Apr 10, 2020

Robust survival-based RNAi using in tandem silencing of adenine phosphoribosyltransferase

BioRxiv : the Preprint Server for Biology
R. G. OrrLuis Vidali

Abstract

RNA interference (RNAi) enables flexible and dynamic interrogation of entire gene families or essential genes without the need for exogenous proteins, unlike CRISPR-Cas technology. Unfortunately, isolation of plants undergoing potent gene silencing requires laborious design, visual screening, and physical separation for downstream characterization. Here, we developed a novel APT-based RNAi technology (APTi) in Physcomitrella patens that simultaneously improves upon the multiple limitations of current RNAi techniques. APTi exploits the pro-survival output of transiently silencing the APT gene in the presence of 2-fluoradenine, thereby establishing survival itself as a reporter of RNAi. To maximize silencing efficacy of gene targets we created vectors that facilitate insertion of any gene target sequence in tandem with the APT silencing motif. The APTi approach resulted in a homogenous population of P. patens mutants specific for our gene target, with zero surviving background plants within 8 days. The observed mutants directly corresponded to a maximal 93% reduction of the tested target protein, substantially exceeding previous dsRNA methods. The positive selection nature of APTi represents a fundamental improvement in RNAi tech...Continue Reading

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Mentioned in this Paper

PIFO gene
Anatomic Bifurcation
Disease Eradication
Anatomical Space Structure
Etiology
Leukemia, Experimental
Primary Malignant Neoplasm
Cancer Remission
Health Care Systems
Body Fluids

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