Exploiting viral vectors to deliver genome editing reagents in plants

Abstract

Genome editing holds great promise for the molecular breeding of plants, yet its application is hindered by the shortage of simple and effective means of delivering genome editing reagents into plants. Conventional plant transformation-based methods for delivery of genome editing reagents into plants often involve prolonged tissue culture, a labor-intensive and technically challenging process for many elite crop cultivars. In this review, we describe various virus-based methods that have been employed to deliver genome editing reagents, including components of the CRISPR/Cas machinery and donor DNA for precision editing in plants. We update the progress in these methods with recent successful examples of genome editing achieved through virus-based delivery in different plant species, highlight the advantages and limitations of these delivery approaches, and discuss the remaining challenges.

Keywords: CRISPR/Cas; Genome editing; Plant genome engineering; Virus-based delivery.

Fig. 1

Cargo delivery in plants using RNA viruses. Scheme for the propagation of RNA viral vectors by agroinfiltration and the use of the propagated virus particles on recipient plants. (1) Construct the recombinant plasmid encoding components of the viral genome and genes of interest. (2) Introduce the plasmid into Agrobacterium tumefaciens and culture the bacterial strain. (3) Infiltrate N. benthamiana leaves with the Agrobacterium suspension to initiate the propagation of the virus. (4) Recover virus particles from the agroinfiltrated N. benthamiana leaves. (5) Infect other recipient plants with the recovered virus particles by rub inoculation or other methods

Fig. 2

Replication and assembly of PSVs, NSVs, and Geminiviruses in plant cells. A The positive-sense RNA genome from a PSV is translated to produce viral proteins, including the RdRP. Replication of the viral genome occurs through a double-stranded RNA intermediate. The amplified viral RNA genome and the capsid protein self-assemble to form the mature virus particles. B The negative-sense RNA genome from an NSV is used as a template to synthesize the complementary positive-sense RNA under the activity of the viral RdRp. The positive-sense RNA is then translated to produce viral proteins and serves as the template for genome replication. Negative-sense RNA assembles with the viral coat protein to form new NSV virus particles. C Geminiviruses complete their genome replication through the rolling circle mechanism. During this process, a double-stranded DNA intermediate is formed. The proliferated single-stranded DNA genome combines with the capsid to form virus particles. PSV, positive-strand RNA virus; NSV, negative-strand RNA virus; RdRP, RNA-dependent RNA polymerase; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA

Fig. 3

Strategies of delivering the CRISPR/Cas9 genome editing reagents into plant cells. A Delivery of CRISPR/Cas9 via Agrobacterium-mediated transformation. The T-DNA is stably integrated into the plant genome and is expressed to produce the CRISPR/Cas9 machinery, resulting in desirable edits at designated genomic targets. B Delivery of guide RNAs using PSVs. Guide RNAs are delivered into plants expressing Cas9 as a transgene. Assembled Cas9-guide RNA ribonucleoproteins target the designated genomic targets for gene edits. C Delivery of the CRISPR/Cas9 machinery using NSVs. Translation of the positive-strand RNA yields the Cas9 protein. Assembled Cas9-guide RNA ribonucleoproteins target the designated genomic targets for gene edits. PSV, positive-strand RNA virus; NSV, negative-strand RNA virus

Fig. 4

Delivering genome editing reagents in plants using PSVs and NSVs for heritable edits. A PSVs are often used to deliver guide RNAs into Cas9-expressing recipient plants. The ability of PSVs to deliver guide RNAs into the germline cells (inset) enables heritable edits to be generated directly in planta. B NSVs have a higher cargo capacity and thus can deliver the entire CRISPR/Cas machinery. However, edits resulting from NSV-based delivery methods reported so far have only occurred in non-germline cells (inset). Therefore, a subsequent tissue culture process is required to convert edited somatic tissue into whole plants carrying heritable edits. PSV, positive-strand RNA virus; NSV, negative-strand RNA virus

Fig. 5

Two strategies of delivering donor DNA into plant cells. A Biolistic delivery of the CRISPR/Cas9 plasmid and the donor plasmid concurrently. A double-strand break (DSB) is incurred by CRISPR/Cas9 at the designated genomic site. The donor plasmid serves as a template for homology-directed repair to introduce specific edits. B Delivery of donor DNA as geminiviral replicons (GVRs). GVRs carrying CRISPR/Cas9 and the donor template are formed via the circularization of DNA molecules delivered by Agrobacterium. Within the host nucleus, GVRs undergo rolling circle replication to reach a high copy number. The increased concentration of the donor template significantly boosts the efficiency of gene targeting