Macrophage-Specific in Vivo Gene Editing Using Cationic Lipid

Jan 9, 2018 - Ying-Li Luo†¶, Cong-Fei Xu‡§¶, Hong-Jun Li‡§ , Zhi-Ting Cao#, Jing ... Xiao-Jiao Du‡§, Xian-Zhu Yang‡§ , Zhen Gu⊥ , an...
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Macrophage-Specific in Vivo Gene Editing Using Cationic Lipid-Assisted Polymeric Nanoparticles Ying-Li Luo,†,¶ Cong-Fei Xu,‡,§,¶ Hong-Jun Li,‡,§ Zhi-Ting Cao,# Jing Liu,† Ji-Long Wang,† Xiao-Jiao Du,‡,§ Xian-Zhu Yang,*,‡,§ Zhen Gu,⊥ and Jun Wang*,†,‡,§,∥ †

School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People’s Republic of China Institutes for Life Sciences and School of Medicine, South China University of Technology, Guangzhou, Guangdong 510006, People’s Republic of China § National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou, Guangdong 510006, People’s Republic of China ∥ Research Institute for Food Nutrition and Human Health, Guangzhou, Guangdong 510006, People’s Republic of China ⊥ Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United States # Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui 230027, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: The CRISPR/Cas9 gene editing technology holds promise for the treatment of multiple diseases. However, the inability to perform specific gene editing in targeted tissues and cells, which may cause off-target effects, is one of the critical bottlenecks for therapeutic application of CRISPR/Cas9. Herein, macrophage-specific promoterdriven Cas9 expression plasmids (pM458 and pM330) were constructed and encapsulated in cationic lipid-assisted PEG-b-PLGA nanoparticles (CLAN). The obtained nanoparticles encapsulating the CRISPR/Cas9 plasmids were able to specifically express Cas9 in macrophages as well as their precursor monocytes both in vitro and in vivo. More importantly, after further encoding a guide RNA targeting Ntn1 (sgNtn1) into the plasmid, the resultant CLANpM330/sgNtn1 successfully disrupted the Ntn1 gene in macrophages and their precursor monocytes in vivo, which reduced expression of netrin-1 (encoded by Ntn1) and subsequently improved type 2 diabetes (T2D) symptoms. Meanwhile, the Ntn1 gene was not disrupted in other cells due to specific expression of Cas9 by the CD68 promoter. This strategy provides alternative avenues for specific in vivo gene editing with the CRISPR/ Cas9 system. KEYWORDS: CRISPR/Cas9, specific gene editing, nanomedicine, specific promoter, type 2 diabetes

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successfully used as CRISPR/Cas9 delivery systems in vivo;8−10 however, high viral immunogenicity and safety concerns have limited their wide therapeutic application.11,12 To address these issues, nonviral vectors have been developed to deliver Cas9 nuclease and sgRNA in the form of plamsid, mRNA, or ribonucleoprotein.13,14 For example, Gu et al. designed a type of DNA nanoclew to deliver Cas9/sgRNA

he CRISPR/Cas9 system can edit genomes in a precise and sequence-dependent manner.1,2 Due to its high specificity, efficiency, and simplicity, the CRISPR/Cas9 system can induce point mutations, gene deletions/insertions, chromosomal translocations, and many other DNA manipulations.3,4 Since the report of mammalian CRISPR-based genetic editing, the system was immediately utilized to correct disease genes through ex vivo approaches.5−7 Subsequently, to achieve clinical application of the CRISPR/Cas9 system, several groups have tried to explore in vivo gene editing technology. Adenovirus and adeno-associated virus (AAV) have been © XXXX American Chemical Society

Received: November 6, 2017 Accepted: January 5, 2018

A

DOI: 10.1021/acsnano.7b07874 ACS Nano XXXX, XXX, XXX−XXX

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ACS Nano ribonucleoproteins for in vivo enhanced green fluorescent protein (EGFP) gene disruption in tumor cells.15 Additionally, lipid-like nanoparticles have been used to deliver Cas9 mRNA and sgRNA into the liver, kidneys, and lungs for gene editing.16,17 Wei and co-workers constructed multifunctional, nucleus-targeting “core−shell” artificial virus nanoparticles to deliver CRISPR/Cas9 plasmid for MTH1 gene disruption in a SKOV3 subcutaneous xenograft tumor model.18 Despite the successful progresses, these CRISPR/Cas9 systems delivered by these nonviral vectors could also work in nontarget cells, resulting in potential nonspecific gene editing in vivo. After systemic administration, these nonviral delivery systems of CRISPR/Cas9 will be internalized by neutrophils, monocytes, macrophages, and some other phagocytes19−21 and will accumulate into nontargeted tissues, including liver, lung, spleen, kidney, and so on.22 Even after reaching the target tissues, these nanoparticle delivery systems may also be internalized by nontargeted cells.23 Several biologists have successfully constructed CRISPR/ Cas9 systems with organism-specific promoters capable of specifically driving gene editing in Caenorhabditis elegans and zebrafish.24−27 Given these reports, we hypothesize that the use of a nonviral gene vector delivery vector as the delivery system of tissue-specific promoter-driven CRISPR/Cas9 plasmid may achieve specific in vivo gene editing in targeted cells. To substantiate our speculation, the human CD68 promoter, which was reported to be capable of driving specific gene expression in monocytes and macrophages,28 was used to replace the original chicken β-actin promoter of pX330 and pX458 (pX458 has a T2A-EGFP tag compared with pX330), and the newly constructed plasmids were denoted pM330 and pM458, respectively. Subsequently, the constructed macrophage-specific Cas9 expression plasmids pM330 and pM458 were encapsulated in our previously reported cationic lipid-assisted PEG-bPLGA nanoparticles (CLAN),29,30 which were denoted CLANpM330 and CLANpM458. Following systemic administration, the CLAN pM330 and CLAN pM458 were efficiently internalized into B cells, neutrophils, monocytes, and macrophages. Under the control of the CD68 promoter, the Cas9 protein was specifically expressed in monocytes and macrophages, but it was not expressed in other types of cells (Scheme 1). Furthermore, to demonstrate the potential of this macrophage-specific gene editing strategy for disease treatment, the Ntn1 gene was chosen for targeted disruption because it has been reported to be a potential therapeutic target in macrophages for the treatment of T2D.31,32 The sgNtn1 gene was added to the pM330 and pM458 plasmids (denoted pM330/sgNtn1 and pM458/sgNtn1) and encapsulated into the CLAN (denoted CLANpM330/sgNtn1 and CLANpM458/sgNtn1). After intravenous injection, CLAN p M 3 3 0 / s g N t n 1 and CLANpM458/sgNtn1 were effectively internalized by diverse cells. However, CD68 promoter-driven Cas9 expression induced the specific deletion of Ntn1 in monocytes and macrophages only. This strategy was shown to be effective for T2D treatment while minimizing off-target effects.

