Activity and specificity of TRV-mediated gene editing in plants

Abstract

Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRV- mediated Cas9 activity for up to 30 d post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no off-target activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.

Keywords: CRISPR/Cas9 system; TRV; plant genome engineering; synthetic site-specific nucleases (SSNs); viral-mediated genome editing.

Figure 1.

Persistence of TRV-mediated CRISPR/Cas9 targeted mutagenesis of the PDS3 gene. (A) Establishment of TRV infection in B14 plants, Nicotiana benthamiana Cas9 overexpression line. Agrobacterium cultures containing RNA1 and engineered RNA2 harboring gRNA for targeting PDS gene were mixed 1: 1 (OD₆₀₀ 0.1 each) and co-infiltrated to 2 fully expanded true leaves. Systemic leaves were collected 7, 15 and 30 d post-infiltration (dpi). (B) T7EI assay for indels detection. Genomic DNA was extracted from the systemic leaves and purified PCR product (200ng) of PDS fragment flanking the targeted locus was subjected to T7EI analysis. All three leaves collected at different time points showed high efficiency of targeted mutagenesis (43– 61 %) compared to vector control systemic leaves collected at 30 dpi. (C) Analysis of progeny plants for the presence of targeted modification using NcoI recognition site loss assay. DNA was extracted from progeny plants (50 seedlings pooled in one tube) and PCR was performed with a primer set to amplify 404 bp fragment encompassing the target site. Purified PCR product (300 ng) was treated with NcoI and separated on 2% agarose gel. Progeny pool 1 and 2 clearly showed a resistant DNA fragment of 404 bp indicating the targeted mutagenesis. (D) Alignment of Sanger sequencing reads showing the presence of indels at the PDS target sequence. Numbers to the right of sequence alignment indicates the number of nucleotides deleted by targeting the PDS genomic target.

Figure 2.

TRV-based CRISPR/Cas9 system exhibited no apparent off-target effects in N. benthamiana genome. (A) Table showing combinatorics approach to identify potential off-target binding of PDS3 gRNA in N. benthamiana genome. The putative off-target binding sites were subjected to further annotation, where sequences were split into 2 groups of conserved and not conserved NcoI restriction site directly preceding the PAM sequence and sites containing mutation in seed and non-seed sequence. Based on dissimilarities of 1 to 7 nucleotides with PDS3 gRNA, a total of 4265 potential off targets were detected, out of which 375 have NcoI site proximal to PAM sequence. (B) T7EI assay for the presence of indels at potential off target sites. DNA fragment flanking potential 13 targets were amplified by PCR with their respective primers. TRV-mediated CRISPR/Cas9 system exhibited no detectable activities at all tested targets using the T7EI assays. The respective contig number of each of 13 potential off target site are represented on the top of gel