Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo
Open accessAbstract
Engineering of the Cpf1 crRNA has the potential to enhance its gene editing efficiency and non-viral delivery to cells. Here, we demonstrate that extending the length of its crRNA at the 5' end can enhance the gene editing efficiency of Cpf1 both in cells and in vivo. Extending the 5' end of the crRNA enhances the gene editing efficiency of the Cpf1 RNP to induce non-homologous end-joining and homology-directed repair using electroporation in cells. Additionally, chemical modifications on the extended 5' end of the crRNA result in enhanced serum stability. Also, extending the 5' end of the crRNA by 59 nucleotides increases the delivery efficiency of Cpf1 RNP in cells and in vivo cationic delivery vehicles including polymer nanoparticle. Thus, 5' extension and chemical modification of the Cpf1 crRNA is an effective method for enhancing the gene editing efficiency of Cpf1 and its delivery in vivo.
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Two weeks after intramuscular injection of PAsp(DET) polymer encapsulating 200 pmol Cpf1 and 200 pmol 5′ extended crRNA on Rosa26tdTomato mice, stop sequences of tdTomato were effectively deleted and robust tdTomato signal was observed.
“Two weeks after intramuscular injection of PAsp(DET) polymer encapsulating 200 pmol Cpf1 and 200 pmol 5′ extended crRNA on Rosa26tdTomato mice, the stop sequences of tdTomato were effectively deleted, and robust tdTomato signal was observed.[157]”
From RNA delivery biomaterials for the treatment of genetic and rare diseases (2019) · cites this paper as [157]
The 5′ extension of crRNA improved serum stability and enhanced nanoparticle formulation with PAsp(DET) polymer, resulting in higher in vivo delivery efficiency than wild type Cpf1 RNPs.
“The 5′ extension of crRNA improved the serum stability and enhanced the nanoparticle formulation with PAsp(DET) polymer, resulting in a higher in vivo delivery efficiency than wild type Cpf1 RNPs.”
From RNA delivery biomaterials for the treatment of genetic and rare diseases (2019) · cites this paper as [157]
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References 72 · Cited by 102
Research works printed but not linked: 4
- [21]Jinek M A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity Science 2012 337 816 822 10.1126/science.1225829 22745249 PMC6286148 · DOI · Paper page
- [29]Yang L RNA-guided human genome engineering via Cas9 Science 2013 339 823 826 10.1126/science.1232033 23287722 PMC3712628 · DOI · Paper page
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- Authors
- Park, H. M., Liu, H., Wu, J., Chong, A., Mackley, V., Fellmann, C., Rao, A., Jiang, F., Chu, H., Murthy, N., & Lee, K.
- Affiliations
- University of California, Berkeley (US)
- Corresponding author
- Murthy, Niren
- Lee, Kunwoo
- ORCID
- Venue
- Nature Communications
- Published
- 2018-08-17
- Volume
- 9
- Issue
- 1
- Pages
- 3313-3313
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC6098076
- OpenAlex
- W2887873381
- Cited by
- 102
- References
- 72
- Citations by year
- 2026: 10 · 2025: 8 · 2024: 13 · 2023: 9 · 2022: 15 · 2021: 12 · 2020: 17 · 2019: 15 · 2018: 2
- FWCI
- 4.09
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion, cc-by)
- Funders
- U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (K99GM118909)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR/Cas12a, genome editing efficiency, non-homologous end joining, homology-directed repair, serum stability, chemical modification, polymeric nanoparticles, in vivo gene delivery, electroporation
- MeSH terms
- Gene Editing, Animals, Bacterial Proteins, Base Sequence, Cations, Humans, Lipids, Polymers, Ribonucleoproteins, RNA, Bacterial, Mice, Nanoparticles, Hep G2 Cells, HEK293 Cells, DNA End-Joining Repair, CRISPR-Cas Systems
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