Scheme 1. CD68 promoter-driven CRISPR/Cas9 plasmids were encapsulated in CLAN vectors using a double emulsion method. After intravenous injection, nanoparticles could be endocytosed by diverse cells, including tissue cells, T cells, B cells, neutrophils, monocytes, and macrophages. However, the CD68 promoter ensured the Cas9 expression occurred only in monocytes and macrophages. This strategy realized specific gene editing in monocytes and macrophages in vivo using the CRISPR/Cas9 system.

which contain the chicken β-actin promoter to express humanized Streptococcus pyogenes Cas9 (hSpCas9) and U6 promoter to transcribe chimeric guide RNA, were used. The pX458 plasmid has a T2A-EGFP tag in comparison to pX330. Additionally, we replaced the original chicken β-actin promoter of pX330 and pX458 with the hCD68 promoter (Figure S1) to obtain pM330 and pM458, respectively. The successful construction of both plasmids is described in the Supporting Information (Figure S2). Meanwhile, fluorescently labeled siRNA was also used as a control to track the CLAN vector in subsequent experiments. Through a double emulsion method, the large CRISPR/Cas9 plasmids were efficiently encapsulated into CLAN vectors, with encapsulation efficiencies of 83.6% and 86.7% for pX458 and pM458, respectively, which were only slightly lower than the encapsulation efficiency of siRNA (90.8%). The obtained CLAN vectors encapsulating siRNA, pM458, and pX458 were denoted CLANsiRNA, CLANpM458, and CLANpX458, respectively. The size and zeta potentials of CLANsiRNA, CLANpM458, and CLANpX458 were determined. The diameters of CLANsiRNA, CLANpM458, and CLANpX458 were 122.3 ± 6.5, 129.7 ± 8.1, and 126.5 ± 7.1 nm, respectively (Figure 1B). The zeta potentials of CLANsiRNA, CLANpM458, and CLANpX458 were 16.6 ± 3.1, 18.5 ± 2.8, and 17.7 ± 2.5 mV, respectively (Figure 1C). The morphology of CLANsiRNA, CLANpM458, and CLANpX458 was further confirmed by cryogenic transmission electron microscope (cryo-TEM) (Figure 1D and Figure S3), revealing a spherical vesicular structure. Collectively, the CLAN vector could efficiently encapsulate the CRISPR/Cas9 plasmid, and the particulate

RESULTS AND DISCUSSION Preparation and Characterization of Nanoparticles Encapsulating Plasmid or siRNA. Previously, we explored CLAN vectors as a highly efficient small interference RNA (siRNA) delivery system in vivo. Herein, we attempted to encapsulate a large CRISPR/Cas9 plasmid into this CLAN vector (Figure 1A). The original pX330 and pX458 plasmids, B

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Figure 1. Characterization of nanoparticles. (A) Schematic illustration of the CLAN vector, which consists of PEG5K-PLGA11K, cationic BHEM-Chol lipid, and plasmid. Diameters (B) and zeta potentials (C) of CLANsiRNA, CLANpM458, and CLANpX458. (D) Representative TEM image of CLANpM458. The scale bar is 100 nm.

Figure 2. Efficient expression of Cas9 in macrophages through transfection with CLANpX458 and CLANpM458. (A) Percentage of EGFP-positive cells in BMDMs after transfection with CLANpM458, CLANpX458, and LipopM458. Data are shown as the means ± SD (n = 3), *p < 0.05, n.s. p > 0.05. (B) Expression of Cas9 protein in BMDMs after treatment with CLANpM458, CLANpX458, and LipopM458. pM458 is a CD68 promoterdriven Cas9-T2A-EGFP expression plasmid; pX458 is a chicken β-actin promoter-driven Cas9-T2A-EGFP expression plasmid.

CLANpM458 at a plasmid dose of 0.5 nM was 25.2%, which was relatively lower than that of CLANpX458 (36.0%) and LipopM458 (41.4%) at the same dose. When transfected with CLANpM458 at a plasmid dose of 1 nM, the percentage of EGFP-positive cells rose to 35.9%. Furthermore, Cas9 protein expression was demonstrated by Western blotting (Figure 2B). After incubation for either 24 or 48 h, Cas9 expressions of CLANpM458 (at a plasmid dose of 1 nM) and CLANpX458 (at a plasmid dose of 0.5 nM) were comparable, slightly lower than that of LipopM458 (at a plasmid dose of 0.5 nM). These results were very consistent with the EGFP expression studies. Based on these results, we can conclude that the CLAN vectors could efficiently transfect pM458 into BMDMs to realize Cas9 and EGFP expression. Although the transcription efficacy of the CD68 promoter was lower than that of the chicken β-actin promoter in pX458, protein expression was improved by increasing the dose of CLANpM458. In Vitro Validation of Macrophage-Specific CD68 Promoter-Driven Cas9-EGFP Expression. We further validated whether the human CD68 promoter could drive the

properties of CLANpM458 and CLANpX458 were similar to that of CLANsiRNA. In Vitro Gene Transfection of Nanoparticles Encapsulating Plasmid. Since efficient Cas9 protein expression is a prerequisite for gene editing, we measured the expression of Cas9 from pM458 in bone-marrow-derived macrophages (BMDMs). To facilitate the assay, pX458 and pM458 both had a Cas9-T2A-EGFP expression cassette, which ensured the simultaneous expression of the EGFP and Cas9 proteins. The difference of pX458 and pM458 is that the EGFP and Cas9 proteins’ expression was driven by the chicken β-actin and CD68 promoter, respectively. After transfection with CLANpX458 or CLANpM458 for 6 h, the cells were further incubated in fresh medium for 24 or 48 h, and then the expression of the EGFP and Cas9 proteins was measured by fluorescence-activated cell sorting (FACS) and Western blotting. As shown in Figure 2A, BMDM transfected with CLANpM458, CLANpX458, and LipopM458 (Lipofectamine 2000 carrying pM458) all showed significant EGFP expression in BMDMs. The percentage of EGFP-positive cells for C

