Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Gene Editing Technologies

Background:

• Clinical application of site-directed RNA editing (SDRE) is hindered by the challenge of designing small, efficient guide RNAs (gRNAs).
• Existing gRNA designs often recruit Adenosine Deaminases that Act on RNA (ADARs) but exceed the size limits of FDA-approved antisense oligonucleotides.

Purpose of the Study:

• To develop an unbiased in vitro selection assay for identifying short gRNAs that promote efficient RNA editing.
• To overcome the size limitations of current gRNA designs for clinical translation of SDRE.

Main Methods:

• An in vitro selection assay using hairpin substrates linking target sequences to partially randomized gRNAs was developed.
• Substrates were incubated with ADAR2, and edited products were selected using amplification refractory mutation system PCR.
• A nine-round selection process was employed to identify superior editing gRNAs.

Main Results:

• After nine rounds of selection, hairpins driving superior RNA editing were identified.
• When delivered in trans, eight of the top ten identified short gRNAs demonstrated enhanced editing efficiency both in vitro and in cellula.

Conclusions:

• The developed selection approach successfully identifies efficient small gRNAs for RNA editing.
• This represents a significant advancement for the clinical application of site-directed RNA editing.