CRISPR-Cas12a delivery by DNA-mediated bioresponsive editing for cholesterol regulation

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Science Advances  20 May 2020:
Vol. 6, no. 21, eaba2983
DOI: 10.1126/sciadv.aba2983


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. When Pcsk9 was selected as the target gene because of its important role in regulating the level of serum cholesterol, efficient Pcsk9 disruption was observed in vivo (~48%), significantly reducing the expression of PCSK9 and gaining the therapeutic benefit of cholesterol control (~45% of cholesterol reduction).


The clustered regularly interspaced short palindromic repeats (CRISPR) system has become a powerful biotechnological tool that found a variety of applications in fundamental research and pharmaceutics (18). However, therapeutic translation of the CRISPR system is severely hampered by the availability of efficient delivery systems (911). Among the class II CRISPR system, Cas12a has distinct properties from Cas9. For example, Cas12a has ribonuclease (RNase) III activity that allows multiplexed gene editing with one single CRISPR RNA (crRNA) array (12). Cas12a requires one single crRNA for target recognition, which is shorter than the engineered single guide RNA (sgRNA) for Cas9 (13). Cas12a recognizes T-rich protospacer adjacent motif (PAM) region, which offers an alternative to the G-rich PAM targeting of Cas9 (14). In addition, Cas12a shows less off-targeting effects in human cells than Cas9 (15). From the perspective of formulation design, the smaller size of Cas12a makes it easier for vector construction and delivery (16).

Current development of delivery systems for CRISPR has been focused on the viral carriers, where the safety concerns with viral carriers call for the development of efficient nonviral delivery systems (1719). There have been a lot of efforts in developing nonviral delivery systems for CRISPR-Cas9 (2028); however, only a few reports demonstrated the delivery of the Cas12a/crRNA ribonucleoprotein (RNP), including the use of gold nanoparticle (29) and electroporation (30) for local therapy. Thus, a biocompatible and efficient delivery method that allows systemic delivery of Cas12a/crRNA RNP to the target tissues is desirable (31). Compared with the plasmid DNA-based delivery of CRISPR (32, 33), the delivery of CRISPR system in the format of RNP allows quick onset of the genome editing process and more stringent control of dosages and reduces the risk of off-target cleavage (34, 35).

Here, we demonstrate a layer-by-layer–based formulation for hepatocyte-targeted delivery of the Cas12a/crRNA RNP, aiming to reduce serum level of cholesterol. We used DNA as building blocks and prepared self-assembled DNA nanoclew (NC) as a core particle to load Cas12a/crRNA RNP (Fig. 1A). The Cas12a/crRNA RNPs were programmed to target enhanced green fluorescent protein (EGFP) in a model cell line or the Pcsk9 gene in mouse hepatocytes. Then, we coated the Cas12a/crRNA/NC with a cationic layer of polyethyleneimine (PEI) for condensing the negatively charged core and promoting endosome escape through “proton-sponge” effect. To allow systemic administration of the assembly, an anionic polymer layer was coated (Fig. 1B), which has a charge reversal behavior upon the exposure to acidic environment (anionic to cationic). Further conjugation of a hepatocyte-targeted ligand, galactose, to the charge reversal layer enables enhanced hepatocyte targeting after systemic administration. Overall, this DNA NC–based charge reversal formulation could circulate in the blood with high biocompatibility because of the negative charge at physiological pH. After binding to the asialoglycoprotein receptor (ASGP-R) on hepatocytes and getting internalized, the acidic endosomal environment can trigger charge conversion of assembly to facilitate endosome disruption, releasing the loaded Cas12a/crRNA cargo into the cytosolic compartment. After nuclear transportation of the RNP through the nuclear localization sequence (NLS) fused on Cas12a, the Cas12a/crRNA complex can cleave the target locus in the genome and introduce insertions/deletions (indels) for gene disruption.

Fig. 1 Design of the DNA NC–based charge reversal assembly for CRISPR-Cas12a delivery.

