CRISPR/Cas9-Mediated Immunity to Geminiviruses: Differential Interference and Evasion

Abstract

The CRISPR/Cas9 system has recently been used to confer molecular immunity against several eukaryotic viruses, including plant DNA geminiviruses. Here, we provide a detailed analysis of the efficiencies of targeting different coding and non-coding sequences in the genomes of multiple geminiviruses. Moreover, we analyze the ability of geminiviruses to evade the CRISPR/Cas9 machinery. Our results demonstrate that the CRISPR/Cas9 machinery can efficiently target coding and non-coding sequences and interfere with various geminiviruses. Furthermore, targeting the coding sequences of different geminiviruses resulted in the generation of viral variants capable of replication and systemic movement. By contrast, targeting the noncoding intergenic region sequences of geminiviruses resulted in interference, but with inefficient recovery of mutated viral variants, which thus limited the generation of variants capable of replication and movement. Taken together, our results indicate that targeting noncoding, intergenic sequences provides viral interference activity and significantly limits the generation of viral variants capable of replication and systemic infection, which is essential for developing durable resistance strategies for long-term virus control.

Figure 1. CRISPR/Cas9-mediated targeting of coding and non-coding sequences of the CLCuKoV genome.

(A) Genome organization of CLCuKoV and CLCuMß. Bidirectional and overlapping ORFs (CP, Rep, Ren, TrAP, V2, and C4) are represented by arrows, the IR by a box, stem loop nonanucleotides by a small circle in the IR and SCR, and targets by arrowheads and individual sequences. The selected targets, one in non-coding IR, one each in coding CP or in the Rep RCRII domain, were analyzed for CRISPR/Cas9-mediated targeting (B) NHEJ repair (indel) analysis via the T7EI assay. Arrow indicates the presence of 255 bp and 191 bp regions only in samples expressing CP-sgRNA, but not in samples with TRV empty vector or virus alone. (C) Alignment of PCR amplicons encompassing the CP region and subjected to Sanger sequencing for indel (NHEJ repair) confirmation. (D) T7EI assay detecting indels in the RCRII domain of CLCuKoV genome. Mutations were detected only in RCRII PCR amplicons from plants infiltrated with TRV containing RCRII-sgRNA, but not in plants infiltrated with TRV empty vector or virus alone. (E) Alignment of PCR amplicons encompassing the RCRII motif and subjected to Sanger sequencing for NHEJ repair confirmation. (F) NHEJ repair analysis at the IR sequence by restriction site loss assay. The arrow indicates the expected SspI-resistant 446 bp DNA fragment; none of the samples produced the SspI-resistant DNA fragment, which is similar to TRV empty vector or virus alone. Arrows in (A), (C) and (E) represent the expected DNA fragments. The indel percentage shown was calculated based on the Sanger sequence reads. In (B) (reverse strand sequence) and (D) the wild-type (WT) sequences are shown at the top (target sequence is shown in red; the protospacer-associated motif [PAM] in green, followed by the various indels formed, as indicated by numbers to the right of the sequence (−, deletion of x nucleotides; + , insertion of x nucleotides; and > , change of x nucleotides to y nucleotides). CP coat protein, Rep replicase, Ren replication enhancer, TrAP transcriptional activator protein, βC1 betasatellite conserved ORF, IR intergenic region, SCR satellite conserved region.

Figure 2. NHEJ repair of coding and non-coding sequences of the MeMV genome.

Non-coding IR, coding CP, and the Rep RCRII domain of MeMV were analyzed for NHEJ repair. (A) NHEJ repair (indel) analysis via an SspI recognition site loss assay. The MeMV IR (453 bp) was analyzed for the loss of the SspI recognition site at the CRISPR/Cas9 target locus. Unlike CLCuKoV, NHEJ-repaired indels are indicated by arrows pointing to the 453-bp SspI-resistant DNA fragments. (B) Alignment of reads of PCR amplicons encompassing the IR of MeMV subjected to Sanger sequencing for NHEJ repair confirmation. (C) T7EI assay to detect indels in the CP of the MeMV genome. The T7EI assay detected indels only in CP PCR amplicons from plants infiltrated with TRV containing the CP-sgRNA, but not in plants infiltrated with TRV empty vector or virus alone. (D) Alignment of reads of the PCR amplicons encompassing the target site and subjected to Sanger sequencing for NHEJ repair confirmation. (E) NHEJ repair analysis at the RCRII motif of MeMV by T7E1 assay. Arrow indicates the expected DNA fragments; TRV empty vector or virus alone did not show similar fragments. All DNA fragments from (A,C and E) were resolved on an 2% agarose gel premixed with ethidium bromide stain. Arrows in (A,C and E) indicate the expected DNA fragments. The indel percentage shown below each gel was calculated based on the Sanger sequence reads. In (B,D and F), the wild-type (WT) sequences are shown at the top (target sequence is shown in red; the protospacer-associated motif [PAM] is indicated in green, followed by the various indels formed, as indicated by numbers to the right of the sequence (−, deletion of x nucleotides; +, insertion of x nucleotides; and >, change of x nucleotides to y nucleotides).

