Harnessing tRNA for Processing Ability and Promoter Activity

David J H F Knapp; Tudor A Fulga

doi:10.1007/978-1-0716-0687-2_7

Harnessing tRNA for Processing Ability and Promoter Activity

Methods Mol Biol. 2021:2162:89-114. doi: 10.1007/978-1-0716-0687-2_7.

Authors

David J H F Knapp¹, Tudor A Fulga²

Affiliations

¹ Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK. david.knapp@umontreal.ca.
² Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

PMID: 32926380
DOI: 10.1007/978-1-0716-0687-2_7

Abstract

Transfer RNA (tRNA) and their associated production and processing machinery can be coopted as a versatile tool for the production of guide RNAs (gRNAs) for Cas9-based genome engineering. Using different tRNA variants enables the production of gRNAs at a variety of steady state levels. Furthermore, engineered tRNAs can be used to process gRNAs from Pol-II transcripts, thus enabling spatial/temporal control of gRNA expression. Here we describe the design, cloning, and testing of tRNA scaffolds for both Pol-III-driven expression of different levels of gRNAs, and for processing gRNAs from Pol-II transcripts.

Keywords: CRISPR; Cas9; Genome editing; Guide RNA; RNA Polymerase-II; Tissue-specific; tRNA.

MeSH terms

CRISPR-Cas Systems / genetics
Gene Editing
Molecular Biology / methods*
Promoter Regions, Genetic / genetics*
RNA Polymerase II / genetics
RNA Processing, Post-Transcriptional / genetics*
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Transfer / genetics*

Substances

RNA, Guide, CRISPR-Cas Systems
RNA, Transfer
RNA Polymerase II