(A) Preparation of the assembly of Cas12a/crRNA/NC/PEI/Gal-PEI-DM. (I) The DNA NC was loaded with Cas12a/crRNA through complementation between DNA NC and crRNA. (II) A PEI layer was coated onto Cas12a/crRNA/NC to condense the assembly and to help induce endosome escape. (III) The Gal-PEI-DM layer was coated for turning the overall charge of the assembly into negative and for targeting hepatocyte with the galactose ligand. Delivery of Cas12a/crRNA by the DNA NC–based charge reversal assembly. (IV) The assembly was administered by intravenous injection. (V) The assembly bound to target hepatocytes through galactose-mediated targeting. (VI) The assembly was internalized through endocytosis, and the charge was reverted to positive in the acidic endosomal environment. (VII) PEI-mediated endosome escape. (VIII) The released Cas12a/crRNA RNP can be transported into the nucleus. (IX) Cas12a/crRNA RNP will search for the target genomic locus for gene editing. (B) Chemical structure of Gal-PEI-DM and mechanism for acidic environment triggered charge reversal.


Preparation of the assembly

To prepare the genome editing assembly, we chose Cas12a isolated from Lachnospiraceae as the cargo (13). Cas12a with a molecular weight of ~145 kDa was expressed in Escherichia coli and purified by the Ni–nitrilotriacetic acid (NTA)–based chromatography (fig. S1A). The crRNA was transcribed and purified in vitro (table S1), and it was later complexed with Cas12a to generate the Cas12a/crRNA RNP. By using EGFP as a model target, the double-stranded DNA cleavage activity of Cas12a/crRNA RNP was confirmed by an in vitro DNA cleavage assay, where a linearized EGFP-encoding plasmid DNA was used as the substrate (fig. S1B). EGFP disruption efficacy was then confirmed in a U2OS.EGFP reporter cell line (36). Using Cas9/sgRNA-EGFP from our previous study as control (37), comparable EGFP disruption efficacy could be observed for Cas12a/crRNA-EGFP (fig. S2).

To load the Cas12a/crRNA-EGFP, we prepared DNA NCs that complement the EGFP-binding region of crRNA-EGFP. The DNA NCs were generated by rolling circle amplification (RCA) (37, 38), and the RCA templates were designed with varying complementarity to the crRNA (table S1) to optimize the interaction between the carrier and the cargo. A sham DNA NC (NC-0) and DNA NCs with 6 base pairs (bp) (NC-6), 12 bp (NC-12), 17 bp (NC-17), and 27 bp (NC-27) of complementation to crRNA-EGFP were synthesized (fig. S1C). To prepare the assembly of Cas12a/crRNA-EGFP/NC, Cas12a was first complexed with crRNA-EGFP at a molar ratio of 1:1. NC-27 was used as the representative DNA NC for investigating the process of the assembly. Cas12a/crRNA-EGFP was loaded into the DNA NC-27 at a molar ratio of ~0.7:1 (Cas12a/crRNA RNP:repeating unit in the DNA NC, weight ratio at 4:1). The Cas12a/crRNA loading reduced the zeta potential of DNA NC from −19.8 ± 1.5 mV to −26.5 ± 2.5 mV and increased the mean hydrodynamic size from 131.4 ± 6.2 nm to 140.2 ± 4.9 nm.

