RPS5A Promoter-Driven Cas9 Produces Heritable Virus-Induced Genome Editing in Nicotiana attenuata

Abstract

The virus-induced genome editing (VIGE) system aims to induce targeted mutations in seeds without requiring any tissue culture. Here, we show that tobacco rattle virus (TRV) harboring guide RNA (gRNA) edits germ cells in a wild tobacco, Nicotiana attenuata, that expresses Streptococcus pyogenes Cas9 (SpCas9). We first generated N. attenuata transgenic plants expressing SpCas9 under the control of 35S promoter and infected rosette leaves with TRV carrying gRNA. Gene-edited seeds were not found in the progeny of the infected N. attenuata. Next, the N. attenuata ribosomal protein S5 A (RPS5A) promoter fused to SpCas9 was employed to induce the heritable gene editing with TRV. The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations at three target genes in N. attenuata seeds with TRV-delivered guide RNA. These monoallelic mutations were found in 2%-6% seeds among M 1 progenies. This editing method provides an alternative way to increase the heritable editing efficacy of VIGE.

Challenges Facing CRISPR/Cas9-Based Genome Editing in Plants

“…The effectiveness of the FT mRNA in raising the frequency of heritable genome editing was independently confirmed with a VIGE system based on cotton leaf crumple virus (CLCrV) in Arabidopsis plants overexpressing SpCas9 ( Lei et al, 2021 ). Surprisingly, in contrast to N. benthamiana and Arabidopsis, SpCas9 expressed from the 35S promoter does not lead to mutations in the germline of coyote tobacco ( N. attenuata ), even when the sgRNA is fused to the FT mRNA ( Oh and Kim, 2021 ), likely due to low expression from the 35S promoter in this species and specific tissue ( Wilkinson et al, 1997 ; Sunilkumar et al, 2002 ; Patro et al, 2012 ). However, transgenic N. attenuata plants expressing SpCas9 from the ribosomal protein S5 A (RPS5A) promoter induced high levels of expression in all plant tissues, including the germline and meristematic tissues; inoculation of leaves with TRV vectors carrying the sgRNA successfully obtained genome-edited seeds ( Oh and Kim, 2021 ).…”

“…(B) In Nicotiana attenuata , infiltration of TRV/sgRNA or TRV2/ FT- sgRNA into N. attenuata plants expressing SpCas9 from the 35S promoter does not lead to genome editing in seeds, as the 35S promoter is insufficient to induce the germline mutation in N. attenuata . However, the RPS5A promoter can induce high gene expression levels in the germline, resulting in genome editing in seeds when leaves are infiltrated with TRV2/sgRNA into N. attenuata plants expressing RPS5A-driven SpCas9 ( Oh and Kim, 2021 ).…”

The Promising Nanovectors for Gene Delivery in Plant Genome Engineering

“…However, the efficiency is far from satisfactory even with the traditional delivery method [ 109 ]. To ensure the efficient and successful editing of desired sites, strong, constitutive, and sometimes virus promoters have often been used to boost the expression level of Cas and sgRNA [ 110 , 111 , 112 , 113 ]. These methods could be used in nanomaterial-mediated genome editing, and also in other nanomaterial-based plant genetic engineering.…”

Fast and Precise: How to Measure Meiotic Crossovers in Arabidopsis

“…These noteworthy technical advances in detecting COs and isolating new CO modifiers will have profound implications for plant breeding and our understanding of meiotic recombination. Furthermore, manipulating the rate and positions of COs using CRISPR/Cas9-based genome editing methods will accelerate plant breeding and QTL (quantitative trait locus) mapping ( Gao, 2021 ; Oh and Kim, 2021 ; Taagen et al, 2020 ).…”