Abstract
BackgroundA key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing.ResultsTo develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected.ConclusionsWith the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.
Highlights
A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs and Cas9, into plants
Since Apple latent spherical virus (ALSV)-RNA2 encodes a polyprotein in a single open reading frame, to avoid disrupting its translation, the downstream of the translation stop codon of the Vp24 protein in ALSVRNA2 was chosen for insertion of guide RNAs (gRNAs) in the Csy4-processed ALSV Carry (CCAC) system
Since the sequences in CCAC have been modified from natural ALSV viral RNAs, which may lead the virus to lose its biological activity, we first tested the ability of the plant to be infected by virus-induced gene silencing (VIGS)
Summary
A key issue for implementation of CRISPR-Cas genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas, into plants. The Cas and gRNA are cloned into a single or two separate binary vectors and subsequently transferred into plant cells usually mediated by Agrobacterium (Feng et al [12]). One disadvantage of this method is that each binary construct is target-specific, and if the target is changed a new construct. Efficient transformation has not been developed for many plant species These inhibit CRISPR/ Cas based genome editing application in many crops, such as soybean
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