Improving CRISPR‐Cas‐mediated RNA targeting and gene editing using SPLCV replicon‐based expression vectors in Nicotiana benthamiana

In this study, we developed Sweet Potato Leaf Curl Virus (SPLCV) replicon-based expression vectors and tested the efficiency of these vectors in CRISPR-Cas-mediated RNA targeting and gene editing by using Nicotiana benthamiana as model plant. Our results showed that the SPLCV-based vectors remarkably enhance the LwaCas13a-mediated RNA targeting activity nd SpCas9/LbCas12a-mediated gene editing efficiency in Nicotiana benthamiana. Our work expands the existing list of geminiviral replicon-based vectors in plants. The SPLCV-based vectors offer an opportunity to enhance the efficiency of CRISPR-Cas-mediated gene knockdown, knockout, and knockin in plants, especially for its host sweetpotato.

Geminiviruses are a family of plant viruses with circular singlestranded DNA genomes. They have been deconstructed by researchers for multiple biotechnological applications, including protein expression, gene silencing and genome editing, in plants (Lozano-Dur an, 2016). Under the deconstructed virus strategy, the coat protein and movement protein genes were removed from the geminiviruses, while the sequences required for replication were retained. The replicons replicate after delivery to plant cells and increase the copy number of carried DNA; this leads to high levels of gene expression (Lozano-Dur an, 2016). Although a few geminiviral replicon-based vectors have been used in gene targeting (Baltes et al., 2014;Cermak et al., 2015;Wang et al., 2017), the list of DNA replicon-based vectors is still limited in plants, especially for food crops. In this study, we developed sweet potato leaf curl virus (SPLCV) replicon-based expression vectors. We tested the efficiency of these vectors in CRISPR-Cas-mediated RNA targeting and gene editing by using Nicotiana benthamiana as model plant.
SPLCV is a monopartite geminivirus belonging to the genus Begomoviruses (Bi and Zhang, 2012). The coding region of SPLCV-JS (accession number: KF040468.1) replication-associated proteins (Rep:1900 bp, encoding four proteins: AC1, AC2, AC3 and AC4) and intergenic region (IR:284 bp) were synthesized and cloned into the binary vector pCambia0390 together with GFP expression cassette (U4:GFP) in an IR-GFP-Rep-IR origination to produce the reporter vector SPLCV-GFP (Figure 1a). A regular T-DNA vector (T-GFP) harbouring the same expression cassette was used as control. N. benthamiana leaves infiltrated with Agrobacterium tumefaciens containing SPLCV-GFP significantly showed stronger GFP fluorescence than T-GFP ( Figure 1b). We confirmed the circularization between the two IRs by PCR with a prime pair facing opposite directions in the SPLCV-GFP-infiltrated leaves ( Figure 1c). The GFP transcripts in SPLCV-GFP-infiltrated leaves were 22.5 times higher than those in T-GFP ( Figure 1d).
After several days of infiltration, evident necrosis was found in most SPLCV-GFP-infiltrated leaves. To decrease cell lethality, three mutational SPLCV vectors were constructed: (i) AC4 m : a T-to-A mutation was introduced in the coding region of Rep, thereby resulting in a premature stop of translation of AC4 (AC4:26T?A (Leu9TAA); AC1:183T?A(Leu61Leu)); (ii) AC2 m : a premature termination codon mutation was introduced in the coding region of AC2, and this mutation changed one amino acid of AC1 (AC2:31A?T(Lys11TAG); AC1:1034A?T(Glu345Val)); and (iii) AC2 m /AC4 m double mutant. All constructs were sequenced to confirm the correct mutation sites. The GFP expression cassette was cloned into the three mutational vectors, and the expression efficiency was compared with that of SPLCV-GFP (WT). No visible decrease in GFP fluorescence was observed in N. benthamiana leaves infiltrated with A. tumefaciens containing these mutational constructs at days 3 and 9 ( Figure 1e). The GFP transcripts exhibited no significant difference between the WT and AC2 m / AC4 m vectors ( Figure 1f). These mutational constructs did not induce necrosis at day 12 ( Figure 1g). We then tested the RNAsilencing suppressor (RSS) activity of these basic viral vectors through a GFP silencing experiment in N. benthamiana (16C) leaves. Figure 1h shows that GFP silencing was only observed in empty and AC2 m /AC4 m vectors. This result implied that the AC2 m /AC4 m construct lost RSS activity. Insertion of up to 9 kb noncoding DNA sequence in AC2 m /AC4 m still resulted in strong GFP expression (Figure 1i). This result suggested that the cargo capacity of SPLCV was sufficient to deliver CRISPR nucleases.