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Figure 3. Delivery of a CD68 promoter-driven CRISPR/Cas9 plasmid by CLAN vector (CLANpM458) enables the specific expression of Cas9 in macrophages in vitro. (A) Cellular uptake of CLANCy5‑siRNA by BMDMs, RAW264.7, 293T, and U87MG cells. Cy5-siRNA fluorescence distribution (left) and mean fluorescence intensity (MFI) of Cy5-siRNA (right). Data are shown as the means ± SD (n = 3), n.s. p > 0.05. (B) Confocal images of BMDMs and RAW264.7, 293T, and U87MG cells treated with CLANCy5‑siRNA (red). The cytoskeletons and nuclei were counterstained with Alexa Fluor 568 (green) and DAPI (blue), respectively. (C) Percentage of EGFP-positive BMDMs and RAW264.7, 293T, and U87MG cells after treatment with CLANpM458 or CLANpX458. Data are shown as the means ± SD (n = 3), ** p < 0.01. (D) Expression of Cas9 protein in BMDMs and RAW264.7, 293T, and U87MG cells after treatment with CLANpM458 or CLANpX458.

expression were measured separately by FACS and Western blotting. As shown in Figure 3C, the percentages of EGFPpositive BMDMs, RAW264.7, 293T, and U87MG cells after transfection with CLANpX458 were similar (24.9%, 31.6%, 31.3%, and 30.4%, respectively). In contrast, after treatment with CLANpM458, the percentages of EGFP-positive BMDMs and RAW264.7 cells were 17.8% and 22.1%, respectively, which were significantly higher than the percentages of EGFP-positive 293T and U87MG cells (1.3% and 1.5%, respectively). Cas9 protein expression, which was detected by Western blotting, exhibited a similar phenomenon. After treatment with CLANpM458, Cas9 expression in BMDMs and RAW264.7 cells was significantly higher than that in 293T, U87MG, 4T1, and B16 cells, while all four types of cells transfected with CLANpX458 showed similar Cas9 expression (Figure 3D and Figure S4B). These results suggested that the CD68 promoter was able to drive the specific expression of Cas9 and EGFP in macrophages (BMDMs and RAW264.7 cells), which supported the hypothesis of macrophage-specific gene editing using CD68 promoter-driven Cas9 expression plasmids. In Vivo Validation of Macrophage-Specific CD68 Promoter-Driven Cas9-EGFP Expression. Encouraged by the in vitro results, we next carried out in vivo animal experiments to confirm our hypothesis that the systemic

specific expression of Cas9 in macrophages. Six types of cells, including BMDMs, macrophage cell line RAW264.7, embryonic kidney cell line 293T, glioma cell line U87MG, mouse breast tumor cell line 4T1, and mouse melanoma cell line B16 were used for transfection assays. First, the cellular uptake of the CLAN vector was measured in the different cells, since the amount of intracellular plasmid significantly affected the expression levels of its encoded proteins. As described in Figure 1, CLAN siRNA possessed properties similar to CLANpM458 and CLANpX458; thus, it was used to replace CLANpM458 and CLANpX458 for the cellular uptake assays. As shown in Figure 3A and Figure S4A, after incubation with CLANCy5‑siRNA, the fluorescence intensities of Cy5-siRNA in BMDMs and RAW264.7, 293T, U87MG, 4T1, and B16 cells were similar. The cellular uptake of CLANCy5‑siRNA was further confirmed through confocal laser scanning microscopy (CLSM) (Figure 3B), which also indicated that the cellular uptake of the CLAN vector was comparable in the four different cell lines. Thus, cellular uptake differences of the CLAN vector by these cells could be excluded, and consequently, the promoter was the main reason for plasmid expression in the four types of cells. Subsequently, the aforementioned cell lines were incubated with CLANpM458 and CLANpX458, and then EGFP and Cas9 D

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Figure 4. Intravenous injection of CLANpM458 results in the specific expression of the EGFP and Cas9 proteins in macrophages and their precursor monocytes in vivo. Percentage of Cy5-positive cells (A) and MFI of Cy5 (B) in T cells, B cells, neutrophils, monocytes, and macrophages. (C) Percentages of EGFP-positive T cells, B cells, neutrophils, monocytes, and macrophages after the intravenous injection of CLANpM458. Data are shown as the means ± SD (n = 5), ** p < 0.01. (D) Cas9 protein expression in neutrophils and monocytes/ macrophages. Neutrophils and monocytes/macrophages were isolated from peripheral blood and spleen, liver, and adipose tissue of C57BL/6 mice 48 h after the injection of CLANpX458 and CLANpM458.

observed to exhibit the highest Cy5 fluorescence, followed by neutrophils and monocytes, while only slight fluorescence signals were detected in B cells and T cells (Figure 4B). From these results, we could conclude that the CLAN vector was more likely to be endocytosed by neutrophils, monocytes, and macrophages. Thereafter, we measured whether the CD68 promoter could drive the specific expression of Cas9 and EGFP in macrophages in vivo. CLANpM458 and the control formulation CLANpX458 were intravenously injected into C57BL/6 mice through the tail vein. The immune cells of peripheral blood, spleen, liver, and adipose tissue were isolated 48 h after injection, and the

administration of CLANpM458 could achieve macrophagespecific Cas9 and EGFP expression in vivo. To do so, we first measured the cellular uptake of the CLAN vector by different immune cells, including T cells, B cells, neutrophils, monocytes, and macrophages. After the intravenous injection of CLANCy5‑siRNA for 24 h, immune cells in peripheral blood and spleen, liver, and adipose tissue were isolated and then analyzed by FACS. As shown in Figure 4A, among these tissues, the percentages of Cy5-positive cells in macrophages, neutrophils, and monocytes were much higher than those of T cells and B cells. In addition, the mean fluorescence intensity (MFI) of Cy5 was further analyzed in these cells. Macrophages were clearly E

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Figure 5. Intravenous injection of CLANpM330/sgNtn1 specifically disrupts the Ntn1 gene in monocytes and macrophages in vivo. (A) Detection of Ntn1 gene disruption efficacy in BMDMs with T7EI cleavage assays after treatment with CLANpM330/sgNtn1, CLANpX330/sgNtn1, or LipopM330/sgNtn1 in vitro. Surveyor products cleaved by T7EI (left) and indels frequency (right) in the Ntn1 locus. Data are shown as the means ± SD (n = 3), **p < 0.01, n.s. p > 0.05. (B) The expression of the Netrin-1 protein in BMDMs after treatment with CLANpM330/sgNtn1, CLANpX330/sgNtn1, or LipopM330/sgNtn1. (C) Representative DNA sequences of modified Ntn1 loci after treatment with CLANpM330/sgNtn1. WT indicates the wild-type Ntn1 sequence. MT-1, -2, -3, and -4 indicate mutant Ntn1 sequences. The sgNtn1 targeting site is marked in red, and the PAM site is marked in green. (D) Detection of Ntn1 gene disruption efficacy in neutrophils and monocytes/macrophages with T7EI cleavage assays after the intravenous injection of CLANpM330/sgNtn1 or CLANpX330/sgNtn1. Surveyor products cleaved by T7EI (left) and indels frequency (right) in the Ntn1 locus. Data are shown as the means ± SD (n = 5), **p < 0.01.

