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Transfer RNA Synthesis

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One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

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Harnessing tRNA for Processing Ability and Promoter Activity.

David J H F Knapp¹, Tudor A Fulga²

¹Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK. david.knapp@umontreal.ca.

Methods in Molecular Biology (Clifton, N.J.)

|September 14, 2020

Summary

This summary is machine-generated.

Transfer RNAs (tRNAs) can be engineered to produce guide RNAs (gRNAs) for genome engineering, allowing adjustable expression levels and spatial-temporal control. This study details tRNA scaffolds for versatile gRNA production in Cas9-based systems.

Keywords:

CRISPR Cas9 Genome editing Guide RNA RNA Polymerase-II Tissue-specific tRNA

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Area of Science:

Molecular Biology
Biotechnology
Genome Engineering

Background:

Transfer RNAs (tRNAs) and their processing machinery offer a novel platform for producing functional RNAs.
Guide RNAs (gRNAs) are essential for Cas9-based genome editing, but their expression control remains a challenge.

Purpose of the Study:

To design and validate tRNA scaffolds for tunable production of gRNAs.
To enable spatial and temporal control over gRNA expression using engineered tRNAs.

Main Methods:

Utilizing tRNA variants for Pol-III driven expression of gRNAs at various steady-state levels.
Engineering tRNAs to process gRNAs from Pol-II transcripts for controlled expression.
Cloning and testing of designed tRNA scaffolds.

Main Results:

Demonstrated successful production of gRNAs using tRNA scaffolds.
Showcased the ability to modulate gRNA levels by selecting different tRNA variants.
Validated spatial/temporal control of gRNA expression through tRNA-mediated processing of Pol-II transcripts.

Conclusions:

Engineered tRNA scaffolds provide a versatile and controllable method for gRNA production in genome engineering.
This approach enhances the toolkit for Cas9-based gene editing by offering tunable and regulated gRNA expression.