Then, a layer of PEI was coated and the ratio of PEI to DNA NC was optimized (Fig. 2A). The weight ratio of PEI to NC-27 was varied between 0:1 and 3:1. When the ratio was increased from 0.5:1 to 1:1, an abrupt change of zeta potential was observed (−9.1 ± 2.3 mV to 17.2 ± 3.1 mV) and the size was reduced from 97.6 ± 4.3 nm to 66.9 ± 2.1 nm. The weight ratio of 1:1 for PEI to NC-27 was chosen for the next step coating of the charge reversal layer. The hepatocyte-targeted charge reversal layer was obtained by modifying PEI with galactose (Gal) and 2,3-dimethylmaleic anhydride (DM) following reported methods (39, 40). Preparation of charge reversal polymer Gal-PEI-DM was confirmed by Fourier transform infrared (FTIR) spectroscopy and 1H nuclear magnetic resonance (NMR) (fig. S3). The polymer Gal-PEI-DM is capable of targeting hepatocytes in vivo and changing its surface charge from negative to positive under an acidic condition. When coating Gal-PEI-DM onto Cas12a/crRNA-EGFP/NC-27, the weight ratio between Gal-PEI-DM and PEI was optimized between 0:1 and 3:1 (Fig. 2B). Increasing the ratio of Gal-PEI-DM to PEI converted the charge of the assembly from positive to negative. The size and charge of the assembly reached an equilibrium when the weight ratio between Gal-PEI-DM and PEI approached 1:1 (80.6 ± 5.1 nm and −25.1 ± 3.4 mV). Assembly of Cas12a/crRNA-EGFP/NC-27/PEI/Gal-PEI-DM was confirmed by confocal laser scanning microscopy (CLSM), where Cas12a was labeled with the infrared dye AF647, Gal-PEI-DM was conjugated with the green dye fluorescein isothiocyanate (FITC), and NC-27 was stained with the blue dye Hoechst 33342 (fig. S4A). Morphology of the assembly was further confirmed with transmission electron microscopy (Fig. 2C), which was consistent with its hydrodynamic size.

Fig. 2 Preparation and characterization of the assembly.

(A) Zeta potential and size changes for coating PEI onto Cas12a/crRNA/NC-27. Data represent mean ± SD (n = 3). (B) Zeta potential and size changes for coating Gal-PEI-DM onto Cas12a/crRNA/NC-27/PEI. Data represent mean ± SD (n = 3). (C) Hydrodynamic size distribution and transmission electron microscopy (TEM) imaging of Cas12a/crRNA/NC-27/PEI/Gal-PEI-DM. Scale bar, 100 nm. (D) Charge reversal profiles of the assembly in buffers with different pH. Data represent mean ± SD (n = 3).

In vitro charge reversal performance

For tissue-targeted delivery of gene editing therapeutics by systemic administration, it is desirable that the carrier remains negatively charged in physiological condition (pH 7.4) but shows positive charge once it reaches intracellular environment so that it will be able to escape from the endosome (pH 4.5 to 6.5). To mimic the process of systemic circulation and intracellular trafficking of the assembly, we used phosphate-buffered saline (PBS) at three pH gradients (pH 7.4, 6.5, and 5.5) to test the charge reversal behavior of the assembly. The negatively charged β-carboxylic amide group on Gal-PEI-DM is stable in alkaline condition, which can be hydrolyzed under acidic environment to generate secondary amines and recover the PEI structure. As shown in Fig. 2D, the assembly Cas12a/crRNA-EGFP/NC-27/PEI/Gal-PEI-DM was stable under pH 7.4, where it remained negatively charged after 24 hours of incubation. Generally, galactosylated nanoparticles administered through intravenous injection could reach hepatocytes in 1 hour (41, 42). Therefore, the stability of Cas12a/crRNA-EGFP/NC-27/PEI/Gal-PEI-DM at pH 7.4 is sufficient for efficient hepatocyte targeting. In contrast, the assembly recovered its positive charge under pH 5.5 within 1 hour of incubation, while those incubated under pH 6.5 started to recover their positive charge after 4 hours of incubation. The rapid charge recovery under acidic pH is favorable for an efficient escape from the endosome once the assemblies are internalized by the target cells. Hydrolysis of the β-carboxylic amide group on Gal-PEI-DM was further confirmed by 1H NMR (fig. S3B).

