (s, -NH2), 5.63 (s, -NH2), 3.71 (s, 6H), and 3.47 (s, 2H). MS mass/charge ratio (m/z) found: 277.15, calculated: 277.12 for C13H16N4O3 M+H+.
Synthesis of polyethylene glycol monooleyl ether-thioketal (BAM-TK-COOH)
To a solution of polyethylene glycol monooleyl ether (n = ~50, 250 mg, 0.1 mmol), DMAP (12 mg, 0.1 mmol) and thioketal (252 mg, 1 mmol) in 20 ml of anhydrous DCM at 0°C were added dropwise with 10 ml of DCC (206 mg, 1 mmol)/DCM solution. After completion of addition of DCC, the reaction mixture was transferred to room temperature and stirred for 72 hours under a nitrogen atmosphere. After that, the insoluble by-product was filtered and the solvents were removed by rotary evaporation. The residue was dissolved in water and dialyzed (MWCO, 1000 Da) against water/methanol (1:1, v/v) for the first 24 hours and water for 48 hours. The product, BAM-TK, was collected and lyophilized for further reaction (193 mg, 70%).
Synthesis of polyethylene glycol monooleyl ether-thioketal (BAM-TK-TMP)
TMP-OH (19 mg, 0.07 mmol) was added to a solution of BAM-TK (190 mg, 0.07 mmol) and DMAP (2 mg, 0.02 mmol) in 20 ml of anhydrous DMF. After stirring for 10 min under a nitrogen atmosphere, DCC (29 mg, 0.14 mmol), which was dissolved in 5 ml of DMF, was added dropwise into reaction mixture. After addition, the mixture was stirred at 30°C for 72 hours. The solvent of crude product was removed by rotary evaporation, and the product was redissolved in DCM. The by-product was filtered, and the filtrate was collected. After removal of DCM by rotary evaporation, the solid was redissolved in methanol. The product, BAM-TK-TMP, was obtained after dialysis against methanol for 24 hours and water for 48 hours (MWCO, 1000 Da) and further lyophilization.
Deep sequencing assay
The schematic illustration of deep sequencing assay was shown in scheme. S3. First, the off-target loci were evaluated and designed at CasOFFinder website (www.rgenome.net/cas-offinder/). Subsequently, the fragments were amplified with corresponding primers, termed first PCR product. After purification by PCR/Gel Extraction and Purification kits (Vazyme Biotech Co., Ltd), the first PCR product was further amplified to obtain PCR fragments less than 250 base pairs (bp) containing corresponding gene loci. Then, the product, termed second PCR product, was further extracted and purified. After that, the second PCR product was amplified with primers containing index sequence and then purified by PCR/Gel Extraction and Purification kits. The products were sent to company for sequencing. The results were analyzed by CRISPResso2 according to the instruction (51).
All data and figures in this paper were analyzed and plotted by GraphPad Prism 8.0. The obtained data are expressed as means ± SD. Biological replicates were used in all experiments unless otherwise stated. The statistical significance was analyzed using one-way analysis of variance (ANOVA) with a Tukey post hoc test. P value less than 0.05 was considered significant (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). ns means no significance. N.D. refers to not detectable.
Article TitleGenome-editing prodrug: Targeted delivery and conditional stabilization of CRISPR-Cas9 for precision therapy of inflammatory disease
Regulation of CRISPR-Cas9 functions in vivo is conducive to developing precise therapeutic genome editing. Here, we report a CRISPR-Cas9 prodrug nanosystem (termed NanoProCas9), which combines the targeted delivery and the conditional activation of CRISPR-Cas9 for the precision therapy of inflammatory bowel disease. NanoProCas9 is composed of (i) cationic poly(β-amino ester) (PBAE) capable of complexing plasmid DNA encoding destabilized Cas9 (dsCas9) nuclease, (ii) a layer of biomimetic cell membrane coated on PBAE/plasmid nanocomplexes for the targeted delivery of PBAE/dsCas9 complexes, and (iii) the stimuli-responsive precursory molecules anchored on the exofacial membrane. The systemic administration of NanoProCas9 enables the targeted delivery of dsCas9 plasmid into inflammatory lesions, where the precursory small molecule can be activated by ROS signals to stabilize expressed dsCas9, thereby activating Cas9 function for inflammatory genome editing. The proposed “genome-editing prodrug” presents a proof-of-concept example to precisely regulate CRISPR-Cas9 functions by virtue of particular pathological stimuli in vivo.