Heritable gene editing using FT mobile guide RNAs and DNA viruses

Abstract

Background: The virus-induced genome editing (VIGE) system can be used to quickly identify gene functions and generate knock-out libraries as an alternative to the virus-induced gene silencing (VIGS). Although plant virus-mediated VIGE has been shown to have great application prospects, edited genes cannot be transferred to the next generations using this system, as viruses cannot enter into shoot apical meristem (SAM) in plants. Results: We developed a novel cotton leaf crumple virus (CLCrV)-mediated VIGE system designed to target BRI1, GL2, PDS genes, and GUS transgene in A. thaliana by transforming Cas9 overexpression (Cas9-OE) A. thaliana. Given the deficiency of the VIGE system, ProYao::Cas9 and Pro35S::Cas9 A. thaliana were transformed by fusing 102 bp FT mRNAs with sgRNAs so as to explore the function of Flowering Locus T (FT) gene in delivering sgRNAs into SAM, thus avoiding tissue culture and stably acquiring heritable mutant offspring. Our results showed that sgRNAs fused with FT mRNA at the 5′ end (FT strategy) effectively enabled gene editing in infected plants and allowed the acquisition of mutations heritable by the next generation, with an efficiency of 4.35–8.79%. In addition, gene-edited offspring by FT-sgRNAs did not contain any components of the CLCrV genome. Conclusions: FT strategy can be used to acquire heritable mutant offspring avoiding tissue culture and stable transformation based on the CLCrV-mediated VIGE system in A. thaliana.