Browsing by Author "Alok, Anshu"

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Compendium of plant-specific CRISPR vectors and their technical advantages
(MDPI AG, 2021) Alok, Anshu; Chauhan, Hanny; Upadhyay, Santosh Kumar; Pandey, Ashutosh; Kumar, Jitendra; Singh, Kashmir
CRISPR/Cas mediated genome editing is a revolutionary approach for manipulating the plant genome. However, the success of this technology is highly dependent on selection of a specific vector and the other components. A plant-specific CRISPR/Cas vector usually consists of a Cas gene, target-specific gRNA, leader sequence, selectable marker gene, precise promoters, and other accessories. It has always been challenging to select the specific vector for each study due to a lack of comprehensive information on CRISPR vectors in one place. Herein, we have discussed every technical aspect of various important elements that will be highly useful in vector selection and efficient editing of the desired plant genome. Various factors such as the promoter regulating the expression of Cas and gRNA, gRNA size, Cas variants, multicistronic gRNA, and vector backbone, etc. influence transformation and editing frequency. For example, the use of polycistronic tRNA-gRNA, and Csy4-gRNA has been documented to enhance the editing efficiency. Similarly, the selection of an efficient selectable marker is also a very important factor. Information on the availability of numerous variants of Cas endonucleases, such as Cas9, Cas12a, Cas12b, Casɸ, and CasMINI, etc., with diverse recognition specificities further broadens the scope of editing. The development of chimeric proteins such as Cas fused to cytosine or adenosine deaminase domain and modified reverse transcriptase using protein engineering enabled base and prime editing, respectively. In addition, the newly discovered Casɸ and CasMINI would increase the scope of genetic engineering in plants by being smaller Cas variants. All advancements would contribute to the development of various tools required for gene editing, targeted gene insertion, transcriptional activation/suppression, multiplexing, prime editing, base editing, and gene tagging. This review will serve as an encyclopedia for plant-specific CRISPR vectors and will be useful for researchers.
CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit
(Elsevier B.V., 2020) Kaur, Navneet; Alok, Anshu; Shivani; Kumar, Pankaj; Kaur, Navjot; Awasthi, Praveen; Chaturvedi, Siddhant; Pandey, Pankaj; Pandey, Ashutosh; Pandey, Ajay K.; Tiwari, Siddharth
Banana is one of the most economically important fruit crops worldwide. Genetic improvement in banana is a challenging task due to its parthenocarpic nature and triploid genome. Genetic modification of crops via the CRISPR/Cas9 module has emerged as a promising tool to develop important traits. In the present work, a CRISPR/Cas9-based approach was used to develop the β-carotene-enriched Cavendish banana cultivar (cv.) Grand Naine (AAA genome). The fifth exon of the lycopene epsilon-cyclase (LCYε) gene was targeted. The targeting specificity of the designed guide-RNA was also tested by its ability to create indels in the LCYε gene at the A genome of cv. Rasthali (AAB genome). Sequence analysis revealed multiple types of indels in the genomic region of Grand Naine LCYε (GN-LCYε). Metabolic profiling of the fruit pulp of selected edited lines showed enhanced accumulation of β-carotene content up to 6-fold (~24 μg/g) compared with the unedited plants. These lines also showed either an absence or a drastic reduction in the levels of lutein and α-carotene, suggesting metabolic reprogramming, without any significant effect on the agro-morphological parameters. In addition, differential expression of carotenoid pathway genes was observed in the edited lines in comparison to unedited plants. Overall, this is the first report in banana to improve nutritional trait by using a precise genome editing approach.
CRISPR/Cas9 edited StbHLH47 lines exhibit altered expression profiling of iron regulating genes and increased iron content in Solanum tuberosum
(Elsevier B.V., 2024) Chauhan, Hanny; Alok, Anshu; Aiana; Upadhyay, Santosh K.; Pandey, Ashutosh; Singh, Kashmir
Iron is an essential plant nutrient, and a continuous supply of it is required as it is a key factor in various metabolic processes, including photosynthesis, chlorophyll synthesis, and respiration. Various transcription factors are known to regulate iron homeostasis in plants, and the bHLH transcription factor family is one of them. The StbHLH47 is a homologue of the Arabidopsis POPEYE (PYE), which is known to repress iron homeostasis-related genes in Arabidopsis. Potato is the most consumed vegetable in the world and is low in iron content. We have generated CRISPR/Cas9-edited StbHLH47 lines and performed a detailed analysis of these lines. The analysis revealed that the roots of StbHLH47 edited lines have decreased ferric chelate reductase (FCR) activity compared to the roots of the wild-type (WT) plant. We also observed that CRISPR/Cas9 edited lines have fewer trichomes when compared to the WT plant. The expression of genes associated with iron homeostasis was also measured. Compared to the control, the expression of StbHLH47 was downregulated in the edited lines, while the expression of StNAS4, StOPT3, and StFRO3 was upregulated. This suggests the negative regulation of StbHLH47 in modulating iron. The iron content was also quantified using inductively coupled plasma mass spectrometry (ICP-MS) and found to be increased in the generated transgenic lines when compared to WT plants. Overall, this study reveals that StbHLH47 negatively regulates the expression of iron homeostasis-related genes. StbHLH47 edited lines exhibited decreased FCR activity, changes in phenotype, and increased iron content in the potato plants.
