Guide RNAs to target N. benthamiana Flowering locus T (NbFT; SolGenomics Niben101Scf01519g10008.1) and Xylosyl transferase 1 (NbXT1; Niben101Scf04205g03008.1) were selected using the CRISPR-P online tool as described by Bernabé-Orts et al. (2019) (Table S1). Nucleotide (nt) sequences of recombinant viral clones are shown in Figs. S1 and S2. These clones were built using the primers shown in Tables S2 and S3.
N. benthamiana wild-type and transformed plants expressing tobacco etch virus (TEV, genus Potyvirus) nuclear inclusion b (NIb) protein (Martí et al., 2020), were grown at 25°C under a 12 h/12 h day/night photoperiod. Plants that were 4-to 6-weeks-old were agroinoculated as previously described (Bedoya et al., 2010; Uranga et al., 2021). Tissue samples (approximately 100 mg) from the first symptomatic upper non-inoculated leaf were collected at different days post inoculation (dpi), as indicated, for virus progeny and plant genome-editing analyses.
Reverse transcription (RT)-polymerase chain reaction (PCR) analysis of viral progeny
RNA was purified from leaf samples using silica gel columns (Uranga et al., 2021). cDNA was synthesized using RevertAid reverse transcriptase (Thermo Scientific) and primer D179 (Table S4). PCR with Thermus thermophilus DNA polymerase (Biotools) was used to amplify the TEV coat protein (CP) cistron (primers D178 and D211; Table S4) or a fragment of the LbCas12a open reading frame (ORF) (primers D3604 and D3605; Table S4). PCR products were separated by electrophoresis in 1% agarose gels followed by staining with ethidium bromide.
Analysis of N. benthamiana genome editing
DNA from leaf samples was purified using silica gel columns (Uranga et al., 2021). N. benthamiana genome fragments were amplified by PCR using high-fidelity Phusion DNA polymerase (Thermo Scientific) (Table S5). PCR products were separated by agarose gel electrophoresis, purified from the gel, and subjected to Sanger sequencing (Table S5). The presence of sequence modifications was analyzed using the inference of CRISPR edits (ICE) software (http://www.synthego.com/products/bioinformatics/crispr-analysis).
Article TitleCRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors
The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR) components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-associated (Cas) nuclease. Here we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors, derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.