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Cited by 17RNA Interference and Gene DeliveryCRISPR and Genetic EngineeringNanoparticle-Based Drug Delivery

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A general genome editing strategy using CRISPR lipid nanoparticle spherical nucleic acids

Open access
Han, Zhenyu, Huang, Chi, Luo, Taokun, Mirkin, Chad A.·2025·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.2426094122PMC PMC12435252
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Abstract

Genome editing with CRISPR–Cas systems hold promise for treating a wide range of genetic disorders and cancers. However, efficient delivery of genome editors remains challenging due to the requirement for the simultaneous delivery or intracellular generation of Cas proteins, guide RNAs, and, in some applications, donor DNAs. Furthermore, the immunogenicity and toxicity of delivery vehicles can limit the safety and efficacy of genetic medicines. Here, we combine two nucleic acid delivery approaches to create CRISPR lipid nanoparticle–spherical nucleic acids (LNP–SNAs) that are both efficient and biocompatible. Compared to lipid nanoparticles (LNPs) lacking a surface-bound DNA shell, CRISPR LNP–SNAs exhibit two- to three-fold higher cellular uptake, reduced cytotoxicity, and improved gene transfection efficiency. Across multiple cell lines and genomic loci, CRISPR LNP–SNAs induce insertion–deletion mutations at average frequencies two- to three-fold higher than those observed with LNPs. When codelivered with donor templates, CRISPR LNP–SNAs enable homology-directed repair at an average efficiency of 21 ± 7%, a 2.5-fold improvement over LNPs (8 ± 4%). The ease of synthesis and biocompatibility of CRISPR LNP–SNAs highlight their potential as a versatile delivery platform for CRISPR–Cas and other gene therapies.

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Crossref · 10.1073/pnas.2426094122

