Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion
The CRISPR-Cas9 system is a powerful gene-editing tool with wide-ranging applications, but the safe and efficient intracellular delivery of CRISPR components remains a challenge. In this study, we utilized biodegradable poly(beta-amino ester) nanoparticles to codeliver plasmid DNA encoding Cas9 and...
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| Format: | Article |
| Language: | English |
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Elsevier
2020-06-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S216225312030113X |
| _version_ | 1818158323274350592 |
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| author | Yuan Rui Mahita Varanasi Shanelle Mendes Hannah M. Yamagata David R. Wilson Jordan J. Green |
| author_facet | Yuan Rui Mahita Varanasi Shanelle Mendes Hannah M. Yamagata David R. Wilson Jordan J. Green |
| author_sort | Yuan Rui |
| collection | DOAJ |
| description | The CRISPR-Cas9 system is a powerful gene-editing tool with wide-ranging applications, but the safe and efficient intracellular delivery of CRISPR components remains a challenge. In this study, we utilized biodegradable poly(beta-amino ester) nanoparticles to codeliver plasmid DNA encoding Cas9 and short guide RNA (sgRNA), respectively, to enable gene knockout following a CRISPR-mediated cleavage at one genomic site (1-cut edit), as well as gene deletion following DNA cleavage at two sites flanking a region of interest (2-cut edits). We designed a reporter system that allows for easy evaluation of both types of edits: gene knockout can be assessed by a decrease in near-infrared fluorescent protein (iRFP) fluorescence, whereas deletion of an expression stop cassette turns on a red-enhanced nanolantern fluorescence/luminescence dual reporter. Nanoparticles enabled up to 70% gene knockout due to small indels, as well as 45% gain-of-function expression after a 600-bp deletion edit. The efficiency of 2-cut edits is more sensitive than 1-cut edits to Cas9 and the sgRNA expression level. We demonstrate promising biodegradable nanoparticle formulations for gene editing. Our findings also provide new insights into the screening and transfection requirements for different types of gene edits, which are applicable for designing nonviral delivery systems for the CRISPR-Cas9 platform. |
| first_indexed | 2024-12-11T15:28:16Z |
| format | Article |
| id | doaj.art-b2baf4fd037649f9a165f0b0af84b132 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-12-11T15:28:16Z |
| publishDate | 2020-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-b2baf4fd037649f9a165f0b0af84b1322022-12-22T01:00:09ZengElsevierMolecular Therapy: Nucleic Acids2162-25312020-06-0120661672Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene DeletionYuan Rui0Mahita Varanasi1Shanelle Mendes2Hannah M. Yamagata3David R. Wilson4Jordan J. Green5Department of Biomedical Engineering, Institute for NanoBioTechnology, Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USADepartment of Biomedical Engineering, Institute for NanoBioTechnology, Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USADepartment of Biomedical Engineering, Institute for NanoBioTechnology, Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USADepartment of Biomedical Engineering, Institute for NanoBioTechnology, Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USADepartment of Biomedical Engineering, Institute for NanoBioTechnology, Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USADepartment of Biomedical Engineering, Institute for NanoBioTechnology, Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Departments of Ophthalmology, Oncology, Neurosurgery, Materials Science & Engineering, and Chemical & Biomolecular Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Bloomberg∼Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Corresponding author: Jordan J. Green, Departments of Ophthalmology, Oncology, Neurosurgery, Materials Science & Engineering, and Chemical & Biomolecular Engineering, Johns Hopkins University School of Medicine, 400 N. Broadway, Smith Building 5017, Baltimore, MD 21231, USA.The CRISPR-Cas9 system is a powerful gene-editing tool with wide-ranging applications, but the safe and efficient intracellular delivery of CRISPR components remains a challenge. In this study, we utilized biodegradable poly(beta-amino ester) nanoparticles to codeliver plasmid DNA encoding Cas9 and short guide RNA (sgRNA), respectively, to enable gene knockout following a CRISPR-mediated cleavage at one genomic site (1-cut edit), as well as gene deletion following DNA cleavage at two sites flanking a region of interest (2-cut edits). We designed a reporter system that allows for easy evaluation of both types of edits: gene knockout can be assessed by a decrease in near-infrared fluorescent protein (iRFP) fluorescence, whereas deletion of an expression stop cassette turns on a red-enhanced nanolantern fluorescence/luminescence dual reporter. Nanoparticles enabled up to 70% gene knockout due to small indels, as well as 45% gain-of-function expression after a 600-bp deletion edit. The efficiency of 2-cut edits is more sensitive than 1-cut edits to Cas9 and the sgRNA expression level. We demonstrate promising biodegradable nanoparticle formulations for gene editing. Our findings also provide new insights into the screening and transfection requirements for different types of gene edits, which are applicable for designing nonviral delivery systems for the CRISPR-Cas9 platform.http://www.sciencedirect.com/science/article/pii/S216225312030113Xpolymernanoparticlenon-viralCRISPR-Cas9gene editinggene delivery |
| spellingShingle | Yuan Rui Mahita Varanasi Shanelle Mendes Hannah M. Yamagata David R. Wilson Jordan J. Green Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion Molecular Therapy: Nucleic Acids polymer nanoparticle non-viral CRISPR-Cas9 gene editing gene delivery |
| title | Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion |
| title_full | Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion |
| title_fullStr | Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion |
| title_full_unstemmed | Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion |
| title_short | Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion |
| title_sort | poly beta amino ester nanoparticles enable nonviral delivery of crispr cas9 plasmids for gene knockout and gene deletion |
| topic | polymer nanoparticle non-viral CRISPR-Cas9 gene editing gene delivery |
| url | http://www.sciencedirect.com/science/article/pii/S216225312030113X |
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