Intrinsic targeting of host RNA by Cas13 constrains its utility

Area of Science:

• Molecular Biology
• Gene Editing
• RNA Biology

Background:

• Cas13 enzymes are RNA-guided nucleases with applications in RNA knockdown, editing, imaging, and therapy.
• Previous understanding of Cas13 function focused on its cis/trans RNA-cleavage activity guided by CRISPR RNA (crRNA).

Purpose of the Study:

• To investigate previously unappreciated mechanisms of Cas13 intrinsic targeting of host RNA in mammalian cells.
• To characterize the transcript-specific and conformation-dependent nature of Cas13-mediated host RNA cleavage.
• To evaluate the impact of intrinsic host RNA targeting on lentiviral delivery and expression of Cas13 systems.

Main Methods:

• RNA immunoprecipitation sequencing (RIP-seq)
• Transcriptome profiling
• Biochemical analysis
• High-throughput screening
• Machine learning
• Lentiviral systems (one-vector and two-vector)

Main Results:

• Cas13 exhibits intrinsic targeting of host RNA in mammalian cells via mechanisms distinct from canonical crRNA-guided cleavage.
• Host RNA cleavage by Cas13 is transcript-specific, crRNA-independent, and dynamically dependent on the Cas13 effector's conformational state.
• Host genes involved in viral processes, when targeted by Cas13, constrain lentiviral delivery and expression of Cas13 systems.
• Several Cas13-family effectors were analyzed within one-vector and two-vector lentiviral systems.

Conclusions:

• Cas13 possesses unappreciated intrinsic RNA targeting capabilities in mammalian cells.
• The findings necessitate caution regarding unintended host RNA targeting in Cas13-based applications, particularly those using lentiviral systems.
• Guidance is provided for the appropriate use of lentiviral Cas13 systems, emphasizing the need to account for intrinsic targeting effects.