Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.

Knockout of CAFFEOYL-COA 3-O-METHYLTRANSFERASE 6/6L enhances the S/G ratio of lignin monomers and disease resistance in Nicotiana tabacum.

Background: Nicotiana tabacum is an important economic crop, which is widely planted in the world. Lignin is very important for maintaining the physiological and stress-resistant functions of tobacco. However, higher lignin content will produce lignin gas, which is not conducive to the formation of tobacco quality. To date, how to precisely fine-tune lignin content or composition remains unclear. Results: Here, we annotated and screened 14 CCoAOMTs in Nicotiana tabacum and obtained homozygous double mutants of CCoAOMT6 and CCoAOMT6L through CRSIPR/Cas9 technology. The phenotype showed that the double mutants have better growth than the wild type whereas the S/G ratio increased and the total sugar decreased. Resistance against the pathogen test and the extract inhibition test showed that the transgenic tobacco has stronger resistance to tobacco bacterial wilt and brown spot disease, which are infected by Ralstonia solanacearum and Alternaria alternata, respectively. The combined analysis of metabolome and transcriptome in the leaves and roots suggested that the changes of phenylpropane and terpene metabolism are mainly responsible for these phenotypes. Furthermore, the molecular docking indicated that the upregulated metabolites, such as soyasaponin Bb, improve the disease resistance due to highly stable binding with tyrosyl-tRNA synthetase targets in Ralstonia solanacearum and Alternaria alternata. Conclusions: CAFFEOYL-COA 3-O-METHYLTRANSFERASE 6/6L can regulate the S/G ratio of lignin monomers and may affect tobacco bacterial wilt and brown spot disease resistance by disturbing phenylpropane and terpene metabolism in leaves and roots of Nicotiana tabacum, such as soyasaponin Bb.

High-throughput creation of Nicotiana tabacum gene-targeted mutants based on CRISPR/Cas9.

KEY MESSAGE: 4382 available sgRNAs targeting 1060 tobacco genes were obtained, and 10,682 targeted mutants were created using high-throughput methods. Four optimization experiments were established to solve problems encountered during genetic transformation.

Effects of editing DFR genes on flowers, leaves, and roots of tobacco.

BACKGROUND: DFR is a crucial structural gene in plant flavonoid and polyphenol metabolism, and DFR knockout (DFR-KO) plants may have increased biomass accumulation. It is uncertain whether DFR-KO has comparable effects in tobacco and what the molecular mechanism is. We employed the CRISPR/Cas9 method to generate a knockout homozygous construct and collected samples from various developmental phases for transcriptome and metabolome detection and analysis. RESULTS: DFR-KO turned tobacco blossoms white on homozygous tobacco (Nicotiana tabacum) plants with both NtDFR1 and NtDFR2 knockout. RNA-seq investigation of anthesis leaf (LF), anthesis flower (FF), mature leaf (LM), and mature root (RM) variations in wild-type (CK) and DFR-KO lines revealed 2898, 276, 311, and 101 differentially expressed genes (DEGs), respectively. DFR-KO primarily affected leaves during anthesis. According to KEGG and GSEA studies, DFR-KO lines upregulated photosynthetic pathway carbon fixation and downregulated photosystem I and II genes. DFR-KO may diminish tobacco anthesis leaf photosynthetic light reaction but boost dark reaction carbon fixation. DFR-KO lowered the expression of pathway-related genes in LF, such as oxidative phosphorylation and proteasome, while boosting those in the plant-pathogen interaction and MAPK signaling pathways, indicating that it may increase biological stress resistance. DFR-KO greatly boosted the expression of other structural genes involved in phenylpropanoid production in FF, which may account for metabolite accumulation. The metabolome showed that LF overexpressed 8 flavonoid metabolites and FF downregulated 24 flavone metabolites. In DFR-KO LF, proteasome-related genes downregulated 16 amino acid metabolites and reduced free amino acids. Furthermore, the DEG analysis on LM revealed that the impact of DFR-KO on tobacco growth may progressively diminish with time. CONCLUSION: The broad impact of DFR-KO on different phases and organs of tobacco development was thoroughly and methodically investigated in this research. DFR-KO decreased catabolism and photosynthetic light reactions in leaves during the flowering stage while increasing carbon fixation and disease resistance pathways. However, the impact of DFR-KO on tobacco growth steadily declined as it grew and matured, and transcriptional and metabolic modifications were consistent. This work offers a fresh insight and theoretical foundation for tobacco breeding and the development of gene-edited strains.