percentage of EGFP-positive cells was analyzed by FACS. As shown in Figure 4C, after treatment with CLANpX458 at a plasmid dose of 1 mg/kg, the percentages of EGFP-positive cells in blood neutrophils (15.0%), monocytes (16.1%), and macrophages (20.1%) were comparable. In contrast, for the CLANpM458 group at the same plasmid dose, the percentages of EGFP-positive cells in blood macrophages and monocytes (precursors of macrophages) were 13.0% and 8.8%, respectively, while only 0.8% of blood neutrophils were EGFPpositive. In addition, when the plasmid dose of CLANpM458 was increased to 2 mg/kg, 20.9% of macrophages and 15.6% of monocytes exhibited EGFP expression, while the percentage of EGFP-expressing neutrophils was only 2.7%. Additionally, the percentage of EGFP-positive T cells and B cells was negligible. Similar results were observed in liver, spleen, and adipose tissue. Therefore, the CD68 promoter of pM458 was concluded

to be able to drive the specific expression of EGFP in macrophages and its precursor monocytes but not in other cells (like neutrophils). On the basis of the macrophage-specific expression of EGFP in vivo, we further detected the expression of Cas9 by Western blotting. After the intravenous injection of the aforementioned formulations for 48 h, the neutrophils, monocytes, and macrophages were purified by FACS from peripheral blood and spleen, liver, and adipose tissue. Cas9 expression in T cells and B cells was not detected due to inefficient uptake of nanomedicine by T cells and B cells (Figure 4A and B). The in vivo cellular uptake of CLAN vector by neutrophils, monocytes, and macrophages was comparable, but their Cas9 expression was completely different. As shown in Figure 4D, after the intravenous injection of CLANpx458 at a plasmid dose of 1 mg/ kg body weight, the efficient expression of Cas9 was clearly F

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Figure 6. Specific disruption of Ntn1 in macrophages by intravenous injection of CLANpM330/sgNtn1 is an effective treatment for T2D in vivo. (A, B) Glucose tolerance tests (A) and insulin tolerance tests (B) of T2D mice after treatment with glyburide, CLANpX330/sgNtn1, or CLANpM330/sgNtn1, as indicated. Data are shown as the means ± SD (n = 10), *p < 0.05, **p < 0.01. (C) Detection of the Ntn1 gene disruption efficacy in monocytes and macrophages via T7EI cleavage assays after treatment. Surveyor products cleaved by T7EI (left) and indels frequency (right) in Ntn1 loci. Data are shown as the means ± SD (n = 5), *p < 0.05. (D) Netrin-1 protein expression in adipose tissue was analyzed after treatment. (E) Representative DNA sequences of modified Ntn1 loci after treatment. WT indicates the wild-type Ntn1 sequence. MT-1, -2, -3, and -4 indicate mutant Ntn1 sequences. The sgNtn1 targeting site is marked in red, and the PAM site is marked in green. NCD indicates mice fed a normal chow diet as healthy controls.

been reported to be a potential therapeutic target in macrophages for the treatment of T2D. We constructed the new plasmid pM330/sgNtn1, which simultaneously expresses Cas9 under the control of the CD68 promoter and a single gRNA targeting the Ntn1 gene. To verify that Ntn1 gene disruption arose from genome modification, we used T7 endonuclease I (T7EI) assays to detect and quantify the frequency of Cas9-mediated genomic insertion/deletion mutations (indels). After transfection with CLANpM330/sgNtn1 in vitro, BMDMs were collected for Ntn1 gene disruption potency assays. As shown in Figure 5A, after incubation with CLANpM330/sgNtn1 at a plasmid dose of 0.5 nM, the frequency of indels in Ntn1 was 15.7%. When the dose of CLANpM330/sgNtn1 was increased to 1.0 nM, the indels frequency in Ntn1 rose to 32.7%, which was similar to that of CLANpX330/sgNtn1 (31.3%) and LipopM330/sgNtn1 (36.7%) at a plasmid dose of 0.5 nM. Furthermore, the expression of netrin-1 (encoded by Ntn1)

observed in the neutrophils, monocytes, and macrophages. In contrast, for CLANpM458 at the same plasmid dose, the neutrophils had no obvious Cas9 expression, while Cas9 was successfully expressed in monocytes and macrophages. Additionally, when the plasmid dose of CLANpM458 was increased to 2 mg/kg, the expression of Cas9 in monocytes and macrophages also increased, reaching a comparable level to 1 mg/kg of CLANpX458. However, the expression of Cas9 in neutrophils was still very low. These results demonstrated that the CD68 promoter ensured the specific expression of Cas9 and EGFP in monocytes and macrophages in vivo after intravenous injection of CLANpM458. Ntn1 Gene Disruption with Nanoparticles Encapsulating pM330/sgNtn1 (CLANpM330/sgNtn1). The macrophagespecific expression of Cas9 could achieve specific gene editing in vivo for disease treatment. To demonstrate the potential of this system, Ntn1 was chosen as a gene target because it has G

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Figure 7. Intravenous injection of CLANpM330/sgNtn1 significantly decreases netrin-1 expression and prevents macrophage retention in adipose tissue for efficient T2D therapy. (A) ELISA detection of Netrin-1 protein expression in the adipose tissue of T2D mice after treatment. (B, C) IL-6 (B) and TNF-α (C) inflammatory cytokine concentrations in serum after treatment. (D) Percentages of CD11b+F4/80+ macrophages in adipose tissue after treatment. Data are shown as the means ± SD (n = 10), *p < 0.05, **p < 0.01.

in monocytes and macrophages in vivo, which could potentially avoid off-target effects during T2D treatment. T2D Treatment with CLANpM330/sgNtn1. The overexpression of netrin-1 (encoded by Ntn1 in macrophages) is an important cause of T2D. The efficient and specific deletion of Ntn1 by CLANpM330/sgNtn1 could be an effective strategy for T2D treatment. Thus, we established a T2D mouse model by feeding C57BL/6 mice a high fat diet (HFD, 60 kcal % fat). Subsequently, the T2D mice were treated with CLANpM330/sgNtn1 at a plasmid dose of 1 or 2 mg/kg body weight via tail vein injection. Mice treated with glyburide (a small-molecule drug for T2D) or CLANpX330/sgNtn1 were used as positive controls. Mice fed a normal chow diet (NCD, 10 kcal % fat) were used as healthy controls. The therapeutic effects were first monitored through glucose tolerance tests (GTTs). As presented in Figure 6A, the healthy mice (NCD group) exhibited a rapid increase in blood glucose levels after intraperitoneal injection of glucose, followed by a gradual decrease to normoglycemia. In contrast, the blood glucose levels of the diabetic mice administered with free pM330/ sgNtn1 or CLANpM330/sgNC were much higher than those of the healthy mice, exhibiting a similar level in comparison to the phosphate-buffered saline (PBS) group. However, the diabetic mice treated with CLANpM330/sgNtn1 showed a delayed increase in blood glucose after glucose injection, followed by a rapid decline to a normal state within 120 min, especially at a dose of 2 mg/kg, making their glucose responsive profiles similar to those of the glyburide group (positive control). Notably, the glucose responsive profiles of the diabetic mice treated with

