Hit and go CAS9 delivered through a lentiviral based self-limiting circuit
Open accessAbstract
In vivo application of the CRISPR-Cas9 technology is still limited by unwanted Cas9 genomic cleavages. Long-term expression of Cas9 increases the number of genomic loci non-specifically cleaved by the nuclease. Here we develop a Self-Limiting Cas9 circuit for Enhanced Safety and specificity (SLiCES) which consists of an expression unit for Streptococcus pyogenes Cas9 (SpCas9), a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self-limiting circuit results in increased genome editing specificity by controlling Cas9 levels. For its in vivo utilization, we next integrate SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Upon delivery into target cells, the lentiSLiCES circuit switches on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, our hit and go system increases safety margins for genome editing.
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In Caco-2 cells, long-term AsCas12a-crRNA+11 expression did not generate unspecific CFTR editing above TIDE background (~1%), whereas AsCas12a-crRNA+11/wt efficiently edited CFTR (86.3%).
“Long-term nuclease expression (10 days after transduction), which has been demonstrated to highly favor non-specific cleavages41, did not generate any unspecific CFTR editing above TIDE background levels (about 1%)38; whereas AsCas12a-crRNA+11/wt efficiently edited the CFTR gene (86.3%, Fig. 2b).”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [41]
Several methods have been described to limit continuous expression of lentiviral transgenes, such as natural promoter silencing and self-limiting CRISPR-Cas genetic circuits.
“Several methods have been described to limit continuous expression of lentiviral transgenes as natural promoter silencing and self-limiting CRISPR-Cas genetic circuits41,59,60.”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [41]
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- Authors
- Petris, G., Casini, A., Montagna, C., Lorenzin, F., Prandi, D., Romanel, A., Zasso, J., Conti, L., Demichelis, F., & Cereseto, A.
- Affiliations
- University of Trento (IT)
- Corresponding author
- Petris, Gianluca
- Cereseto, Anna
- ORCID
- Venue
- Nature Communications
- Published
- 2017-05-22
- Volume
- 8
- Issue
- 1
- Pages
- 15334-15334
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC5458126
- OpenAlex
- W2619894622
- Cited by
- 98
- References
- 14
- Citations by year
- 2026: 7 · 2025: 3 · 2024: 10 · 2023: 8 · 2022: 12 · 2021: 16 · 2020: 7 · 2019: 14 · 2018: 15 · 2017: 5 · 2013: 1
- FWCI
- 4.58
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Institutional Research Information System (University of Udine) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Iris (University of Trento) (submittedVersion, other-oa)
- Funders
- Università degli Studi di Trento
- H2020 European Research Council (648670)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- CRISPR/Cas9, slices, lentiviral delivery, SpCas9, genome editing specificity, off-target cleavage, in vivo gene editing, viral particle production
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