Figure 3. Variable efficiencies of indel formation at the IR sequences of different strains of TYLCV.

(A) Stem-loop structure of the different geminiviruses used in this study. The conserved nonanucleotide motif is flanked on each side by a short stretch of complementary sequences. (B) Restriction site loss assay for detecting NHEJ-based indels at the IR of TYLCSV. The TYLCSV IR (562 bp) was analyzed for the loss of the SspI recognition site at the targeting locus. The arrow indicates the location of the expected 562-bp SspI-resistant DNA fragment in samples with IR-sgRNA, but like TRV empty vector and virus alone, no SspI-resistant fragment was observed. (C) SspI recognition site loss assay for detecting indels at the IR in TYLCV2.3. The variant TYLCSV-IRsgRNA (two lanes after marker) and authentic TYLCV2.3-IRsgRNA (last three lanes) were used to target the IR of TYLCV2.3. Arrows indicate the presence of the expected 269 bp SspI-resistant DNA fragments in samples with TYLCV2.3-IR-sgRNA or TYLCSV-IRsgRNA, but no SspI-resistant fragment was observed in TRV empty vector. (D) T7EI assay for detecting NHEJ-based indels in the CP and RCRII domain of the TYLCSV genome. T7EI assay detected high rates of indel formation both in CP and RCRII PCR amplicons from plants infiltrated with TRV containing CP or RCRII-sgRNA compared with TRV empty vector. DNA fragments of (B,C and D) were resolved on a 2% agarose gel stained with ethidium bromide. Arrows represent the expected DNA fragment in T7EI-digested DNA. The indel percentage shown below each gel was calculated based on the Sanger sequence reads.

Figure 4. IR targeting by CRISPR/Cas9 interferes with genome accumulation of both CLCuKoV and TYLCSV.

(A) DNA blot analysis assaying CLCuKoV (A) and TYLCSV (B) genomic DNA accumulation in Cas9OE plants expressing CLCuKoV-IRsgRNA and TYLCSV-IRsgRNA, respectively. CLCuKoV and TYLCSV genomic DNA was detected with DIG-labeled probe produced against the respective IRs of CLCuKoV and TYLCSV. All individual plants with IR-sgRNA that were infiltrated with CLCuKoV and TYLCSV exhibited reduced accumulation of the genomes relative to plants inoculated with TRV empty vector and virus only. Arrowheads in (A,B) indicate detection of the expected size of the TYLCV genome. (C) Alignment of cloned Sanger-sequenced PCR amplicons encompassing the IR of CLCuKoV. Alignment of sequence reads encompassing the IR shows only long deletions. The wild-type (WT) CLCuKoV sequence is shown at the top; the various indels formed are indicated by numbers in the middle of the sequence reads.

Figure 5. NHEJ-repaired CP sequence evades CRISPR/Cas9.

(A) Evasion of repaired CP sequence of TYLCV genomes, as revealed by the T7EI assay. Sap from TYLCV-infected plants with an established CRISPR/Cas9 system against the CP region was applied to WT N. benthamiana plants. Total genomic DNA was isolated from top (young) leaves of sap-inoculated WT plants at 15 DAI. CP targets flanking PCR amplicons were subjected to T7EI. Arrows indicate the presence of the expected digested DNA fragment from samples of CP-targeted sap-infected plants compared to TRV empty vector with TYLCV. (B) BsmBI-recognition site loss assay for detecting escapees. BsmBI-treated PCR fragments were used as template in another round of PCR with the same primers. Purified DNA from this PCR was again subjected to BsmBI digestion. Arrowheads indicate the expected BsmBI-resistant DNA fragments compared to WT PCR amplicons from TRV empty vector. (C) Alignment of Sanger-sequencing reads of PCR amplicons encompassing the CP region of TYLCVfor mutation at the CRISPR/Cas9 targeting site. The wild-type (WT) TYLCV sequences are shown at the top (target sequence is shown in red, the BsmBI site by a line, and the protospacer-associated motif [PAM] is indicated in green; the various indels formed are shown in enlarged, bold, and blue font at their respective sites. (D) Evasion analysis of TYLCSV genomes with repaired CP sequences via the T7EI assay. For TYLCSV samples were prepared as in (A) CP targets flanking the PCR amplicons were subjected to T7EI. Arrows indicate the presence of expected digested DNA fragments from samples of CP-targeted sap-infected plants compared to TRV empty vector with TYLCSV. (E) Evasion analysis of genomes of CLCuKoV genomes with repaired CP sequences via the T7EI assay. Wild-type scions were grafted to the stocks of CLCuKoV-infected plants with an established CRISPR/Cas9 system against the CP region. Total genomic DNA was isolated from top (young) leaves of WT scions at 21 DAI. CP-target-flanking PCR amplicons were subjected to T7EI. Arrows indicate the presence of the expected digested DNA fragment from samples of CP-targeted CLCuKoV compared to TRV empty vector with CLCuKoV.