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.

Non-GM Genome Editing Approaches in Crops

“…When transgenic N. benthamiana plants overexpressing Cas9 were infected with TRV vectors containing FT augmented gRNAs, up to 65% of the progeny contained CRISPR-generated mutations ( Ellison et al, 2020 ). Since the initial report, gRNA FT augmentation has been used with other dicotyledon viruses such as PVX and a DNA virus, Cotton leaf crumple virus, generating heritable edits of 22% in N. benthamiana and >4% in A. thaliana seedlings, respectively ( Lei et al, 2021 ; Uranga et al, 2021a ). This strategy has also been implemented in monocot crops such as wheat with surprising results.…”

CRISPR/Cas systems versus plant viruses: engineering plant immunity and beyond

“… Ellison et al (2020) modified the TRV-based sgRNA expression system ( Ali et al, 2015a ; Ellison et al, 2020 . Lei et al (2021) modified Cotton leaf crumple virus replicons by fusing an mRNA FT-translocation signaling motif ( Wigge, 2011 ) or tRNA ( Zhang et al, 2016 ) to the 3' end of sgRNA to enhance its translocation into germline cells ( Lei et al, 2021 ). Very recently, simple mechanical inoculation of PYX and negative-strand RNA viruses vectors were used to deliver sgRNAs for simultaneous multisite genome editing or the whole CRISPR/Cas machinery into plant tissue to increase (up to 90%) the recovery of foreign DNA free genome-edited plants ( Ariga et al, 2020 ; Ma et al, 2020 ; Uranga et al, 2021 ).…”

“… Ellison et al (2020) modified the TRV-based sgRNA expression system ( Ali et al, 2015a ; Ellison et al, 2020 . Lei et al (2021) modified Cotton leaf crumple virus replicons by fusing an mRNA FT-translocation signaling motif ( Wigge, 2011 ) or tRNA ( Zhang et al, 2016 ) to the 3' end of sgRNA to enhance its translocation into germline cells ( Lei et al, 2021 ).…”

CRISPR-Cas12a Genome Editing at the Whole-Plant Level Using Two Compatible RNA Virus Vectors

“…These strategies are commonly termed ''virus-induced genome editing'' (VIGE) and have focused on the delivery of one or more sgRNAs using RNA or DNA virus vectors in transgenic plants that stably express the Cas nuclease. [11][12][13][14][15][16][17][18][19] Expression of a Cas nuclease using a plant virusderived vector able to move systemically through a plant was long considered unachievable due to cargo constraints. However, the innovative work by Ma et al 20 demonstrated efficient genome editing by delivering both the sgRNA and SpCas9 at the whole-plant level using a vector derived from sonchus yellow net virus (SYNV; family Rhabdoviridae).…”