Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsFT 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.
Highlights
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)
Design of Cotton leaf crumple virus (CLCrV)‐mediated single guide RNAs (sgRNAs) delivery system The feasibility of CLCrV-mediated VIGE was analyzed in A. thaliana to determine whether the geminivirus CLCrV could be used to deliver sgRNAs for targeted genes in plants
The results showed that the Flowering Locus T (FT)-sgRNA strategy in A. thaliana could cause heritable mutations in the form of heterozygotes, while homozygotes could only be acquired in the M2 generation
Summary
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). Unlike first-generation genome editing tools, Type II CRISPR/Cas genome editing, which has been widely applied for crop improvement, involves simple designing and cloning methods [2,3,4,5,6]. During this process, single guide RNAs (sgRNAs) is used to guide the endonuclease Cas to cut a double-stranded. This process leads to DSBs repair through nonhomologous end-joining (NHEJ) or homologous recombination (HR). The application of CRISPR/Cas in plants may be challenging because the acquisition of heritable mutations via CRISPR/Cas usually requires a pathway of stable transformation, which is both time-consuming and laborious
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