High efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus

Abstract

Delivery of gene editing components such as the Cas nuclease and single guide RNAs (sgRNAs) into plant cells is commonly accomplished by agrobacterium-mediated transformation. Although Arabidopsis is easy to transform, generation of biallelic edited plants requires screening a large number of plants in subsequent generations. Here, we describe optimization of the Tobacco rattle virus (TRV) for in planta delivery of sgRNAs fused to a tRNAIleu that induces efficient multiplex somatic and biallelic heritable editing in Arabidopsis. Inclusion of tRNAIleu enhances the systemic movement of TRV and the mutant phenotype is visible in the initial TRV::sgRNA-tRNAIleu infected Arabidopsis, which allows for the uncovering of lethal phenotypes. Mutant progeny are recovered in the next generation (M1) at frequencies ranging from 30-60%, with 100% mutant recovery in the following (M2) generation. TRV::tRNAIleu system described here allows generation of biallelic edited plants in a single generation and is amenable for large-scale high throughput CRISPR screens.

Barley stripe mosaic virus-mediated somatic and heritable gene editing in barley (Hordeum vulgare L.)

“…The fusion of tRNAs or mobile RNA elements such as flowering locus T (FT) to gRNAs delivered by TRV can increase the heritable editing frequency in A. thaliana (Nagalakshmi et al, 2022) and N. benthamiana (Ellison et al, 2020). In wheat, both increased and decreased heritability of induced mutations upon fusion of RNA elements to gRNAs in BSMV were reported (Li et al, 2021;Chen et al, 2022a;Wang et al, 2022).…”

“…Plant viruses are engineered to deliver either guide RNAs into plants stably expressing endonucleases or even the complete genome engineering components (Cody and Scholthof, 2019;Metje-Sprink et al, 2019;Tsanova et al, 2021;Varanda et al, 2021;Gentzel et al, 2022). VIGE has been applied in dicot plants including Nicotiana benthamiana, Arabidopsis thaliana, and Glycine max using plant RNA viruses such as Tobacco rattle virus (TRV) (Ellison et al, 2020;Nagalakshmi et al, 2022), Pea early browning virus (PEBV) (Ali et al, 2018), Beet necrotic yellow vein virus (BNYVV) (Jiang et al, 2019), Potato virus X (PVX) (Ariga et al, 2020), Barley yellow striate mosaic virus (BYSMV) (Gao et al, 2019) or Sonchus yellow net rhabdovirus (SYNV) (Ali et al, 2015;Zaidi and Mansoor, 2017;Cody and Scholthof, 2019;Ellison et al, 2020;Ma et al, 2020;Nagalakshmi et al, 2022). In monocots, Foxtail mosaic virus in maize (FoMV) (Mei et al, 2019) or BSMV in wheat, cotton, and maize (Hu et al, 2019;Li et al, 2021;Chen et al, 2022a;Chen et al, 2022b;Wang et al, 2022) were employed for VIGE.…”

“…BSMV was harnessed to deliver sgRNAs into plants that ectopically express Cas9, such as monocots (wheat and maize) and the dicot N. benthamiana, for eliciting Cas9-mediated targeted genome editing in somatic tissues (Hu et al, 2019;Li et al, 2021;Chen et al, 2022a;Wang et al, 2022). In wheat, different frequencies of BSMV-mediated heritable gene editing were observed, ranging from 0.8 to 3.0% (Chen et al, 2022a;Chen et al, 2022b), 12.9 to 100% (Li et al, 2021), and 0 to 19% (Wang et al, 2022), depending on the genotype, type of sgRNA (with/without mobile RNA elements), and the target site (Notaguchi et al, 2014;Zhang et al, 2016;Ellison et al, 2020;Nagalakshmi et al, 2022).…”