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Figures

Fig. 1. Design of CRISPR lipid nanoparticle–spherical nucleic acids (LNP–SNAs). (A) Lipid components that assemble into LNPs. (B) Plasmid construct encoding a single-guide (sg)RNA and a spCas9 protein tagged with 3 × FLAG epitopes and nuclear localization signals (NLSs). (C) Synthetic scheme for CRISPR LNP–SNAs. (D) Scheme for CRISPR LNP–SNA cellular delivery and genome engineering.
Fig. 1. Design of CRISPR lipid nanoparticle–spherical nucleic acids (LNP–SNAs). (A) Lipid components that assemble into LNPs. (B) Plasmid construct encoding a single-guide (sg)RNA and a spCas9 protein tagged with 3 × FLAG epitopes and nuclear localization signals (NLSs). (C) Synthetic scheme for CRISPR LNP–SNAs. (D) Scheme for CRISPR LNP–SNA cellular delivery and genome engineering.CC BY-NC-ND · PubMed Central
Fig. 2. Characterization of CRISPR LNP–SNAs synthesized via the thiol-maleimide reaction. (A) Agarose (0.5%) gel electrophoresis showing the migration of Cy5-labeled 5′-thiol-DNA, CRISPR LNP–SNAs, and CRISPR LNP–SNAs ruptured using a surfactant (1% Triton X-100). (B) Hydrodynamic diameters of LNPs, CRISPR LNPs, and CRISPR LNP–SNAs measured using DLS. (C) Zeta potentials of LNPs, CRISPR LNPs, and CRISPR LNP–SNAs. The mean ± SD from three biological replicates is shown.
Fig. 2. Characterization of CRISPR LNP–SNAs synthesized via the thiol-maleimide reaction. (A) Agarose (0.5%) gel electrophoresis showing the migration of Cy5-labeled 5′-thiol-DNA, CRISPR LNP–SNAs, and CRISPR LNP–SNAs ruptured using a surfactant (1% Triton X-100). (B) Hydrodynamic diameters of LNPs, CRISPR LNPs, and CRISPR LNP–SNAs measured using DLS. (C) Zeta potentials of LNPs, CRISPR LNPs, and CRISPR LNP–SNAs. The mean ± SD from three biological replicates is shown.CC BY-NC-ND · PubMed Central
Fig. 3. Cellular uptake, biocompatibility, and functional delivery of LNP–SNAs (prepared using thiol-maleimide chemistry). (A) Flow cytometry analysis of rhodamine-labeled CRISPR LNPs and CRISPR LNP–SNAs incubated with HaCaT cells over a 4-h period. (B) Confocal imaging of rhodamine- and Cy5-labeled CRISPR LNP–SNAs in HaCaT cells. (C) Cell viability assays for CRISPR LNPs and CRISPR LNP–SNAs across four different cell lines at different particle concentrations (0 to 40 nM) after a 24-h incubation. (D) Flow cytometry analysis of EGFP fluorescence following the incubation of HaCaT cells with an EGFP plasmid (pEGFP), EGFP LNPs, and EGFP LNP–SNAs (with encapsulated pEGFP). (E) Quantification of protein expression levels, as determined by EGFP fluorescence intensity. The mean ± SD from three biological replicates is shown.
Fig. 3. Cellular uptake, biocompatibility, and functional delivery of LNP–SNAs (prepared using thiol-maleimide chemistry). (A) Flow cytometry analysis of rhodamine-labeled CRISPR LNPs and CRISPR LNP–SNAs incubated with HaCaT cells over a 4-h period. (B) Confocal imaging of rhodamine- and Cy5-labeled CRISPR LNP–SNAs in HaCaT cells. (C) Cell viability assays for CRISPR LNPs and CRISPR LNP–SNAs across four different cell lines at different particle concentrations (0 to 40 nM) after a 24-h incubation. (D) Flow cytometry analysis of EGFP fluorescence following the incubation of HaCaT cells with an EGFP plasmid (pEGFP), EGFP LNPs, and EGFP LNP–SNAs (with encapsulated pEGFP). (E) Quantification of protein expression levels, as determined by EGFP fluorescence intensity. The mean ± SD from three biological replicates is shown.CC BY-NC-ND · PubMed Central
Fig. 4. Genome engineering with CRISPR LNP–SNAs. (A) Scheme illustrating the gene editing strategies, including indel mutations and HDR-mediated gene modification with a repair template. (B) Indel frequencies at the GRIN2B target in hBMSCs and HaCaT cells. Cells were treated with CRISPR LNPs or LNP–SNAs conjugated via either DBCO–azide (Azide) or thiol-maleimide (Mal) chemistry, and indel rates were assessed by T7EI or sequencing (Seq). The mean ± SD across three to four biological replicates is shown. Statistical significance was determined by two-tailed Welch’s t test. (C) Indel frequencies at the GRIN2B target in HEK293T cells. Cells were treated with CRISPR LNPs or LNP–SNAs with DBCO–azide formulation, and indel rates were assessed by T7EI. The mean ± SD across three biological replicates is shown, and statistical significance was determined by two-tailed Welch’s t test. (D) Indel frequencies at the DNAse I and Grin2b targets using CRISPR LNPs or LNP–SNAs formulated via thiol-maleimide chemistry (in hBMSC and HaCaT cells) or DBCO–azide chemistry (in HEK293T and RAW 264.7 cells). Indel rates were assessed by T7EI. The mean ± SD across three to four biological replicates is shown, and statistical significance was determined by two-tailed Welch’s t test. (E) HDR efficiencies at the GRIN2B and DNAse I targets using lipofectamine, CRISPR LNPs, or LNP–SNAs formulated via DBCO–azide chemistry. HDR efficiencies were assessed by HindIII digestion. The mean ± SD across three biological replicates is shown, and statistical significance was determined by two-tailed Welch’s t test. (F) Surveyor assay in HEK293T cells targeting the EGFP site. (G) Confocal microscopy images of untreated and CRISPR LNP–SNA-treated cells, with arrows indicating cells whose fluorescence decreased following SNA treatment. (H) Flow cytometry histogram of HEK293T/EGFP cells treated with CRISPR LNPs or CRISPR LNP–SNAs.
Fig. 4. Genome engineering with CRISPR LNP–SNAs. (A) Scheme illustrating the gene editing strategies, including indel mutations and HDR-mediated gene modification with a repair template. (B) Indel frequencies at the GRIN2B target in hBMSCs and HaCaT cells. Cells were treated with CRISPR LNPs or LNP–SNAs conjugated via either DBCO–azide (Azide) or thiol-maleimide (Mal) chemistry, and indel rates were assessed by T7EI or sequencing (Seq). The mean ± SD across three to four biological replicates is shown. Statistical significance was determined by two-tailed Welch’s t test. (C) Indel frequencies at the GRIN2B target in HEK293T cells. Cells were treated with CRISPR LNPs or LNP–SNAs with DBCO–azide formulation, and indel rates were assessed by T7EI. The mean ± SD across three biological replicates is shown, and statistical significance was determined by two-tailed Welch’s t test. (D) Indel frequencies at the DNAse I and Grin2b targets using CRISPR LNPs or LNP–SNAs formulated via thiol-maleimide chemistry (in hBMSC and HaCaT cells) or DBCO–azide chemistry (in HEK293T and RAW 264.7 cells). Indel rates were assessed by T7EI. The mean ± SD across three to four biological replicates is shown, and statistical significance was determined by two-tailed Welch’s t test. (E) HDR efficiencies at the GRIN2B and DNAse I targets using lipofectamine, CRISPR LNPs, or LNP–SNAs formulated via DBCO–azide chemistry. HDR efficiencies were assessed by HindIII digestion. The mean ± SD across three biological replicates is shown, and statistical significance was determined by two-tailed Welch’s t test. (F) Surveyor assay in HEK293T cells targeting the EGFP site. (G) Confocal microscopy images of untreated and CRISPR LNP–SNA-treated cells, with arrows indicating cells whose fluorescence decreased following SNA treatment. (H) Flow cytometry histogram of HEK293T/EGFP cells treated with CRISPR LNPs or CRISPR LNP–SNAs.CC BY-NC-ND · PubMed Central

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Citations

Cites

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Metadata

Authors
Han, Z., Huang, C., Luo, T., & Mirkin, C. A.
Affiliations
Northwestern University (US)
Corresponding author
  • Han, Zhenyu
  • Huang, Chi
  • Mirkin, Chad A.
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2025-09-04
Volume
122
Issue
36
Pages
e2426094122-e2426094122
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.2426094122
PMC
PMC12435252
OpenAlex
W4413980850
Cited by
17
References
8
Citations by year
2026: 13 · 2025: 4
FWCI
5.11
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • DOD | AF | AMC | AFRL | Air Force Office of Scientific Research (FA9550-22-1-0300)
  • National Science Foundation (DMR-2428112, DMR-2308691, NSF ECCS-2025633)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
Keywords
CRISPR/Cas genome editing, lipid nanoparticles, spherical nucleic acids, nucleic acid delivery, gene therapy, homology-directed repair, insertion-deletion mutations, cellular uptake, biocompatibility
MeSH terms
Gene Editing, RNA, Guide, CRISPR-Cas Systems, Humans, Lipids, Liposomes, Nucleic Acids, Transfection, Nanoparticles, HEK293 Cells, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems

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