was detected by Western blotting. As presented in Figure 5B, CLANpM330/sgNtn1 significantly inhibited the expression of netrin-1 at both plasmid doses, and the inhibition efficacy of netrin-1 expression with CLANpM330/sgNtn1 transfection at a plasmid dose of 1.0 nM was similar to that of CLANpX330/sgNtn1 and LipopM330/sgNtn1 at a plasmid dose of 0.5 nM. Some representative mutant alleles harboring indels in the Ntn1 locus are presented in Figure 5C and Figure S5, furthering proving the sequence-specific gene editing of the CRISPR/Cas9 system. Collectively, the CLAN vector efficiently delivered pM330/ sgNtn1 into macrophages for Ntn1 gene disruption in vitro. To corroborate this specific Ntn1 disruption ability in vivo, CLANpM330/sgNtn1 was intravenously injected into C57BL/6 mice, and the neutrophils, monocytes, and macrophages were sorted from peripheral blood and spleen, liver, and adipose tissue by FACS. The in vivo Ntn1 disruption efficacy was also detected using T7EI assays. As shown in Figure 5D, treatment with CLANpM330/sgNtn1 resulted in the cleavage of Ntn1 in monocytes and macrophages but not neutrophils. The indels frequency in monocytes/macrophages was 10.1% (at a dose of 1 mg/kg) and 19.6% (at a dose of 2 mg/kg), which was significantly higher than that in neutrophils (1.2% at a dose of 1 mg/kg and 2.3% at a dose of 2 mg/kg). In contrast, the cleavage of Ntn1 was clearly demonstrated in neutrophils and monocytes/macrophages for CLANpX330/sgNtn1; the indels frequency in neutrophils and monocytes/macrophages reached 20.1% and 23.2%, respectively. These results verified that CLANpM330/sgNtn1 was able to specifically disrupt the Ntn1 gene H

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CONCLUSIONS In summary, we constructed macrophage-specific CRISPR/ Cas9 plasmids (pM330 and pM458) by replacing the original chicken β-actin promoter of pX330 and pX458 with the CD68 promoter. In addition, these CD68 promoter-driven CRISPR/ Cas9 plasmids could be encapsulated in a CLAN vector, resulting in the specific expression of Cas9 in macrophages and their precursor monocytes in vitro and in vivo. Furthermore, after encapsulation of the CRISPR/Cas9 plasmids encoding Cas9 and sgNtn1, the obtained CLANpM330/sgNtn1 could disrupt the Ntn1 gene in monocytes and macrophages but not in other cells (like neutrophils). After intravenous injection, CLANpM330/sgNtn1 improved the glucose tolerance and insulin sensitivity of T2D mice by inhibiting netrin-1 expression in macrophages and by subsequently reducing macrophage retention in adipose tissue. In addition, CLANpM330/sgNtn1 avoided off-target effects due to the CD68 promoter. This strategy provides a promising avenue for specific in vivo gene editing with the CRISPR/Cas9 system in targeted cells or tissues.

CLANpX330/sgNtn1 at a dose of 1 mg/kg were also comparable to those of the glyburide group, demonstrating efficient glucose tolerance. Insulin tolerance tests (ITTs) were further performed to detect the therapeutic efficacy of these formulations. As shown in Figure 6B, insulin sensitivity was remarkably improved by treatment with CLANpM330/sgNtn1 at a dose of 1 or 2 mg/kg (Figure 6B), and the insulin sensitivity of the diabetic mice treated with 2 mg/kg CLANpM330/sgNtn1 was similar to that of the glyburide and CLANpX330/sgNtn1 groups, demonstrating the therapeutic effectiveness of CLANpM330/sgNtn1 as a T2D therapy. To further confirm that the improvement of T2D symptoms observed with CLANpM330/sgNtn1 was indeed caused by Ntn1 gene disruption, macrophages were isolated for T7EI assays after completion of the experiment. As shown in Figure 6C, cleavage bands were visible after CLANpM330/sgNtn1 and CLANpX330/sgNtn1 treatment. The indels frequency of mice treated with CLANpM330/sgNtn1 at a dose of 1 mg/kg was 8.8%. When the plasmid dose of CLANpM330/sgNtn1 was increased to 2 mg/kg, the indels frequency increased to 18.1%, reaching a similar level to that of CLANpX330/sgNtn1 at a dose of 1 mg/kg (21.8%). Subsequently, the expression of netrin-1 was detected by Western blotting. As presented in Figure 6D, treatment with CLANpM330/sgNtn1 or CLANpX330/sgNtn1 resulted in the downregulation of netrin-1, which was in accordance with the results of the T7EI assays. Additionally, some representative mutant alleles harboring indels in the Ntn1 locus after CLANpM330/sgNtn1 treatment are presented in Figure 6E and Figure S6. Collectively, these results demonstrated that CLANpM330/sgNtn1 was effective in the treatment of T2D via specific Ntn1 disruption in macrophages. Cytokine Secretion, Adipose Macrophage Retention, and Off-Target Effect Evaluation after CLANpM330/sgNtn1 Treatment. Finally, we assessed cytokine secretion and adipose macrophage retention after treatment. Consistent with the above results, the production of netrin-1 in adipose tissue was significantly inhibited by CLANpM330/sgNtn1 treatment (Figure 7A). In addition, other macrophage-associated inflammatory cytokines (including IL-6 and TNF-α) were also dramatically downregulated by CLANpM330/sgNtn1 treatment (Figure 7B and C), with levels comparable to those of the glyburide-treated positive control group. As described above, netrin-1 promotes T2D by increasing macrophage retention in adipose tissue. We measured the percentage of CD11b+F4/80+ macrophages in adipose tissue by FACS. As shown in Figure 7D, treatment with CLANpM330/sgNtn1 led to significantly reduced percentages of CD11b+F4/80+ macrophages. These results confirmed that treatment with CLANpM330/sgNtn1 specifically disrupted Ntn1 in macrophages, which subsequently inhibited netrin-1 secretion and reduced macrophage retention in adipose tissue, resulting in efficient T2D therapy. Moreover, to demonstrate the specificity of such CD68 promoter-driven CRISPR/Cas9 gene editing, five potential offtarget sites of sgNtn1 predicted by the online CRISPR Design Tool (Table S2) were measured. The indels frequencies of five potential off-target sites were within 0.5% and were thus significantly lower than those caused by CLANpM330/sgNtn1 offsite targeting (Figure S7). These results demonstrated that a CLAN vector carrying a specific promoter-driven CRISPR/ Cas9 is capable of specific gene editing in target cells, indicating its great potential as a highly specific and safe strategy for disease treatment.

