CRISPR/Cas9-mediated viral interference in plants

CRISPR/Cas9-mediated interference with TYLCV

In this study, we investigated whether the CRISPR/Cas9 system can be used in plants
to impart molecular immunity against DNA viruses. To this end, we used our recently
developed system for genome editing, which involves systemic delivery of sgRNA molecules
via tobacco rattle virus (TRV) into N. benthamiana plants overexpressing Cas9 endonuclease (NB-Cas9OE) 36]. We designed sgRNAs specific for different TYLCV coding and non-coding sequences
(Fig. 1a), and inserted them into the TRV RNA2 genome. Next, we delivered the sgRNAs via agroinfection
of TRV into NB-Cas9OE plants. Seven days post-infiltration (dpi) with TRV, we challenged
the NB-Cas9OE plants with an infectious TYLCV clone via agroinfection (Fig. 1b) 37]. Ten days later, we isolated total RNA and DNA from the NB-Cas9OE plant systemic
leaves for various molecular studies. To determine TYLCV titer, we performed semi-quantitative
PCR using primers encompassing the IR region (Table S1 in Additional file 1). The titer was lower in samples co-infiltrated with sgRNA targeting the IR region
than in those infiltrated with TRV vector controls and TYLCV (Fig. 1c). TYLCV replicates through a RCA mechanism that exploits the plant machinery 38]. An RCA assay revealed that targeting of the IR via the CRISPR/Cas9 system prevented
accumulation of the TYLCV genome (Fig. 1d). Because TYLCV is a ssDNA virus that is converted to dsDNA inside the plant cell
nucleus, interference with TYLCV replication by targeting the IR within replicating
viral dsDNA should significantly reduce the accumulation of both the ssDNA and dsDNA
forms. To test for interference with TYLCV replication, we performed dot blot assays.
The results showed that the titer of TYLCV in IR-sgRNA plants was lower than that
of the TRV vector control (Figure S1 in Additional file 2). In addition, we validated our dot blot results by Southern blotting, which confirmed
that targeting the IR of TYLCV prevented accumulation of both ssDNA and dsDNA (Fig. 1e).

We further confirmed our findings by using a different method for TYLCV inoculation,
namely, the sap transmission method (Additional file 3). Sap from young leaves of TYLCV-infected wild-type N. benthamiana plants was directly applied to N. benthamiana Cas9OE plants 7 days after infection with TRV-sgRNA. DNA was extracted from the systemic
leaves after 21 days of sap application and then subjected to different types of molecular
analysis. Non-specific sgRNA (possessing no sequence similarity to the TYLCV genome;
Supplementary sequence 9 in Additional file 4) rather than an empty TRV vector was used in the sap transmission experiments. The
RCA results revealed a reduction in TYLCV genome accumulation in both samples treated
with IR-sgRNA or CP-IR-sgRNA compared with samples treated with non-specific sgRNA
or TYLCV alone (Figure S2a in Additional file 2). To confirm the RCA results, we next performed semi-quantitative PCR to amplify
a 560-bp fragment encompassing the TYLCV IR. The results revealed lower amplification
of TYLCV with specific sgRNAs than with controls (Figure S2b in Additional file 2), thereby confirming the RCA results. Both the RCA and semi-quantitative PCR assays
are based on the amplification of available TYLCV genome. To further confirm these
data, we next performed Southern blotting, which confirmed lower accumulation of TYLCV
in the presence of specific sgRNAs than in the presence of controls (Figure S2c in
Additional file 2).