Developing an efficient and visible prime editing system to restore tobacco 8-hydroxy-copalyl diphosphate gene for labdane diterpene Z-abienol biosynthesis.

Prime editing (PE) is a versatile CRISPR-Cas based precise genome-editing platform widely used to introduce a range of possible base conversions in various organisms. However, no PE systems have been shown to induce heritable mutations in tobacco, nor in any other dicot. In this study, we generated an efficient PE system in tobacco that not only introduced heritable mutations, but also enabled anthocyanin-based reporter selection of transgene-free T1 plants. This system was used to confer Z-abienol biosynthesis in the allotetraploid tobacco cultivar HHDJY by restoring a G>T conversion in the NtCPS2 gene. High levels of Z-abienol were detected in the leaves of homozygous T1 plants at two weeks after topping. This study describes an advance in PE systems and expands genome-editing toolbox in tobacco, even in dicots, for use in basic research and molecular breeding. And restoring biosynthesis of Z-abienol in tobacco might provide an efficient way to obtain Z-abienol in plants.

Isochromenes from the Nicotiana tabacum-derived endophytic fungus Aspergillus versicolor and their anti-tobacco mosaic virus activities.

Three new isochromenes, (5-methoxy-7-prenyl-1H-isochromen-3-yl)methanol (1), 3-(3-(hydroxymethyl)-5-methoxy-1H-isochromen-7-yl)propan-1-ol (2), and (5-methoxy-7-methyl-1H-isochromen-3-yl)methanol (3), along with three known analogues (4-6) were isolated from the fermentation products of a Nicotiana tabacum-derived endophytic fungus Aspergillus versicolor. Their structures were elucidated by spectroscopic methods, including extensive 1 D and 2 D NMR techniques. Compounds 1-3 and 6 were evaluated for their anti-tobacco mosaic virus (anti-TMV) activities. The results showed that compound 2 exhibited high anti-TMV activity with inhibition rate of 46.4%, and this rate is higher than that of positive control. Compounds 1, 3, and 6 also showed potential anti-TMV activity with inhibition rates of 28.6, 30.5, and 26.2%, respectively. The IC50 of compounds 1-3 and 6 were also tested, and showed IC50 values of 49.3, 22.4, 42.2, and 54.1 µM, respectively.

Highly efficient transgene-free genome editing in tobacco using an optimized CRISPR/Cas9 system, pOREU3TR.

CRISPR/Cas9 genome-editing technology has revolutionized plant science and holds enormous promise for crop improvement. The exploration of this system received much attention regarding plant genome editing. Here, by editing the NtPDS gene in tobacco, we first verified that incorporating an OsU3-tRNA promoter combination into the CRISPR/Cas9 system contributed to the highest editing efficiency, as the sgRNA expression level was greater than that resulting from the AtU6-tRNA and AtU6 promoters. Then, we optimized the existing tobacco CRISPR/Cas9 system, pORE-Cas9, by using the OsU3-tRNA promoter combination instead of AtU6 and by fusing an AtUb10-Ros1 expression cassette to the T-DNA to monitor the transgene events. The new system was named pOREU3TR. As expected, 49 transgene-free and homozygous gene-edited green plants were effectively screened in the T1 generation as a result of editing the NtLHT1 gene in tobacco, and the plant height and the contents of most free amino acids in the leaves of the T2 mutant plants were significantly different from those in the leaves of WT plants, demonstrating the high efficiency of the new editing system. This OsU3-tRNA-sgRNA/AtUb10-Ros1 system provides essential improvements for increasing the efficiency of plant genome editing.

The anti-TMV potency of the tobacco-derived fungus Aspergillus versicolor and its active alkaloids, as anti-TMV activity inhibitors.