MATERIALS AND METHODS Materials. The polymer mPEG5K-b-PLGA11K (molar ratio: LA/GA = 75/25) and cationic lipid N,N-bis(2-hydroxyethyl)-N-methyl-N-(2cholesteryloxycarbonylaminoethyl) ammonium bromide (BHEMChol) were synthesized according to our previous reports. Human codon-optimized Cas9 and guide RNA expression plasmids pX330 and pX458 were purchased from Addgene (Cambridge, MA, USA). The only difference between pX330 and pX458 is that pX458 has a 2AEGFP tag. DH5α competent cells were purchased from Takara (Dalian, China). A NucleoBond Xtra Maxi endotoxin-free plasmid DNA purification kit was purchased from Macherey-Nagel (Düren, Germany). All of the primers used in this study were synthesized by General Biosystems (Anhui, China), and the sequences of these primers are shown in the Supporting Information. Fluorescently labeled Cy5-siRNA and unlabeled negative control siRNA were provided by Suzhou Ribo Life Science Co., Ltd. (Kunshan, China). The Cy5-siRNA or NC-siRNA is a scramble sequence (antisense strand, 5′-ACGUGACACGUUCGGAGAAdTdT-3′). Dulbecco’s modified Eagle’s medium (DMEM) was from Gibco BRL Life Technologies (Eggenstein, Germany). Fetal bovine serum (FBS) was from Shanghai ExCell Bio, Inc. (Shanghai, China). Lipofectamine 2000 transfection reagent was from Invitrogen Co. (Carlsbad, CA, USA), and it was used as suggested by the manufacturer. The flow cytometry antibodies (anti-CD3, anti-CD19, anti-CD11b, anti-F4/80, anti-Ly6C, and anti-Ly6G) were purchased from Biolegend (San Diego, CA, USA). The Cas9 antibody was from Abcam (Shanghai, China). Netrin-1 and β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). ELISA detection kits for TNF-α and IL-6 were purchased from RayBiotech (Norcross, GA, USA). The Netrin-1 ELISA detection kit was purchased from CUSABIO Life Science (MD, USA). Construction of Macrophage-Specific CRISPR/Cas9 Plasmids. A macrophage-specific promoter (the CD68 promoter) was first synthesized by General Biosystems (Anhui, China). The CD68 promoter was used to replace the original chicken β-actin promoter of pX330 and pX458 via the SnaBI and AgeI restriction enzymes. The two newly constructed CD68 promoter-driven plasmids were denoted pM330 and pM458, respectively. To generate the Ntn1 knockout plasmids, CRISPR/Cas9 target sites were designed using the online CRISPR Design Tool (http://tools.genome-engineering.org). One pair of 25-bp complementary oligonucleotides (Ntn1 F1:5′CACCGCACCACGCAGTAGCGCGCCG-3′; Ntn1 R1:5′AAACCGGCGCGCTACTGCGTGGTGC-3′) with a 20-bp target sequence was synthesized by General Biosystems (Anhui, China). Then, the complementary oligonucleotides were annealed to generate double-stranded DNA (dsDNA) with 4-bp overhangs, which was I

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BMDMs or RAW264.7, 293T, U87MG, 4T1, or B16 cells (2 × 105 cells/well) were seeded in six-well plates for 24 h. Then, the cells were incubated with CLANCy5‑siRNA at an siRNA dose of 50 nM. After incubation for 6 h, the cells were washed three times with cold PBS (0.01 M, pH 7.4), trypsinized, and collected for FACS analyses. For CLSM observation, BMDMs or RAW264.7, 293T, or U87MG cells (5 × 104 cells/well) were seeded on coverslips in 24-well plates for 24 h. After incubation for 6 h, the cells were treated with the formulations described above, and then the cytoskeletons and nuclei were counterstained with Alexa Fluor 568 (green) and DAPI (blue), respectively, according to the standard protocols provided by the suppliers. Then, the cellular uptake behavior was visualized under CLSM (CLSM 710, Carl Zeiss Inc., Jena, Germany). In Vitro Validation of Macrophage-Specific CD68 PromoterDriven Cas9-EGFP Expression. To confirm that the CD68 promoter could drive macrophage-specific Cas9 expression, BMDMs or RAW264.7, 293T, U87MG, 4T1, or B16 cells (2 × 105 cells/well) were seeded in six-well plates for 24 h and then incubated with CLANpM458 or CLANpX458 at a plasmid dose of 0.5 nM for 6 h. The medium was then replaced with fresh medium. After further incubation for 48 h at 37 °C, the cells were washed, trypsinized, and collected to determine the percentage of EGFP-positive cells and Cas9 protein expression as described above. In Vivo Cellular Uptake of CLANCy5‑siRNA by Different Immune Cells. Briefly, CLANCy5‑siRNA was intravenously injected into C57BL/6 mice (6−8 weeks). The dose of Cy5-siRNA was 1 mg/kg body weight. At 24 h postinjection, the peripheral blood, spleen, liver, and adipose tissue were harvested. The immune cells of peripheral blood were isolated with red blood cell lysis buffer (Stemcell Technologies, Vancouver, Canada) as instructed by the manufacturer. Spleen immune cells were isolated by filtering spleen through nylon-woolglass bead to get a single-cell suspension. Liver and adipose tissues were first digested with 2 mg/mL collagenase I/II for 45 min at 37 °C. Then the immune cells of liver and adipose tissue were purified with 40% Percoll (GE Healthcare, UK) as instructed by the manufacturer. Subsequently, the immune cells of peripheral blood, spleen, liver, and adipose tissue were resuspended in red blood cell lysis buffer for 5 min and centrifuged at 350g for 5 min to remove erythrocyte fragments. Finally, the immune cells were stained with flow cytometry antibodies at a concentration of 1 × 107 cells/mL in 100 μL for 30 min following the standard staining protocol from Biolegend. The surface markers of T cells, B cells, neutrophils, monocytes, and macrophages were CD3+, CD19+, CD11b+Ly6G+, CD11b+Ly6C+, and CD11b+F4/80+, respectively. The in vivo cellular uptake of CLANCy5‑siRNA by each kind of immune cells was measured by FACS. The percentage of Cy5-positive cells and MFI of Cy5 were analyzed with Flowjo 7.6.1 (BD Biosciences, Bedford, MA, USA). In Vivo Validation of Macrophage-Specific Expression of CD68 Promoter-Driven Cas9-EGFP. CLANpM458 was intravenously injected into C57BL/6 mice (6−8 weeks) at a plasmid dose of 1 or 2 mg/kg body weight. CLANpX458 at the plasmid dose of 1 mg/kg body weight was used as the positive control, while mice injected with PBS or free pM458 were used as negative control. Forty-eight hours postinjection, peripheral blood, spleen, liver, and adipose tissue were harvested. The immune cells of different tissues were isolated and stained with antibodies as described above. Finally, the percentage of EGFP-positive cells and Cas9 protein expression in different tissues were analyzed by FACS and Western blot assay as mentioned above. In Vitro Ntn1 Gene Disruption with CLANpM330/sgNtn1. Briefly, BMDMs (2 × 105 cells/well) were seeded in six-well plates for 24 h and then incubated with CLANpM330/sgNtn1 at a plasmid doses of 0.5 or 1 nM. The CLANpX330/sgNtn1 and Lipofectamine 2000 carrying pM330/sgNtn1 (LipopM330/sgNtn1) at a plasmid dose of 0.5 nM were used as positive controls, while free pM330/sgNtn1, empty CLAN, and CLAN encapsulating pM330 with scramble sgRNA sequence (CLANpM330/sgNC) were used as negative controls. After incubation for 6 h, the medium was replaced with fresh medium. Then the cells were collected by trypsinization after further incubation for 72 h. To detect Ntn1 gene disruption efficiency with the T7EI assay, genomic DNA was exacted by an AxyPrep multisource genomic DNA

