Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo
Open accessAbstract
Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.
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Cationic lipid-based delivery is a relatively easy, low-cost process to deliver CRISPR components into cells.
“Cationic lipid-based delivery is a relatively easy, low-cost process to deliver CRISPR components into cells 75 .”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [75]
The Introduction and Results report that cationic lipids deliver Cas9:sgRNA complexes into cultured human cells. The Online Methods describe a short preparation using commercially available lipid reagents, which supports the characterization as relatively easy. The paper does not establish that the process is low-cost. (LLM, conf=0.85)
Experimental — accuracy not yet measuredLNP is currently the most popular vector for in vivo gene editing.
“thus LNP is currently the most popular vector for in vivo gene editing”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Zuris et al., 2015
The Results section ('In vivo delivery of Cre recombinase and Cas9:sgRNA') describes cationic lipid-mediated delivery for gene editing in the mouse inner ear, and the Discussion calls protein delivery a viable approach to in vivo genome editing. Neither passage says that lipid nanoparticles are currently the most popular vector or compares their popularity with other vectors. (LLM, conf=0.86)
Experimental — accuracy not yet measuredConcerns regarding potential for DNA damage remain if Cas9 is present for an extended period of time.
“Moreover, concerns regarding potential for DNA damage remain if Cas9 is present for an extended period of time14.”
From Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo (2016) · cites this paper as [14]
Effective delivery of Cas9/sgRNA ribonucleoprotein complexes (RNPs) can be difficult due to the large size of Cas9 protein (~160 kD), the positive charge of Cas9 protein and the strong negative charge of sgRNA.
“However, due to the large size of Cas9 protein (~ 160 kD), the positive charge of Cas9 protein and the strong negative charge of sgRNA, the effective delivery of Cas9/sgRNA ribonucleoprotein complexes (RNPs) can be difficult [88–91].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [88]
First obstacle: effective encapsulation of editing tools in delivery vectors is hindered by large size and different charge properties of Cas9 protein, mRNA and donor DNA.
“The first obstacle is effective encapsulation of the editing tools in delivery vectors due to the large size and different charge properties of Cas9 protein, mRNA and donor DNA.”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [88]
Native Cas9 protein is positively charged, while conventional proteins are negatively charged.
“Meanwhile, native Cas9 protein is positively charged, while the conventional proteins are negatively charged [88, 97, 103, 139].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [88]
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“Prolonged expression of CRISPR components may lead to adverse effects and increased off-target activity [28,52–54].”
- Authors
- Zuris, J. A., Thompson, D. B., Shu, Y., Guilinger, J. P., Bessen, J. L., Hu, J. H., Maeder, M. L., Joung, J. K., Chen, Z.-Y., & Liu, D. R.
- Affiliations
- Howard Hughes Medical Institute (US)
- Massachusetts Eye and Ear Infirmary (US)
- Harvard University (US)
- Corresponding author
- Liu, David R
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2014-10-30
- Volume
- 33
- Issue
- 1
- Pages
- 73-80
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC4289409
- OpenAlex
- W2073217307
- Cited by
- 1,460
- References
- 50
- Citations by year
- 2026: 63 · 2025: 93 · 2024: 114 · 2023: 106 · 2022: 141 · 2021: 148 · 2020: 164 · 2019: 156 · 2018: 150 · 2017: 137 · 2016: 117 · 2015: 66 · 2014: 3 · 2012: 1
- FWCI
- 44.17
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- Funders
- Howard Hughes Medical Institute
- National Institute on Deafness and Other Communication Disorders (R01DC006908)
- National Institute of General Medical Sciences (F32 GM 106601-2, DP1GM105378, R01GM095501, F32GM106601)
- National Center for Chronic Disease Prevention and Health Promotion (DP1 GM105378)
- 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
- cationic lipid transfection, protein delivery, genome editing, CRISPR/Cas9, Cre recombinase, endosomal escape, serum tolerance, inner ear, hair cells, therapeutic proteins
- MeSH terms
- Cations, Lipids, Proteins, Transfection, Trans-Activators, In Vitro Techniques
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