CRISPR/Cas9 mediates targeted cleavage of the TYLCV genome

We subsequently investigated whether the attenuated replication of TYLCV was indeed
due to targeted cleavage or modification of the genome, rather than simply to interference
with the replication machinery resulting from binding by the CRISPR/Cas9 complex.
To this end, we employed T7EI and restriction site loss assays to confirm targeting
and determine the efficiency of modifications within the selected sequences. The 20-nucleotide
target sequence of the IR of the TYLCV contains a recognition sequence for SspI endonuclease at the predicted cleavage site 3 bp upstream of the PAM sequence. We
isolated genomic DNA at 10 dpi with the TYLCV infectious clone and PCR amplified a
560-bp fragment encompassing the IR target sequence, which contains two additional
SspI sites (Supplementary sequence 1 in Additional file 4). Complete SspI digestion of the wild-type sequence produced four fragments of 53, 92, 189, and
216 bp; targeted modification of the IR sequence and subsequent repair via non-homologous
end joining eliminated the SspI site within the IR, generating a 269-bp SspI-resistant band. We observed the 269-bp band only in the IR-sgRNA samples, indicating
successful targeted modification of the IR by the CRISPR/Cas9 system (Fig. 2a). To confirm the presence of indels, we cloned the 560-bp PCR amplicons into the
pJET 2.1 cloning vector and performed Sanger sequencing. Alignment of the sequencing
reads of 300 clones indicated that 42 % of the clones carried targeted modifications
within the IR sequence (Fig. 2c; Table S2 in Additional file 1). Furthermore, to determine whether targeting ORFs could also mediate interference
with TYLCV, we designed sgRNAs targeting the CP and RCRII motif of the Rep ORF. The
T7EI assays and Sanger sequencing indicated that different ORFs could be targeted
for modification to interfere with TYLCV accumulation (Fig. 2b, d; Figure S3 in Additional file 2; Table S2 in Additional file 1). We confirmed the results of the T7EI assays by performing RCA and Southern blotting
assays (Figures S4 and S5 in Additional file 2). In nature, DNA viruses are transmitted by different means and vectors. Therefore,
we wondered whether our system was capable of targeting the sap-transmitted TYLCV
genome. DNA was extracted from sap-transmitted TYLCV and used to infect N. benthamiana Cas9OE plants expressing IR-sgRNA, CP-IR-sgRNA, or controls. The corresponding CP
fragment (642 bp) from CP-IR-sgRNA and the IR fragment (560 bp) from CP-IR-sgRNA or
IR-sgRNA were PCR amplified and subjected to BsmBI (CP) and SspI (IR) recognition site loss assays. DNA fragments resistant to BsmBI in CP amplicons and to SspI in IR amplicons were detected in CP-IR-sgRNA samples but not in controls (Figure
S2d in Additional file 2). The corresponding SspI-resistant fragment was also observed in IR-sgRNA samples but not in controls (Figure
S2e in Additional file 2).

Fig. 2. CRISPR/Cas9-mediated targeted cleavage of the TYLCV genome. a Mutation analysis using a restriction site loss assay. The TYLCV IR (560 bp) was
analyzed for loss of the SspI recognition site at the targeted locus. The arrow indicates the presence of a 269-bp SspI-resistant DNA fragment only in samples harboring IR-sgRNA, but not in samples harboring
the TRV empty vector. b T7EI assay for detecting indels in the RCRII domain of the TYLCV genome. The T7EI
assay detected mutations only in RCRII PCR amplicons from plants infiltrated with
TRV containing RCRII-sgRNA, but not in plants infiltrated with TRV empty vector. DNA
fragments A and B were resolved on a 2 % agarose gel and stained with ethidium bromide.
Arrows show the expected DNA fragments. c Alignment of reads from PCR amplicons encompassing the IR region, which were subjected
to Sanger sequencing. d Alignment of reads from the PCR amplicons encompassing the RCRII motif, which were
subjected to Sanger sequencing. The wild-type (WT) TYLCV sequences are shown at the top. The target sequence is shown in red, the SspI site is indicated by a line, and the protospacer-associated motif (PAM) is indicated in green. This is followed by the various indels, which are indicated by the numbers to the
right of the sequence (?x indicates deletion of x nucleotides; +x indicates insertion
of x nucleotides; and T??G indicates change of T to G). Arrows indicate the expected
sizes of the cleavage products

CRISPR/Cas9 system mediates specific and multiple targeting of viral genomes

We next asked whether the CRISPR/Cas9 is capable of mediating specific interference
with TYLCV. Notably, the RCRII motifs of the Rep ORFs of geminiviruses are conserved
at the amino acid level but variable at the nucleotide level. To confirm that our
RCRII-sgRNA targeted only the TYLCV RCRII region and interfered specifically with
TYLCV genome replication, we co-infiltrated another monopartite geminivirus, beet
curly top virus (BCTV) strain Worland (Supplementary sequence 7 in Additional file
4), along with the TYLCV-RCRII-sgRNA. We tested for modifications of the RCRII sequences
of both TYLCV and BCTV using T7EI assays. The results confirmed that TYLCV-RCRII-sgRNA
specifically targeted the TYLCV genome, but not the BCTV genome (Fig. 3a). We confirmed that BCTV-RCRII-sgRNA targeted the BCTV genome but not the TYLCV genome
(Fig. 3a). Sanger sequencing data confirmed the results of T7EI assays with regard to specific
targeting of each genome (Fig. 3b, c).

