Efficient and reversible Cas13d-mediated knockdown with an all-in-one lentivirus-vector

Area of Science:

• Molecular Biology
• Gene Regulation
• Biotechnology

Background:

• Type VI CRISPR effector RfxCas13d mediates RNA-guided RNA knockdown.
• Existing inducible RfxCas13d systems require separate expression of CRISPR RNA and Cas13d.
• This presents handling challenges and limits applications.

Purpose of the Study:

• To engineer an all-in-one lentivirus vector for RfxCas13d-mediated inducible gene knockdown.
• To achieve doxycycline-inducible and dosage-dependent gene silencing.
• To enable rapid reversal of gene knockdown.

Main Methods:

• Design and construction of a single lentivirus vector encoding RfxCas13d and its guide RNA.
• Transduction of mammalian cells with the lentivirus vector.
• Induction and reversal of gene knockdown using doxycycline treatment and withdrawal.

Main Results:

• The all-in-one lentivirus vector enabled efficient and inducible gene knockdown in mammalian cells.
• Gene knockdown exhibited a doxycycline dosage-dependent response.
• Cas13d's short half-life facilitated prompt knockdown reversal upon doxycycline withdrawal.

Conclusions:

• The developed all-in-one RfxCas13d lentivirus vector provides a user-friendly and efficient system for inducible gene knockdown.
• This system is particularly valuable for applications involving essential genes or cells that are difficult to transduce.
• The rapid reversibility enhances its utility for dynamic gene expression studies.