To evaluate the biocompatibility of Cas12a/crRNA-EGFP/NC-27/PEI/Gal-PEI-DM under physiological pH and its membrane-lytic activity in acidic environment, we preincubated the assembly in PBS buffers of different pH for 8 hours and then tested their hemolysis capability in vitro. As shown in Fig. 3A, Cas12a/cRNA-EGFP/PEI/Gal-PEI-DM pretreated in pH 5.5 showed ~20% hemolysis activity, and those pretreated in pH 6.5 showed ~7% hemolysis, while the assemblies incubated in pH 7.4 PBS did not cause significant hemolysis, confirming the biocompatibility and acid-triggered membrane-lytic activity of the charge reversal nanoparticle.

Fig. 3 In vitro characterization of the assembly.

(A) Hemolysis property of the charge reversal assembly Cas12a/crRNA-EGFP/NC27/EPI/Gal-PEI-DM. The assembly was preincubated in PBS of different pH to induce charge conversion. Data represent mean ± SD (n = 3). (B) Intracellular localization of the assembly and endosome escape in U2OS cells, characterized by CLSM imaging. Green, LysoTracker Green for the endo/lysosome; red, AF647-labeled Cas12a; blue, Hoechst-stained nuclei. Scale bar, 20 μm. (C) Quantitative analysis by flow cytometry of EGFP disruption. Dosages of the formulations were optimized in terms of Cas12a. Data represent mean ± SD (n = 3). (D) EGFP disruption in U2OS.EGFP cell line by Cas12a/crRNA-EGFP/NC/PEI/Gal-PEI-DM. DNA NCs with different complementarities were tested for the performance in delivering Cas12a/crRNA. Green, EGFP; blue, nuclei stained with Hoechst 33342. Scale bar, 50 μm. Representative images from fluorescent microscope and flow cytometry analysis of the gene disruption were shown for assemblies with Cas12a at 200 nM. (E) T7 endonuclease I (T7EI) assay of U2OS.EGFP cells treated with different EGFP-disrupting assemblies (Cas12a at 200 nM). (1) Untreated, (2) NC-27, (3) NC-17, (4) NC-12, (5) NC-6, (6) NC-0, (7) NC-free, (L) DNA ladder.

GFP disruption in vitro

To evaluate the performance of the assembly Cas12a/crRNA/NC/PEI/Gal-PEI-DM in vitro, we used U2OS cell lines for the investigation. We examined the intracellular trafficking profile of the assembly with an EGFP-free U2OS cell line that allows the tracking of the endo/lysosomes with LysoTracker Green (Fig. 3B and fig. S4B). After 2 hours of incubation, the assemblies reached intracellular compartment but most of them were visualized in the endo/lysosome. After 4 hours of incubation, it could be observed that the assemblies started to escape from the endo/lysosome and reach the nuclei. After 6 hours of incubation, the assemblies efficiently escaped from the endo/lysosome and accumulated in the nuclei.

After confirming the efficient endosome escape of the assembly, we studied the gene editing (EGFP disruption) efficacy of the assembly. We used U2OS.EGFP cell for this study, which is an engineered reporter cell line that has an integrated copy of destabilized EGFP in the genome, making it a suitable reporter for the genomic disruption of EGFP (36). The Cas12a/crRNA-EGFP RNP was designed to target the open reading frame (ORF) of EGFP, where successful cleavage of EGFP gene could introduce indels to the ORF of EGFP and cause the disruption of EGFP. To optimize the interaction between Cas12a/crRNA RNP and the DNA NC, we prepared various versions of the assembly, where the DNA NCs were programmed with different complementarity to crRNA-EGFP (NC-27, NC-17, NC-12, NC-6, and NC-0). Dosages of the assemblies were also optimized in terms of the concentration of Cas12a (25 to 200 nM). Quantitative analysis of EGFP disruption by flow cytometry (Fig. 3C) showed a Cas12a concentration–dependent EGFP disruption profile, where higher dosages of Cas12a/crRNA-EGFP lead to increased EGFP disruption. Furthermore, the complementation with the DNA NCs significantly affected the delivery efficacy of Cas12a/crRNA. NC-17–cored assembly showed the highest EGFP disruption efficiency, followed by NC-12 and NC-6. Considering the fact that the annealing temperature of RNA/DNA hybrids is mainly affected by the length of complementation, suggesting that the phenomenon of medium complementation between DNA NC and Cas12a/crRNA leads to better Cas12a/crRNA delivery might be caused by a balance of Cas12a/crRNA loading into and release from the DNA NC. High complementation (NC-27) might hamper the release of Cas12a/crRNA from the DNA NC, while low complementation (NC-0) might lead to inefficient loading of Cas12a/crRNA into the assembly.