We then tested whether the SPLCV vector can enhance LwaCas13a-mediated RNA targeting activity in plants (Abudayyeh et al., 2017). The AC2 m /AC4 m construct was used for this series of experiments. We artificially synthesized the DNA sequence of plant codon-optimized LwaCas13a and cloned into AC2 m /AC4 m and regular vectors with 39Flag fusion on the C terminus and a dual-flanking nuclear localization sequence (NLS) under the expression of the U4 promotor. Moreover, we designed two gRNAs against the mGFP5 transcript and recombined the targeting vectors (SPLCV-based vector: SV; T-DNA vector: TV; Figure 1j). The same vectors containing a non-targeting (NT) spacer were used as control. After 4 days of infiltration, a higher accumulation of LwaCas13a and gRNA transcripts and the LwaCas13a protein was found in SV-infiltrated leaves than in TV-infiltrated ones (Figure 1k [1993][1994][1995] right side infiltrated with SVs at day 4. This phenomenon was not apparent in TVs (Figure 1m). The SVs resulted in significantly higher levels of knockdown of mGFP5 (67% for gRNA1, 70% for gRNA2) than TVs (25% for gRNA1, 32% for gRNA2; Figure 1n). The SVs-infiltrated samples accumulated less mGFP5 protein than TVs (Figure 1o). In addition, we obtained the similar results when targeting an endogenous transcript NbPDS1 (Figure 1p). These results clearly showed that SPLCV-based RNA-targeting vectors were more efficient than regular ones.
We further tested whether SPLCV vector can enhance SpCas9and LbCas12a-mediated gene editing in plants. The AC4 mderived constructs could be efficient for gene editing in plants due to the loss of cell lethality but maintained RSS activity (Figure 1h; Mao et al., 2018). Thus, the AC4 m vector was used in this series of experiments. We cloned plant codon-optimized SpCas9 (with NLS fusion on the C terminus) and LbCas12a (with 39HA and NLS fusion on the C terminus) into AC4 m and regular vectors under the expression of the U4 promotor. We designed several crRNAs against NbPDS1 and mGFP5 in wild type and 16C N. benthamiana, respectively, and recombined the SVs and TVs (Figure 1q). The same vectors containing NT crRNA were used as control. After 6 days of infiltration, a higher accumulation of nucleases, crRNA transcripts and the nuclease protein was found in SV-infiltrated leaves than in TV-infiltrated ones (Figures 1r and  s). The average relative amplification values of the target sites obtained using the qPCR-based method were approximately 1.01 and 0.90 in NT-infiltrated and TV-infiltrated samples, respectively (Figures 1t and u;Peng et al., 2018). Such a notion suggests that the mutation frequency for TVs was approximately 10%, and this result was similar to a previous report (Bernab e-Orts et al., 2019). However, the average relative amplification values of five target sites were approximately 0.65 in SV-infiltrated samples (Figures 1t  and u). Thus, the mutation frequencies for SVs were 35% and much higher than those of TVs.
In summary, our work expands the existing list of geminiviral replicon-based vectors in plants. The SPLCV-based vectors offer an opportunity to enhance the efficiency of CRISPR-Cas-mediated gene knockdown, knockout and knockin in plants, especially for its host sweet potato; gene editing through de novo induction of meristems may also be conducted in this important hexaploid crop in the near future (Maher et al., 2020).