Improving CRISPR-Cas-mediated RNA targeting and gene editing using SPLCV replicon-based expression vectors in Nicotiana benthamiana.
Improving CRISPR-Cas-mediated RNA targeting and gene editing using SPLCV replicon-based expression vectors in Nicotiana benthamiana.
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DOI：
10.1111/pbi.13384
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发表时间：
2020-10
影响因子：
13.8
通讯作者：
Sun J
中科院分区：
文献类型：
--
作者：
Yu Y;Wang X;Sun H;Liang Q;Wang W;Zhang C;Bian X;Cao Q;Li Q;Xie Y;Ma D;Li Z;Sun J
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-Durán, 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-Durán, 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) AC4m: 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) AC2m: 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) AC2m/AC4m 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 AC2m/AC4m vectors (Figure 1f). These mutational constructs did not induce necrosis at day 12 (Figure 1g). We then tested the RNA-silencing 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 AC2m/AC4m vectors. This result implied that the AC2m/AC4m construct lost RSS activity. Insertion of up to 9 kb noncoding DNA sequence in AC2m/AC4m still …
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影响因子：
12.3
作者：
Mao Y;Yang X;Zhou Y;Zhang Z;Botella JR;Zhu JK
通讯作者：
Zhu JK
影响因子：
12.3
作者：
Čermák T;Baltes NJ;Čegan R;Zhang Y;Voytas DF
通讯作者：
Voytas DF
影响因子：
64.8
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Abudayyeh OO;Gootenberg JS;Essletzbichler P;Han S;Joung J;Belanto JJ;Verdine V;Cox DBT;Kellner MJ;Regev A;Lander ES;Voytas DF;Ting AY;Zhang F
通讯作者：
Zhang F
影响因子：
46.9
作者：
Maher, Michael F.;Nasti, Ryan A.;Voytas, Daniel F.
通讯作者：
Voytas, Daniel F.
影响因子：
2.7
作者：
Bi, Huiping;Zhang, Peng
通讯作者：
Zhang, Peng