Nicotiana tabacum (tobacco) has attracted interest as one of the most economically important industrial crops widely cultivated in China, whose dried leaves are popularly consumed medicinally and recreationally by human societies. In this study, five undescribed alkaloids derivatives, isoaspergillines A-E, together with eight known alkaloids, notoamide D, (1R,4S)-4-benzyl-1-isopropyl-2,4-dihydro-1H-pyrazino-[2,1-b]quinazoline-3,6-dione, protuboxepin K, notoamide C, notoamide M, deoxybrevianamide E, cyclo (D-Pro-L-Trp), and versicolamide B, were obtained from the culture of the Nicotiana tabacum-derived fungus Aspergillus versicolor. Their structures were mainly elucidated through comprehensive analyses of spectroscopic data. Bioactivity evaluation of all isolated compounds revealed that isoaspergilline A and notoamide M exhibited anti-TMV activities with IC50 values of 20.0 and 22.8 µM, respectively. Molecular docking suggested that isoaspergilline A and notoamide M were well located into the active site of anti-TMV by interacting with SER138, SER143, and ASN73 residues. This study enlightens the therapeutic potential of the endophytic fungus A. versicolor and it is helpful to find undescribed anti-TMV activity inhibitors, as well as searching for new anti-TMV candidates from natural sources.

The Agronomic Traits, Alkaloids Analysis, FT-IR and 2DCOS-IR Spectroscopy Identification of the Low-Nicotine-Content Nontransgenic Tobacco Edited by CRISPR-Cas9.

In this study, the agricultural traits, alkaloids content and Fourier transform infrared spectroscopy (FT-IR) and two-dimensional correlation infrared spectroscopy (2DCOS-IR) analysis of the tobacco after Berberine Bridge Enzyme-Like Proteins (BBLs) knockout were investigated. The knockout of BBLs has limited effect on tobacco agricultural traits. After the BBLs knockout, nicotine and most alkaloids are significantly reduced, but the content of myosmine and its derivatives increases dramatically. In order to identify the gene editing of tobacco, principal component analysis (PCA) was performed on the FT-IR and 2DCOS-IR spectroscopy data. The results showed that FT-IR can distinguish between tobacco roots and leaves but cannot classify the gene mutation tobacco from the wild one. 2DCOS-IR can enhance the characteristics of the samples due to the increased apparent resolution of the spectra. Using the autopeaks in the synchronous map for PCA analysis, we successfully identified the mutants with an accuracy of over 90%.

Cyclopiazonic acid type indole alkaloids from Nicotiana tabacum-derived fungus Aspergillus versicolor and their anti-tobacco mosaic virus activities.

Indole alkaloids have attracted widespread attention of chemists and biologists. Therefore, the aim of this study is to screen more bioactivities indole alkaloids from the microorganisms. In this study, five undescribed CPA-type indole alkaloids, aspergillines F-J, and three known CPA-type indole alkaloids, aspergilline A, aspergilline C, and cyclopiamide E, were obtained from the Nicotiana tabacum-derived fungus Aspergillus versicolor. Notably, aspergillines F and G represent the first examples of indole alkaloids with a benzo[cd]indol-2(1H)-one skeleton, and aspergilline J is also the firstly obtained indole alkaloids bearing a N-1-(2-(1H-imidazole-5-yl)ethyl) moiety. Aspergillines F-J and cyclopiamide E were tested for their anti-TMV activities, and the results revealed that aspergillines G and J exhibited obvious anti-TMV activities with inhibition rates of 41.2 and 56.8% at the concentration of 20 µM, respectively. These rates are high than that of positive control (with inhibition rate of 32.5%). In addition, the molecular docking studies for the isolated CPA-type indole alkaloids may also reveal that the benzo[cd]indol-2(1H)-one substructure is the fundamental for anti-TMV activity and the oxygen-containing substituent groups at C-19 also increases the inhibitory activity. This study of structure-activity relationship is helpful to find new anti-TMV activity inhibitors.

