Methods for in vitro CRISPR/CasRx-Mediated RNA Editing
Open accessAbstract
Specific changes in the genome have been accomplished by the revolutionary gene-editing tool known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. The advent of programmable RNA editing CRISPR/Cas nucleases has made this gene-editing tool safer and more precise. Specifically, CasRx, a family member of the Cas13d family, has shown great therapeutic potential. Here, we describe the in vitro methods of utilizing this powerful RNA editing platform and determine the RNA editing efficiencies for CasRx with different forms of guide RNAs (also known as gRNA or sgRNA).
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A previously described CasRx RNA editing system for ocular neovascularization was validated in vitro in HEK293FT and MIO-M1 cells, demonstrating more than 90% and 50% VEGFA mRNA knockdown, respectively.
“This approach was validated in vitro in HEK293FT and MIO-M1 cells, demonstrating more than 90% and 50% VEGFA mRNA knockdown, respectively (19).”
From Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina (2024) · cites this paper as [19]
The Results section (Figure 3; page unavailable) confirms in vitro VEGFA mRNA testing in HEK293FT and MIO-M1 cells. It reports 47 ± 4% and 68 ± 3% knockdown for the all-in-one constructs in HEK293FT cells, not more than 90%; it describes suppression in MIO-M1 cells without giving the claimed 50% figure. A separate HEK293FT experiment reports 90 ± 2% with a single pre-sgRNA, but that does not establish the stated pair of percentages. (LLM, conf=0.88)
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- Authors
- Chuang, Y.-F., Wang, P.-Y., Kumar, S., Lama, S., Lin, F.-L., & Liu, G.-S.
- Affiliations
- University of Tasmania (AU)
- Chinese Academy of Sciences (CN)
- Corresponding author
- Wang, Peng-Yuan
- Liu, Guei-Sheung
- Venue
- Frontiers in Cell and Developmental Biology
- Publisher
- Frontiers Media SA
- Published
- 2021-06-11
- Volume
- 9
- Pages
- 667879-667879
- ISSN
- 2296-634X (ISSN-L)
- PMC
- PMC8226256
- OpenAlex
- W3170941595
- Cited by
- 21
- References
- 29
- Citations by year
- 2026: 2 · 2025: 4 · 2024: 5 · 2023: 3 · 2022: 6 · 2021: 1
- FWCI
- 1.18
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Frontiers in Cell and Developmental Biology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- UTAS Research Repository (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Funders
- National Health and Medical Research Council (1185600)
- Shenzhen Key Laboratory Fund (ZDSYS20190902093409851)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- RNA editing, Cas13d, guide RNA, single guide RNA, genome editing
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