CRISPR/Cas9 is a powerful tool for precise genome editing of legume crops: a review.

Legumes are an imperative source of food and proteins across the globe. They also improve soil fertility through symbiotic nitrogen fixation (SNF). Genome editing (GE) is now a novel way of developing desirable traits in legume crops. Genome editing tools like clustered regularly interspaced short palindromic repeats (CRISPR) system permits a defined genome alteration to improve crop performance. This genome editing tool is reliable, cost-effective, and versatile, and it has to deepen in terms of use compared to other tools. Recently, many novel variations have drawn the attention of plant geneticists, and efforts are being made to develop trans-gene-free cultivars for ensuring biosafety measures. This review critically elaborates on the recent development in genome editing of major legumes crops. We hope this updated review will provide essential informations for the researchers working on legumes genome editing. In general, the CRISPR/Cas9 novel GE technique can be integrated with other techniques like omics approaches and next-generation tools to broaden the range of gene editing and develop any desired legumes traits. Regulatory ethics of CRISPR/Cas9 are also discussed.

Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands.

BACKGROUND: G-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. In a previous study, we identified 1998 G-quadruplex-forming sequences using a mouse CpG islands DNA microarray with a fluorescent-labeled G-quadruplex ligand. Among these putative G-quadruplex-forming sequences, G-quadruplex formation was verified for 10 randomly selected sequences by CD spectroscopy and DMS footprinting analysis. In this study, the biological function of the 10 G-quadruplex-forming sequences in the transcriptional regulation has been analyzed using a reporter assay. RESULTS: When G-quadruplex-forming sequences from the Dele and Cdc6 genes have been cloned in reporter vectors carrying a minimal promoter and the luciferase gene, luciferase expression is activated. This has also been detected in experiments applying a promoterless reporter vector. Mutational analysis reveals that guanine bases, which form the G-tetrads, are important in the activation. In addition, the activation has been found to decrease by the telomestatin derivative L1H1-7OTD which can bind to the G-quadruplex DNA. When Dele and Cdc6 CpG islands, containing the G-quadruplex-forming sequence, have been cloned in the promoterless reporter vector, the luciferase expression is activated. Mutational analysis reveals that the expression level is decreased by mutation on Dele G-quadruplex; however, increased by mutation on Cdc6 G-quadruplex. CONCLUSION: Dele and Cdc6 G-quadruplex formation is significant in the transcriptional regulation. Dele and Cdc6 G-quadruplex DNA alone possess enhancer and promotor function. When studied in more complex CpG islands Dele G-quadruplex also demonstrates promotor activity, whereas Cdc6 G-quadruplex may possess a dual function of transcriptional regulation.