Tissue-specific genome editing of laminA/C in the posterior silk glands of Bombyx mori.

The RNA-guided CRISPR/Cas9 system has been shown to be a powerful tool for genome editing in various organisms. A comprehensive toolbox for multiplex genome editing has been developed for the silkworm, Bombyx mori, a lepidopteran model insect of economic importance. However, as previous methods mainly relied on delivery of transient Cas9/guide RNA (gRNA), they could not be used in loss-of-function studies of essential genes. Here, we report a simple and versatile tissue-specific genome editing strategy. We perform a proof-of-principle demonstration by establishing and crossing two transgenic B. mori lines, one expressing Cas9 protein in the posterior silk glands (PSGs) and the other constitutively expressing BmlaminA/C (BmLMN) gRNA. All BmLMN alleles in the PSG cells were edited precisely at the target genome region, resulting in diverse mutations. mRNA expression of BmLMN was reduced by up to 75%, and only very low levels of BmLaminA/C protein were detected. Knockout of BmLMN produced obvious defects in gland cell development and cocoon production. In this study, we developed an efficient strategy for spatially controlled genome editing, providing unprecedented opportunities for investigating the function of essential/lethal genes in B. mori, with potential application for other insects.

Genome editing of BmFib-H gene provides an empty Bombyx mori silk gland for a highly efficient bioreactor.

Evolution has produced some remarkable creatures, of which silk gland is a fascinating organ that exists in a variety of insects and almost half of the 34,000 spider species. The impressive ability to secrete huge amount of pure silk protein, and to store proteins at an extremely high concentration (up to 25%) make the silk gland of Bombyx mori hold great promise to be a cost-effective platform for production of recombinant proteins. However, the extremely low production yields of the numerous reported expression systems greatly hindered the exploration and application of silk gland bioreactors. Using customized zinc finger nucleases (ZFN), we successfully performed genome editing of Bmfib-H gene, which encodes the largest and most abundant silk protein, in B. mori with efficiency higher than any previously reported. The resulted Bmfib-H knocked-out B. mori showed a smaller and empty silk gland, abnormally developed posterior silk gland cells, an extremely thin cocoon that contain only sericin proteins, and a slightly heavier pupae. We also showed that removal of endogenous Bmfib-H protein could significantly increase the expression level of exogenous protein. Furthermore, we demonstrated that the bioreactor is suitable for large scale production of protein-based materials.

Highly efficient multiplex targeted mutagenesis and genomic structure variation in Bombyx mori cells using CRISPR/Cas9.

Bombyx mori is an economically important insect and a model organism for studying lepidopteran and arthropod biology. Using a highly efficient CRISPR/Cas9 system, we showed that this system could mediate highly efficient targeted genome editing of a single gene locus, large chromosomal deletion or inversion, and also multiplex genome editing of 6 genes simultaneously in BmNs cell line derived from B. mori. The simplicity and high efficiency of our system provide unprecedented possibilities for researchers to implement precise and sophisticated manipulation of a chosen B. mori gene in BmNs cells easily in a limited time course, and perhaps new opportunities for functional genomics of B. mori and other lepidopteran insects.

CRISPR/Cas9 mediated multiplex genome editing and heritable mutagenesis of BmKu70 in Bombyx mori.

CRISPR/Cas9, a bacterial adaptive immune system derived genome-editing technique, has become to be one of the most compelling topics in biotechnology. Bombyx mori is an economically important insect and a model organism for studying lepidopteran and arthropod biology. Here we reported highly efficient and multiplex genome editing in B. mori cell line and heritable site-directed mutagenesis of Bmku70, which is required for NHEJ pathway and also related to antigen diversity, telomere length maintenance and subtelomeric gene silencing, using CRISPR/Cas9 system. We established a simple and practicable method and obtained several Bmku70 knockout B. mori lines, and showed that the frequency of HR was increased in embryos of the Bmku70 knockout B. mori. The mutant lines obtained in this study could be a candidate genetic resource for efficient knock-in and fundamental research of DNA repair in B. mori. We also provided a strategy and procedure to perform heritable genome editing of target genes with no significant phenotype effect.