Knockout of the virus replication-related genes UbEF1B and CCR4/NOT3 by CRISPR/Cas9 confers high-efficiency and broad-spectrum resistance to geminiviruses in Nicotiana benthamiana.
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SUBMITTER: Li H
PROVIDER: S-EPMC10955485 | biostudies-literature | 2024 Apr
REPOSITORIES: biostudies-literature
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Knockout of the virus replication-related genes UbEF1B and CCR4/NOT3 by CRISPR/Cas9 confers high-efficiency and broad-spectrum resistance to geminiviruses in Nicotiana benthamiana.
Li Hao H Gong Pan P Xu Xiongbiao X Zhou Xueping X Li Fangfang F
Plant biotechnology journal 20231226 4
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Project description:BackgroundSingle-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)-transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency.ResultsHere, we systematically investigate the effect of these two elements on knockout efficiency and showed that modifying the sgRNA structure by extending the duplex length and mutating the fourth thymine of the continuous sequence of thymines to cytosine or guanine significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the optimized sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss of function in non-coding genes.ConclusionsBy a systematic investigation of sgRNA structure we find that extending the duplex by approximately 5 bp combined with mutating the continuous sequence of thymines at position 4 to cytosine or guanine significantly increases gene knockout efficiency in CRISPR-Cas9-based genome editing experiments.
| S-EPMC4699467 | biostudies-literature
Project description:Provided data came from a detailed study on Nicotiana benthamiana 16c plants where we use Tobacco Rattle Virus (TRV) as a molecular switch to change the chromatin state of a reporter gene (P35S::GFP) from an actively transcribed to a transcriptionally silenced state. Our approach enables us to interrogate different chromatin states of the same locus with the same set of CRISPR/Cas9 genome editing reagents and systematically describe the effect of chromatin state on the frequency and type of mutations induced at various Cas9 targets in a huge set of independently edited cells.
2022-05-04 | E-MTAB-10256 | biostudies-arrayexpress
Project description:CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons.
| S-EPMC6508695 | biostudies-literature
Project description:The selection of resistant crops is an effective method for controlling geminivirus diseases. ty-5 encodes a messenger RNA surveillance factor Pelota with a single amino acid mutation (PelotaV16G), which confers effective resistance to tomato yellow leaf curl virus (TYLCV). No studies have investigated whether ty-5 confers resistance to other geminiviruses. Here, we demonstrate that the tomato ty-5 line exhibits effective resistance to various geminiviruses. It confers resistance to two representative begomoviruses, tomato yellow leaf curl China virus/tomato yellow leaf curl China betasatellite complex and tomato leaf curl Yunnan virus. The ty-5 line also exhibits partial resistance to a curtovirus beet curly top virus. Importantly, ty-5 confers resistance to TYLCV with a betasatellite. Southern blotting and quantitative polymerase chain reaction analyses showed that significantly less DNA of these geminiviruses accumulated in the ty-5 line than in the susceptible line. Moreover, knockdown of Pelota expression converted a Nicotiana benthamiana plant from a geminivirus-susceptible host to a geminivirus-resistant host. Overall, our findings suggest that ty-5 is an important resistance gene resource for crop breeding to control geminiviruses.
| S-EPMC9415776 | biostudies-literature
Project description:The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant ΔXTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and ΔXTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.
| S-EPMC9433777 | biostudies-literature
Project description:Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N-linked glycans, including the presence of β-1,2-xylose and core α-1,3-fucose residues in plants, can affect the activity, potency and immunogenicity of plant-derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N-glycosylation machinery to allow the synthesis of complex N-glycans lacking β-1,2-xylose and core α-1,3-fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant-specific α-1,3-fucosyltransferase and β-1,2-xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry-based N-glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64-binding affinity of 2G12 glycovariants produced in wild-type N. benthamiana, the newly generated FX-KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco-engineered antibody performed as well as its CHO-produced counterpart.
| S-EPMC6335070 | biostudies-literature
Project description:BackgroundCRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS).ResultsIn this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger.ConclusionsThe tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.
| S-EPMC8943966 | biostudies-literature
Project description: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.
| S-EPMC4881029 | biostudies-literature
Project description:Key messageTobacco etch virus accumulation declined in Nicotiana benthamiana eEF1Bγ gene-edited lines, suggesting that eEF1Bγ may be a host factor for this virus. Viruses use host factors to replicate and move from cell to cell. Therefore, the editing of genes encoding viral host factors that are not essential for plant survival enables the rapid development of plants with durable virus resistance. Eukaryotic initiation factors, such as eIF4E and eIF4G, function as host factors for viral infection, and loss-of-function mutations of these factors lead to virus resistance. Broadening the spectrum of host factor targets would help expand resources for engineering virus resistance. In this study, we tested whether editing the eukaryotic translation elongation factor gene eEF1Bγ would produce virus-resistant plants. Accordingly, we targeted the four eEF1Bγ genes in Nicotiana benthamiana for editing using virus-induced gene editing (VIGE) with Tobacco rattle virus (TRV). Although we attempted to obtain plants edited for all four eEF1Bγ homologs, we failed to identify such plants. Instead, we obtained plants with three of the four homologs knocked out, harboring 1-bp insertion/deletions resulting in premature stop codons. These eEF1Bγ-edited plants did not exhibit resistance to Potato virus X (PVX), Tobacco mosaic virus (TMV), or Tomato bushy stunt virus (TBSV) but showed reduced accumulation of Tobacco etch virus (TEV) compared to wild-type plants. These findings demonstrate the feasibility of conferring resistance in plants through gene editing of eEF1Bγ, underscoring the importance of exploring diverse host factor targets for comprehensive virus resistance.
| S-EPMC11846736 | biostudies-literature
Project description: Not available
| S-EPMC9491447 | biostudies-literature