Fig. 3. Specific targeting of different viral genomes. RCRII sgRNAs specific for TYLCV and
BCTV sequences target only the TYLCV and BCTV genomes, respectively. a T7EI assays showing specific targeting of the TYLCV or BCTV genomes. b Alignment of Sanger sequenced reads from the TYLCV-targeted RCRII region. c Alignment of Sanger sequenced reads from the BCTV-targeted RCRII region. The various
indels are indicated by the numbers to the right of the sequences (?x indicates deletion
of x nucleotides; +x indicates insertion of x nucleotides; and X??Y indicates change
of nucleotide X to nucleotide Y). Arrows indicate the expected sizes of the cleavage
products. WT wild type

Because the stem-loop sequence of the origin of replication in the IR is conserved
in all geminiviruses, we investigated the possibility of targeting different viruses
with a single sgRNA. We designed an IR-sgRNA that contains the invariant TAATATTAC
sequence common to all geminiviruses (Fig. 4a) 39] and tested this IR-sgRNA against TYLCV and BCTV. Sanger sequencing confirmed the
presence of indels and targeted modifications in both viruses (Fig. 4b, c; Table S2 in Additional file 1). Because monopartite and bipartite geminiviruses share the same conserved stem-loop
sequence in the origin of replication within the IR (Fig. 4a), we next targeted a bipartite virus, the Merremia mosaic virus (MeMV) (Supplementary
sequence 8 in Additional file 4) 40]. Sanger sequencing confirmed that IR-sgRNA specific to TYLCV but containing the invariant
TAATATTAC sequence targeted a similar sequence in the MeMV genome (Fig. 4d; Table S2 in Additional file 1). Thus, a single sgRNA is capable of targeting multiple viruses.

Fig. 4. Targeting of different geminivirus genomes using a single sgRNA. A single IR-sgRNA
was capable of targeting the TYLCV, BCTV, and MeMV genomes. a IR-sgRNA (upper sequence) identical to the TYLCV IR sequence but harboring mismatches with the BCTV and MeMV
IR sequences (blue) was used to target all three viral genomes. b–d Alignment of Sanger sequenced reads from the IR-targeted region in the TYLCV, BCTV,
and MeMV genomes showing the respective targeted modifications. The wild-type (WT) sequence is shown at the top (red) and the PAM is shown in green

The CRISPR/Cas9 system attenuates or represses TYLCV symptoms

Interference with TYLCV replication by the CRISPR/Cas9 machinery is predicted to eliminate
or reduce TYLCV symptoms, reminiscent of the CRISPR/Cas9 system’s originally evolved
function in natural immunity of bacteria against phages. Accordingly, we assessed
and evaluated TYLCV symptoms in NB-Cas9OE plants expressing sgRNAs against TYLCV coding
and non-coding sequences. In these experiments, we challenged three groups of NB-Cas9OE
plants, which expressed sgRNAs specific to the IR, CP, or Rep regions, with an infectious
TYLCV clone. NB-Cas9OE plants expressing sgRNA targeting the IR exhibited significantly
reduced TYLCV symptoms relative to the TRV vector control (Figure S6 in Additional
file 2; Table S2 in Additional file 1). Moreover, NB-Cas9OE plants expressing sgRNA targeting the CP or Rep ORF also exhibited
a reduction of TYLCV symptoms, but the magnitude of this reduction was smaller than
that achieved by targeting the IR-sgRNA (Figure S7 in Additional file 2; Table S2 in Additional file 1). In a second set of experiments, we investigated whether targeting more than one
sequence of the TYLCV genome would lead to greater reduction of TYLCV symptoms. To
test this, we co-infiltrated a pair of TRV RNA2 genomes carrying sgRNAs targeting
the CP region and IR. Targeting two different sequences did not have an additive effect
on the reduction of TYLCV symptoms (Figure S8 in Additional file 2; Table S2 in Additional file 1).

Because co-infiltration of two RNA2 genomes carrying two different sgRNAs does not
ensure the delivery of both sgRNAs into a single cell (and their subsequent activities
against single molecules of the viral genome), we multiplexed our sgRNA delivery using
the recently developed polycistronic tRNA–gRNA (PTG) system 41]. Subsequently, to determine the activity of both gRNAs in this system, we delivered
a single RNA2 genome carrying both IR-sgRNA and CP-sgRNA. This TRV system was capable
of expressing two sgRNAs that can target both IR and CP sequences. Restriction site
loss assays (Figure S9 in Additional file 2) and Sanger sequencing confirmed the targeted modification of both IR and CP sequences
(Figure S10 in Additional file 2). Furthermore, simultaneous targeting of two sequences by PTG-based expression led
to greater reduction of virus titer and recovery of disease symptoms in NB-Cas9OE
plants (Figure S11 in Additional file 2; Table S2 in Additional file 1). Next, we performed Southern blotting to confirm the absence or reduced accumulation
of the TYLCV genome in IR-CP-sgRNA infiltrated plants relative to that in plants infiltrated
with the vector control. The molecular analyses showed a significant reduction in
viral genome levels when the IR and CP region were simultaneously targeted for cleavage.
This confirmed the phenotypic data regarding the TYLCV symptoms (Figure S12 in Additional
file 2).