Overall, assemblies that contained the DNA NC core, irrespective of the complementarity, showed enhanced performance compared to the formulation without the DNA NC core (NC free). Under the optimal condition where NC-17 was used in the Cas12a/crRNA-EGFP/NC-17/PEI/Gal-PEI/DM assembly and Cas12a was administered at 200 nM, approximately 82% of EGFP disruption was observed in U2OS.EGFP. Representative fluorescent microscopy and flow cytometry images were shown in Fig. 3D and fig. S5.

Under the same optimized condition, we used a control crRNA that does not target any gene in U2OS.EGFP and confirmed that the EGFP disruption was due to the specific targeting of crRNA-EGFP (fig. S6). To evaluate biocompatibility of the assembly, we stained the cells with a TO-PRO-3 live/dead stain. No significant cytotoxicity against U2OS.EGFP cells could be observed, indicating the high biocompatibility of the charge reversal assembly and confirming that the efficient EGFP disruption was not caused by cytotoxicity. To assess the formation of indels at the targeted genomic locus of U2OS.EGFP cells, we applied the T7 endonuclease I (T7EI) assay that can detect the mismatches between mutated and wild-type (WT) genetic sequences. As shown in Fig. 3E, efficient cut of the amplicons was detected that paralleled the analysis by flow cytometry.

Pcsk9 disruption in vitro

PCSK9 (proprotein convertase subtilisin/kexin type 9) is a liver-secreted protein that binds low-density lipoprotein (LDL) receptor, a key receptor that mediates endocytosis of cholesterol (43). Naturally occurring loss-of-function mutation of PCSK9 in some families significantly reduced their risk of cardiovascular disease (~88%) (44, 45), which is a leading cause of death in the United States (46). Furthermore, recent evidence also showed that molecules inhibiting PCSK9 are promising candidates for lowering cholesterol in the blood circulation (47, 48). The PCSK9 inhibitors alirocumab and evolocumab have received U.S. Food and Drug Administration (FDA) approval for controlling cholesterol, where the traditional statin therapy may not work satisfactorily (49).

For efficient Pcsk9 gene disruption, we optimized the crRNA selection by designing two crRNAs targeting exon2 (crRNA-exon2) and exon3 (crRNA-exon3) of Pcsk9. DNA NCs that have 17 bp of complementation to the crRNAs-exon2 and crRNA-exon3 were synthesized, generating NC-exon2 and NC-exon3. Following the formulation optimized for EGFP disruption, Cas12a/crRNA-exon2/NC-exon2/PEI/Gal-PEI-DM and Cas12a/crRNA-exon3/NC-exon3/PEI/Gal-PEI-DM were incubated with the model mouse cell line 3T3-L1. The assembly prepared with the control crRNA that does not target any locus of the genome (Cas12a/crRNA-control/NC-control/PEI/Gal-PEI-DM) was used as the control. With the T7EI assay, approximately 75% indel formation was detected with the crRNA-exon3–containing assembly, while the crRNA-exon2–containing assembly induced ~44% indels (Fig. 4, A and B, and fig. S7). Thus, crRNA-exon3 was chosen for the following studies.

Fig. 4 Disruption of Pcsk9 for serum cholesterol control.

