DUSP11 is an RNA triphosphatase that limits PspCas13b activity by destabilizing gRNA abundance in mammalian cells

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• The CRISPR-Cas13 system offers programmable RNA targeting for research and therapeutics.
• PspCas13b shows high RNA knockdown after transient transfection but limited activity with stable lentiviral delivery.
• This limitation hinders the therapeutic and research utility of PspCas13b.

Purpose of the Study:

• To identify mammalian host factors that restrict PspCas13b activity.
• To understand the mechanism by which PspCas13b activity is suppressed.
• To develop strategies for enhancing PspCas13b efficacy in mammalian cells.

Main Methods:

• Conducted a genome-wide CRISPR-Cas9 knockout screen to find factors inhibiting PspCas13b.
• Investigated the role of identified factors, specifically DUSP11, in regulating guide RNA stability.
• Quantified guide RNA levels and PspCas13b-mediated knockdown efficiency in DUSP11 knockout cells.

Main Results:

• Discovered DUSP11, an RNA triphosphatase, suppresses PspCas13b by dephosphorylating and degrading guide RNAs (gRNAs).
• DUSP11 knockout increased gRNA levels 2.5-4 fold, significantly enhancing PspCas13b-mediated RNA knockdown.
• Enhanced activity was sustained for over 27 days and enabled targeting of previously refractory transcripts.

Conclusions:

• Host factor DUSP11 restricts bacterial CRISPR-Cas13 systems, with gRNA levels being a key limiting factor.
• DUSP11 knockout provides a simple strategy to improve PspCas13b activity in mammalian cells.
• Findings have implications for developing PspCas13b-based therapeutics and enhancing genome editing technologies.