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Efficient and reversible Cas13d-mediated knockdown with an all-in-one lentivirus-vector
- Source :
- Frontiers in Bioengineering and Biotechnology, Vol 10 (2022)
- Publication Year :
- 2022
- Publisher :
- Frontiers Media S.A., 2022.
-
Abstract
- Type VI CRISPR effector Cas13d from Ruminococcus flavefaciens XPD3002 (RfxCas13d) is an RNA-guided RNA endonuclease. RfxCas13d has been harnessed to knockdown gene expression with high specificity in various systems including mammalian cells. While inducible knockdown is advantageous over constitutive knockdown in many scenarios, current inducible systems of RfxCas13d express CRISPR RNA and Cas13d separately. Such systems could be cumbersome to handle and may hamper the application of RfxCas13d in some scenarios. Here, we design an all-in-one Cas13d lentivirus vector which renders efficient and inducible knockdown in a doxycycline dosage-dependent manner. Furthermore, we find that Cas13d has a short half-life in mammalian cells. As a result, knockdown can be promptly reversed after doxycycline withdrawal. This vector is particularly useful for applications involving indispensable genes and/or in cells hard to transduce.
- Subjects :
- Cas13d
lentivirus
CasRX
CRISPR
Tet-ON
SV40
Biotechnology
TP248.13-248.65
Subjects
Details
- Language :
- English
- ISSN :
- 22964185
- Volume :
- 10
- Database :
- Directory of Open Access Journals
- Journal :
- Frontiers in Bioengineering and Biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.5dc487132bac4a52b2b1d9c7d2b13421
- Document Type :
- article
- Full Text :
- https://doi.org/10.3389/fbioe.2022.960192