CRISPR/dCas9-KRAB mediated transcriptional suppression of NtbHLH47 enhances tolerance to iron stress and modulates iron content in tobacco
(Elsevier B.V., 2025) Alok, Anshu; Chauhan, Hanny; Rout, Biswaranjan; Pandey, Ashutosh; Singh, Kashmir
Iron homeostasis is a multifaceted regulatory process that needs to be studied to elucidate iron distribution, uptake, and storage in plants. NtbHLH47, a homologue to AtbHLH47, is a negative regulator of iron. The current study deploys CRISPR interference-dCas9-KRAB (Krüppel-associated box) in the transcriptional suppression of NtbHLH47 and its effect on iron uptake by plants. The pHSN6I01 harbouring dCas9-KRAB and gRNA targeting NtbHHLH47 was constructed. Four gRNAs were designed, G1, G2, G3, and G4, located at + 19, + 111, + 232, and + 335 bp upstream from the ATG start codon in the promoter region of NtbHLH47. The NtbHLH47 was repressed in the developed transgenic lines of tobacco and the qRT-PCR analysis showed that target sites G1 and G2 suppressed NtbHLH47 effectively. The transgenic pHSN6I01 +G1 plants were tolerant to the elevated levels of iron, copper, zinc, and magnesium. The root Ferric chelate reductase activity of pHSN6I01 +G1 lines was reduced against wild type. The Perl staining showed high iron content in the roots of the pHSN6I01 +G1 plants. ICP-MS analysis showed increased Fe content in the roots of pHSN6I01 +G1 line suggesting that NtbHLH47 modulates it. The expression of NtbHLH38, NtbHLH100, NtbHLH101, and NtFIT was found to be upregulated in the pHSN6I01 +G1 line. This is the first report of using CRISPRi based on dCas9-KRAB in tobacco and its application in the functional validation of a gene. Using this, NtbHLH47 was transcriptionally suppressed and the generated lines expressed increased levels of iron in the roots of N. tabacum and gave insight in the iron homeostasis.
Heterologous expression of Chlorophytum borivilianum Squalene epoxidase in tobacco modulates stigmasterol production and alters vegetative and reproductive growth
(Springer Nature Publishing AG, 2023) Kaushal, Nishant; Verma, Deepika; Alok, Anshu; Pandey, Ashutosh; Singh, Kashmir
Chlorophytum borivilianum is a high-value medicinal plant with many promising preclinical applications that include saponins as a major active ingredient. Squalene epoxidase (SE) is one of the major rate-limiting enzymes of the saponin biosynthetic pathway. Here, we functionally characterized C. borivilianum SE (CbSE) by over-expressing heterologously in Nicotiana tabacum. The heterologous expression of CbSE resulted in stunted pant growth with altered leaf and flower morphology. Next, RT-qPCR analysis of transgenic plants overexpressing CbSE revealed increased expression levels of Cycloartenol synthase (CAS), Beta amyrin synthase (βAS), and cytochrome P450 monooxygenase 51 (CYP51) (Cytochrome P450), which encode key enzymes for triterpenoid and phytosterol biosynthesis in C. borivilianum. Further, Methyl Jasmonate (MeJa) treatment upregulated Squalene synthase (SQS), SE, and Oxidosqualene cyclases (OSCs) to a significant level. GC–MS analysis of the leaf and hairy roots of the transformants showed an increased stigmasterol content (0.5–1.0 fold) compared to wild type (WT) plants. These results indicate that CbSE is a rate-limiting gene, which encodes an efficient enzyme responsible for phytosterol and triterpenoid production in C. borivilianum.
Targeted metabolite profiling of five cultivars of Vitis vinifera L. fruits
(American Chemical Society, 2021) Alok, Anshu; Tyagi, Shivi; Singh, Kashmir; Pandey, Ashutosh
Grapes (Vitis vinifera L.) are a very popular fruit and are produced around the world. They are consumed as a fresh ripe fruit, dried fruit, juice, and processed product such as wine and are a good source of human nutrition. We aimed to investigate and analyze the nutritional and phytochemical components of grape cultivars, mostly cultivated in different regions of India. The targeted metabolic profiling of these cultivars was examined. A total of 58 metabolites, including three major sugars, nine organic acids, 14 amino acids, nine vitamins, and 23 phenolic compounds, were quantified in ripe berries of these grapes. Mineral quantification showed that these varieties contain major elements (K, Mg, and Ca) and minor elements (Mn, Fe, Zn, Cu, and Na). Principal component analysis showed that the contents of sugars, organic acids, and phenolic compounds are almost similar in cultivars FS and BJ, whereas the vitamin contents are nearly similar in TS and GS. Overall, these findings reveal that there is a differential in the accumulated content of primary and specialized metabolites as well as minerals in ripe fruits, providing a source for commercial value and food industry applications.