(A) Schematic of the target site of Pcsk9 exon 3. (B) T7EI assay for the disruption of Pcsk9 exon 3 by (1) Cas12a/crRNA-control/NC-control/PEI/Gal-PEI-DM and (2) Cas12a/crRNA-exon3/NC-exon3/PEI/Gal-PEI-DM. (C) Ex vivo fluorescence imaging of the major organs collected from C57BL/6 mice. The mice were intravenously injected with (1) free Cas12a-Cy5.5/crRNA-exon3 and (2) Cas12a-Cy5.5/crRNA-exon3/NC-exon3/PEI/Gal-PEI-DM. (D) Region-of-interest analysis of fluorescence intensity of the Cas12a/crRNA accumulation in the major organs. Data represent mean ± SD (n = 3). ***P < 0.001. (E) T7EI assay of the on-target disruption of Pcsk9 exon 3. The cells were treated with (1) Cas12a/crRNA-control/NC-control/PEI/Gal-PEI-DM or (2) Cas12a/crRNA-exon3/NC-exon3/PEI/Gal-PEI-DM. (F) T7EI assay at three potential off-target sites. Lanes (1), (3), and (5) represent OT-1, OT-2, and OT-3 sites assayed from 3T3-L1 cells treated with PBS. Lanes (2), (4), and (6) represent OT-1, OT-2, and OT-3 sites assayed from 3T3-L1 cells treated with Cas12a/crRNA-exon3/NC-exon3/PEI/Gal-PEI-DM. Analysis of serum PCSK9 (G) and cholesterol levels (H) after the treatment. Data represent mean ± SD (n = 5). ***P < 0.001.

After confirming the efficacy of the assembly in inducing Pcsk9 disruption in vitro, we tested the performance of the nanoparticle in vivo. PCSK9 is mostly secreted from hepatocytes, making liver the organ of interest. In the design of the charge reversal layer, we have conjugated galactose as a hepatocyte-specific targeting ligand on the polymer. Galactose could bind to ASGP-R specifically overexpressed by hepatocytes (50), which enables rapid removal of galactose-terminated nanoparticles from the circulation (51). To demonstrate the liver-targeted accumulation of the delivered Cas12a/crRNA RNP, ex vivo imaging of fluorescently labeled nanoparticles was carried out. We labeled Cas12a with Cy5.5 (Cas12a-Cy5.5), free Cas12a-Cy5.5/crRNA-exon3 RNP and Cas12a-Cy5.5/crRNA-exon3/NC-exon3/PEI/Gal-PEI-DM were administered. One hour after the intravenous injection, the mice were sacrificed for analyzing the distribution of the nanoparticles by fluorescence-based in vivo imaging (IVIS). It can be observed that free Cas12a/crRNA has a much higher rate of renal clearance and more Cas12a/crRNA was delivered by the assembly to the liver after 1 hour of administration (Fig. 4C). Quantification of IVIS imaging by the region of interest analysis (Fig. 4D) showed that 2.6-fold more Cas12a/crRNA reached the liver when it was delivered by the assembly.