ligated to BbsI-predigested pM330 or pX330 to generate sgRNAtargeting Ntn1 expression plasmids. The obtained plasmids were denoted pM330/sgNtn1 and pX330/sgNtn1, respectively. Preparation and Characterization of CLAN Vector Encapsulating Plasmid or siRNA. First, mPEG5K-b-PLGA11K (25 mg in 400 μL of chloroform), BHEM-Chol (2 mg in 100 μL of chloroform), and plasmid or siRNA (200 mg in 25 mL of DNase/RNase free water) were emulsified in a 50 mL centrifuge tube for 1 min at 80 W over an ice bath using a Vibra-Cell VCX130 (Sonics & Materials, Inc., Newtown, CT, USA). Subsequently, 5 mL of DNase/RNase free water was added to the primary emulsion and further emulsified for 1 min at 80 W. Then, chloroform was removed via rotary evaporation at room temperature using a Rotavapor R-3 from Buchi Co. (New Castle, DE, USA). The siRNA, pM330, and pM458-encapsulated CLAN vectors were denoted CLANsiRNA, CLANpM330, and CLANpM458, respectively. The diameters and zeta potentials of the nanoparticles were determined by a dynamic light scattering (DLS) method using a Malvern Zetasizer Nano ZS90 (Worcestershire, UK). The morphology of the nanoparticles was examined using an FEI Tecnai cryo-TEM (Hillsboro, OR, USA) at an accelerating voltage of 200 kV after freezedrying. The encapsulation efficiency of each plasmid was measured via PicoGreen assays according to the manufacturer’s instruction. The encapsulation efficiency of siRNA was measured by high-performance liquid chromatography (HPLC) analysis as previously reported. Cell Culture. Primary bone-marrow-derived macrophages were prepared by flushing the bone marrow of the tibia and femur of 6- to 8-week-old C57BL/6 mice, followed by culturing in DMEM supplemented with 10% FBS. Mouse macrophage cell line RAW264.7, human embryonic kidney cell line 293T, human glioma cell line U87MG, mouse breast tumor cell line 4T1, and mouse melanoma cell line B16 were obtained from the American Type Culture Collection (ATCC). All cell lines were cultured in DMEM supplemented with 10% FBS at 37 °C with 5% CO2. Animals and the High-Fat-Diet-Induced T2D Model. C57BL/ 6 (6−8 weeks old) mice were purchased from Beijing HFK Bioscience Co., LTD (Beijing, China). All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. All procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. C57BL/6 mice were placed on a high fat diet (D12492, Research Diets, New Brunswick, NJ, USA) with 60 kcal % fat for 12 weeks to induce T2D. Mice placed on a normal chow diet (D12450, Research Diets) with 10 kcal % fat were used as healthy controls. The body weight of each mouse was recorded weekly. In Vitro Gene Transfection with CLANpM458. The pM458 plasmid, which simultaneously expresses Cas9 and EGFP under the control of the CD68 promoter, was used to determine the plasmid transfection efficiency of the CLAN vector, and the pX458 plasmid with the chicken β-actin promoter was used as a control. Briefly, BMDMs (2 × 105 cells/well) were seeded in six-well plates for 24 h, and the cells were then incubated with CLANpM458 at plasmid doses of 0.5 or 1 nM. CLANpX458 and Lipofectamine 2000 carrying pM458 (LipopM458) at a plasmid dose of 0.5 nM were used as positive controls; free pM458 and empty CLAN vector were used as negative controls. After incubation for 6 h, the medium was replaced with fresh medium and further incubated for 24 or 48 h. Subsequently, after further incubation for 48 h, the percentage of EGFP-positive cells was analyzed by a flow cytometric assay (FACS Calibur flow cytometer, BD Biosciences, USA), and after further incubation for 24 or 48 h, Cas9 protein expression was analyzed by Western blotting. A monoclonal antibody against Cas9 (Abcam, Shanghai, China) was used at a dilution of 1:1000, and goat anti-mouse IgG-HRP (1:5,000, EMD Millipore, USA) was used as the secondary antibody. The results were visualized using an ImageQuant LAS 4000 mini system (GE Healthcare, UK). In Vitro Cellular Uptake of CLANCy5‑siRNA by Different Cell Types. The cellular uptake of the CLAN vector by different cell types (BMDMs and the RAW264.7, 293T, U87MG, 4T1, and B16 cell lines) was analyzed. Cy5-siRNA was encapsulated into the CLAN vector (CLANCy5‑siRNA) to facilitate detection. For flow cytometric analysis, J

