RNA silencing suppressor-influenced performance of a virus vector delivering both guide RNA and Cas9 for CRISPR gene editing

Kelvin T Chiong,Herman B Scholthof,Will B Cody

doi:10.1038/s41598-021-85366-4

Kelvin T Chiong, Herman B Scholthof + Show 1 more

Open Access

https://doi.org/10.1038/s41598-021-85366-4

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Abstract

We report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver CRISPR/Cas9 components into plants. First, production of a Cas9 (HcoCas9) protein from a binary plasmid increased when co-expressed in presence of suppressors of gene silencing, such as the TMV 126-kDa replicase or the Tomato bushy stunt virus P19 protein. Such suppressor-generated elevated levels of Cas9 expression translated to efficient gene editing mediated by TRBO-G-3′gGFP expressing GFP and also a single guide RNA targeting the mgfp5 gene in the Nicotiana benthamiana GFP-expressing line 16c. Furthermore, HcoCas9 encoding RNA, a large cargo insert of 4.2 kb, was expressed from TRBO-HcoCas9 to yield Cas9 protein again at higher levels upon co-expression with P19. Likewise, co-delivery of TRBO-HcoCas9 and TRBO-G-3′gGFP in the presence of P19 also resulted in elevated levels percentages of indels (insertions and deletions). These data also revealed an age-related phenomenon in plants whereby the RNA suppressor P19 had more of an effect in older plants. Lastly, we used a single TRBO vector to express both Cas9 and a sgRNA. Taken together, we suggest that viral RNA suppressors could be used for further optimization of single viral vector delivery of CRISPR gene editing parts.

Highlights

We report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver clustered regularly interspaced palindromic repeat (CRISPR)/Cas[9] components into plants
Co-expression of P19 resulted in detectable levels of Cas[9] with increased protein expression from 4 to 6 dpi, followed by a slow decrease in expression through 9 dpi. These results firmly demonstrate the additive advantage of delivering P19, in addition to the endogenous P126 suppressor encoded by the TMV vector itself, to obtain readily detectable levels of Cas[9] expression from TRBO using our experimental conditions
The results showed that the TRBO vector can replicate with the large Cas[9] insert, and the protein is catalytically active as demonstrated through the formation of indels in treated tissue

Summary

Introduction

We report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver CRISPR/Cas[9] components into plants. Production of a Cas[9] (HcoCas9) protein from a binary plasmid increased when co-expressed in presence of suppressors of gene silencing, such as the TMV 126-kDa replicase or the Tomato bushy stunt virus P19 protein Such suppressor-generated elevated levels of Cas[9] expression translated to efficient gene editing mediated by TRBO-G-3′gGFP expressing GFP and a single guide RNA targeting the mgfp[5] gene in the Nicotiana benthamiana GFP-expressing line 16c. In a similar experimental design, GFP expressed using a P19-defective TBSV vector in N. benthamiana resulted in an antiviral silencing response and low level of GFP expression, whereas GFP expression recovered significantly when a separate P19 construct was infiltrated in the same leaves[25] Another viral suppressor of RNA silencing (VSR) is the P126 replicase subunit of TMV, which is produced when translation of the full replicase prematurely terminates at an amber stop codon. Experiments support the notion that P126 interferes with the RNAi pathway by binding to siRNA duplexes and physically blocking the vital HEN1-dependent methylation process, inhibiting their incorporation into R ISC28,29

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