Transposase-assisted target-site integration for efficient plant genome engineering.

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops1,2. Often considered to be genome 'parasites', transposable elements (TEs) evolved to insert their DNA seamlessly into genomes3-5. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type6-9. Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria10-12, we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean-a major global crop in need of targeted insertion technology. We have engineered a TE 'parasite' into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.

Nitrate supply regulates tissue calcium abundance and transcript level of Calcineurin B-like (CBL) gene family in wheat.

Calcium ion (Ca2+) is the most ubiquitous signalling molecule and is sensed by different classes of Ca2+ sensor proteins. Recent evidences underscore the role of calcium signalling in plant response to nitrogen/nitrate supply. Recently we found that under nitrate deficiency, a short-term supply of calcium could improve the plant biomass, nitrate assimilation, anthocyanin accumulation and expression of nitrate uptake and signalling genes. Long-term calcium supply, on the other hand, was not beneficial. Calcineurin B-like (CBL) proteins are one of the vital plant Ca2+ sensory protein family which is essential for stress perception and signaling. To understand the dynamics of CBL-mediated stress signalling in bread wheat, we identified CBL genes in bread wheat (Triticum aestivum) and its progenitors, namely Triticum dicoccoides, Triticum urartu and Aegilops tauschii with the aid of newly available whole-genome sequence. The expression of different CBLs and the changes in root Ca2+ localization in response to nitrate provision or deficiency were analysed. Expression of the CBLs were studied in two bread wheat genotypes with comparatively higher (B.T. Schomburgk, BTS) and lower (Gluyas early, GE) nitrate responsiveness and nitrogen use efficiency. High N promoted the expression of CBLs in seedling leaves while in roots the expression was promoted by N deficiency. At the 5 days after anthesis stage, nitrate starvation downregulated the expression of CBLs while nitrate supply enhanced the expression. At anthesis stage, expression of CBL6 was significantly promoted by HN in panicles of both the genotypes, the highest expression was recorded in BTS. Expression of CBL6 was significantly upregulated by short term nitrate treatment also suggesting its role in Primary nitrate response (PNR) in wheat. There was a significant down regulation of CBL6 expression post nitrate starvation, making it a probable regulator of nitrogen starvation response (NSR) as well. In seedling roots, the tissue localization of Ca2+ was increased both by high and low nitrate treatments, albeit at different magnitudes. Our results suggest that calcium signalling might be a major signalling pathway governing nitrogen responsiveness and CBL6 might be playing pivotal role in NSR and PNR in wheat.

Genome Editing Targets for Improving Nutrient Use Efficiency and Nutrient Stress Adaptation.

In recent years, the development of RNA-guided genome editing (CRISPR-Cas9 technology) has revolutionized plant genome editing. Under nutrient deficiency conditions, different transcription factors and regulatory gene networks work together to maintain nutrient homeostasis. Improvement in the use efficiency of nitrogen (N), phosphorus (P) and potassium (K) is essential to ensure sustainable yield with enhanced quality and tolerance to stresses. This review outlines potential targets suitable for genome editing for understanding and improving nutrient use (NtUE) efficiency and nutrient stress tolerance. The different genome editing strategies for employing crucial negative and positive regulators are also described. Negative regulators of nutrient signalling are the potential targets for genome editing, that may improve nutrient uptake and stress signalling under resource-poor conditions. The promoter engineering by CRISPR/dead (d) Cas9 (dCas9) cytosine and adenine base editing and prime editing is a successful strategy to generate precise changes. CRISPR/dCas9 system also offers the added advantage of exploiting transcriptional activators/repressors for overexpression of genes of interest in a targeted manner. CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) are variants of CRISPR in which a dCas9 dependent transcription activation or interference is achieved. dCas9-SunTag system can be employed to engineer targeted gene activation and DNA methylation in plants. The development of nutrient use efficient plants through CRISPR-Cas technology will enhance the pace of genetic improvement for nutrient stress tolerance of crops and improve the sustainability of agriculture.