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ACS Nano miniprep kit according to the manufacturer’s instructions. Genomic regions flanking the target sites (486 bp) were amplified by the corresponding primers (Ntn1 F primer: 5′-CTTAGCATGTTCGCCGGCCAGGCGG-3′, Ntn1 R primer: 5′GTGCGGCCGGTTGTACATTTTGCGG-3′). Ntn1 gene disruption efficiency was detected with the T7EI assay with T7 endonuclease I (New England Biolabs, Beijing, China) following the protocol from New England Biolabs. Ntn1 gene disruption efficiency was calculated using the following formula: Indels frequency (%) = 100% × (1 − 1 − fraction cleaved ). The PCR products of the genomic region flanking the target sites were cloned into T-clone vector for DNA sequencing by General Biosystems (Anhui, China). To detect netrin-1 protein expression after Ntn1 gene disruption, total protein was extracted with RIPA buffer (Sigma, Saint Louis, MO, USA) and analyzed with Western blot. Monoclonal antibody against netrin-1 (Santa Cruz Biotechnology) was used at a dilution of 1:1000, and goat anti-rabbit IgG-HRP (1:5,000, EMD Millipore, USA) was used as the secondary antibody. The result was visualized using the ImageQuant LAS 4000 mini system (GE Healthcare, UK). In Vivo Ntn1 Gene Disruption with CLANpM330/sgNtn1. CLANpM330/sgNtn1 was intravenously injected into C57BL/6 mice (6−8 weeks) at a plasmid dose of 1 or 2 mg/kg body weight. Mice injected with CLANpX330/sgNtn1 at the plasmid dose of 1 mg/kg body weight were used as positive control, while PBS or free pM330/ sgNtn1 was used as negative control. At 48 h postinjection, peripheral blood, spleen, liver, and adipose tissue were harvested. Then, immune cells of different tissues were isolated and stained with antibodies as described above. Neutrophils (CD11b + Ly6G + ), monocytes (CD11b+Ly6C+), and macrophages (CD11b+F4/80+) were sorted by FACSAria II (BD Biosciences). Genomic DNA of neutrophils or monocytes/macrophages was exacted by an AxyPrep multisource genomic DNA miniprep kit according to the manufacturer’s instructions. Ntn1 gene disruption efficiency in neutrohils or monocytes/macrophages was detected with the T7EI assay as described above. T2D Treatment with CLANpM330/sgNtn1. HFD-induced T2D mice were established as described above and randomly divided into seven groups (n = 10). CLANpM330/sgNtn1 was intravenously injected once every other day for 2 weeks at a plasmid doses of 1 or 2 mg/kg body weight. Mice injected with CLANpX330/sgNtn1 once every other day for 2 weeks at a plasmid dose of 1 mg/kg body weight were used as positive control, while mice injected with PBS, free pM330/sgNtn1, or CLANpM330/sgNC were used as negative control. Besides, glyburide was intraperitoneally injected daily for 30 days at a dose of 500 mg/kg body weight to evaluate the therapeutic efficacy of CLANpM330/sgNtn1. Mice placed on a normal chow diet of the same age were used as healthy control. Glucose tolerance and insulin tolerance tests were performed once a week. The final GTTs and ITTs were performed on the 30th day after the first injection of CLANpM330/sgNtn1. For the GTT, mice were injected intraperitoneally with glucose at a dose of 1.5 g/kg body weight after fasting for 8 h. For the ITT, mice were injected with 1.5 IU/kg body weight of recombinant human insulin (Gibco, Eggenstein, Germany) after fasting for 6 h. Blood samples were obtained at different time points (0, 15, 30, 60, 120 min) for glucose measurements by using a glucosemeter (Roche, Basel, Switzerland). Detecting Ntn1 Gene Disruption Efficacy after CLANpM330/sgNtn1 Treatment. Peripheral blood, spleen, liver, and adipose tissue were harvested after the final GTT and ITT measurements. As described above, monocytes/macrophages were isolated and sorted by FACS. Genomic DNA of monocytes/ macrophages was exacted for DNA sequencing and T7EI assay to detect gene disruption efficacy in Ntn1. To examine potential offtarget effects of CLANpM330/sgNtn1, we identified five potential off-target sites in the mouse genome. The potential off-target sites predicted by the online CRISPR Design Tool (http://tools.genome-engineering. org) and corresponding primers are listed in Table S2 and Table S3. Genomic regions flanking the off-target sites were amplified by PCR and used for off-target effects detection.

The expression of netrin-1 in adipose tissue was measured by Western blot and ELISA. Briefly, the total protein of adipose tissue was extracted with RIPA buffer (Sigma). The Western blot assay was performed with netrin-1 monoclonal antibody as described above. Quantitative netrin-1 expression was measured by netrin-1 ELISA detection kit (CUSABIO Life Science) according to the manufacturer’s instructions. To measure IL-6 and TNF-α expression in blood, serum was isolated from the peripheral blood samples by centrifugation. IL-6 and TNF-α expression were measured by ELISA detection kits (RayBiotech) as instructed by the manufacturer. To measure percentage of macrophages (CD11b+F4/80+) in adipose tissue, immune cells of adipose tissue were isolated and stained with antibodies as described above. Then, stained immune cells were analyzed by a FACSCalibur flow cytometer (BD Biosciences). The percentage of CD11b+F4/80+ macrophages in adipose tissue was calculated by Flowjo 7.6.1 (BD Biosciences). Statistical Analysis. All data are shown as means ± standard deviations (SD). Statistical analysis was performed by one-way ANOVA. Significant differences between groups were indicated by *p < 0.05 and **p < 0.01, respectively. p < 0.05 was considered statistically significant in all analyses (95% confidence level).

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b07874. Figures S1−S7 and Tables S1−S3 (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected] (X. Z. Yang). *E-mail: [email protected] (J. Wang). ORCID

Hong-Jun Li: 0000-0002-1765-8445 Xian-Zhu Yang: 0000-0002-1006-0950 Zhen Gu: 0000-0003-2947-4456 Jun Wang: 0000-0001-9957-9208 Author Contributions ¶

Y. L. Luo and C. F. Xu contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Key R&D Program of China (2017YFA0205602), the National Natural Science Foundation of China (51633008, 51390482, and 51728301), the National Basic Research Program of China (2015CB932100), and the 111 Project (B17018). REFERENCES (1) Cong, L.; Ran, F. A.; Cox, D.; Lin, S. L.; Barretto, R.; Habib, N.; Hsu, P. D.; Wu, X. B.; Jiang, W. Y.; Marraffini, L. A.; Zhang, F. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 2013, 339, 819−823. (2) Ran, F. A.; Hsu, P. D.; Wright, J.; Agarwala, V.; Scott, D. A.; Zhang, F. Genome Engineering Using the CRISPR-Cas9 System. Nat. Protoc. 2013, 8, 2281−2308. (3) Sander, J. D.; Joung, J. K. CRISPR-Cas Systems for Editing, Regulating and Targeting Genomes. Nat. Biotechnol. 2014, 32, 347− 355. (4) Pelletier, S.; Gingras, S.; Green, D. R. Mouse Genome Engineering via CRISPR-Cas9 for Study of Immune Function. Immunity 2015, 42, 18−27. K

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DOI: 10.1021/acsnano.7b07874 ACS Nano XXXX, XXX, XXX−XXX