Pcsk9 disruption in vivo and therapeutic efficacy

Then, we investigated the in vivo Pcsk9 disruption efficacy of Cas12a/crRNA-exon3/NC-exon3/PEI/Gal-PEI-DM. PBS and Cas12a/crRNA-control/NC-control/PEI/Gal-PEI-DM were used as the controls. The assemblies were administered by intravenous injection into C57BL/6 mice every other day for three times. One week after the last drug administration, peripheral blood and livers were collected for analysis. T7EI assay identified around 53% disruption at the on-target site (Fig. 4E), while examination of the top three potential off-target sites (OT-1, OT-2, and OT-3) of crRNA-exon3 showed no detectable off-target cleavage (Fig. 4F). The serum levels of secreted PCSK9 protein were significantly reduced (~88%) when the mice were treated with the assemblies delivering crRNA-exon3, while the mice treated with the assemblies delivering crRNA-control did not show significant changes to serum PCSK9 levels (Fig. 4G). The augmented PCSK9 reduction in serum over the gene disruption efficacy has also been observed in other studies (52). It is possibly due to the increased expression of LDL receptor after Pcsk9 gene disruption, which facilitated the removal of PCSK9 from the serum (53). As a result of the reduced PCSK9 level, serum cholesterol level showed a ~45% reduction after the gene disruption (Fig. 4H). Then, we looked into the biocompatibility of the assemblies by monitoring liver damage–associated biomarkers. Examination of the levels of serum albumin and alanine aminotransferase (ALT) showed that the assemblies are highly biocompatible for delivering either crRNA-control or crRNA-exon3, indicating that neither the assembly nor the targeted gene disruption caused any observable hepatic toxicity (Fig. 5, A and B). Biocompatibility of the liver targeted assemblies was also evaluated by hematoxylin and eosin (H&E) staining of the liver sections. No observable differences between the PBS and assembly-treated groups could be detected (Fig. 5C and fig. S8), suggesting that the assemblies are biocompatible to the liver and other major organs. Deep sequencing of the on-target editing region showed an overall indel frequency of 48%, which is consistent with the T7EI assay. Mapping the deep sequencing results to WT sequence showed that most of the indels were deletions at the 3′ end of the protospacer sequence. Cas12a/crRNA-exon3 caused deletions around the target site, which confirmed the high specificity of the delivered RNP (Fig. 5D). Size of the deleted nucleotides varied widely and −19 deletion was detected to be the most frequent indel (Fig. 5E), and the representative deletions were shown in Fig. 5F.

Fig. 5 Analysis of in vivo Pcsk9 disruption.

Analysis of serum-related biomarkers for liver toxicity. (A) Albumin levels. (B) ALT levels. Data represent mean ± SD (n = 5). (C) Histological analysis of livers after the treatment by H&E staining. Scale bar, 100 μm. (D to F) Deep sequencing for targeted disruption of Pcsk9 exon 3. (D) Nucleotide deletion distribution around the cut site of Pcsk9 exon 3. (E) Distribution of the sizes of deleted nucleotide fragments. (F) Eight most common deletions detected by deep sequencing in the order of descending frequency. The mutated sequences were aligned to the WT sequence.


Here, we have developed a new nonviral carrier for targeted delivery of the CRISPR-Cas12a system for therapeutic genome editing. Our core-shell formulation based on the DNA NC core and the charge reversal polymeric shell demonstrates a highly biocompatible delivery system for CRISPR-Cas12a. The DNA NCs provided an anionic core with high charge density to load the Cas12a/crRNA RNP and to enable the layer-by-layer assembly of the cationic and charge reversal layers. Interactions between the DNA NC and Cas12a/crRNA RNP allowed efficient cargo loading into the carrier, and partial complementation led to enhanced gene disruption efficacy possibly through an optimized drug loading/release process. Coating of the cationic layer enabled endosome escape of the assembly, and further coating of a charge reversal layer allowed systemic administration of the assembly and boosted endosome escape when its charge was reverted to positive in the acidic endosomes. Using Pcsk9 as a model target, the Cas12a/crRNA delivering assembly showed efficient gene disruption both in vitro and in vivo, gaining therapeutic benefits by lowering cholesterol levels. Compared with the PCKS9 inhibitor or statin-based cholesterol therapies, Cas12a RNP–mediated Pcsk9 disruption could cause permanent PCSK9 reduction, generating sustained therapeutic efficacy. This delivery carrier provides a platform strategy that could be adapted to target other organs by applying appropriate targeting ligands. The crRNA could also be reprogrammed to target other genes of interest for treating different diseases, and the high multiplexity of CRISPR-Cas12a could make it convenient to target multiple genes simultaneously (12).



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 ( 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.

T7EI assay. 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.

Deep sequencing. 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).

Statistics. 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.


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Acknowledgments: Funding: This work was supported by grants from start-up packages of UCLA and UNC/NC State. A.K. was supported by the NIH (R01GM126571 and R01HL140951). Author contributions: W.S. and Z.G. designed the experiments. W.S., J.W., Q.H., and X.Z. performed experiments. W.S., J.W., Q.H., X.Z., A.K., and Z.G. analyzed the data and wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.
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