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

Abstract

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).

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.

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).

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.

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.

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.