Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold.

Area of Science:

• Molecular Biology
• RNA Biology
• Biotechnology

Background:

• Precise regulation of RNA processing and metabolism is crucial for cellular integrity and function.
• CRISPR-Cas13 systems allow targeted RNA engineering, but simultaneous modulation of multiple RNA processing steps and off-target effects remain challenges.

Purpose of the Study:

• To develop a novel platform for simultaneous, multi-functional RNA modulation.
• To overcome limitations of existing RNA engineering tools, including off-target events.

Main Methods:

• Developed Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST) platform.
• Appended RNA scaffolds to Cas13 gRNA and fused cognate RNA binding proteins with enzymatic domains.
• Created bifunctional and tri-functional CREST systems for RNA alternative splicing and base editing (A-to-G, C-to-U).
• Utilized a split-design approach for enzyme reconstitution to minimize off-target events.

Main Results:

• Demonstrated simultaneous RNA manipulation of alternative splicing and base editing.
• Achieved significant reduction (nearly 99%) in off-target events using a split-enzyme design.
• Successfully reconstituted enzyme activity at target sites.

Conclusions:

• CREST platform enables simultaneous execution of multiple RNA modulation functions on different RNA targets.
• The split-design strategy effectively minimizes off-target effects, improving specificity.
• CREST expands the transcriptome engineering toolbox for RNA biology research.