Opportunities and challenges with CRISPR-Cas mediated homologous recombination based precise editing in plants and animals.

KEY MESSAGE: We summarise recent advancements to achieve higher homologous recombination based gene targeting efficiency in different animals and plants. The genome editing has revolutionized the agriculture and human therapeutic sectors by its ability to create precise, stable and predictable mutations in the genome. It depends upon targeted double-strand breaks induction by the engineered endonucleases, which then gets repaired by highly conserved endogenous DNA repair mechanisms. The repairing could be done either through non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The HDR-based editing can be applied for precise gene targeting such as insertion of a new gene, gene replacement and altering of the regulatory sequence of a gene to control the existing protein expression. However, HDR-mediated editing is considered challenging because of lower efficiency in higher eukaryotes, thus, preventing its widespread application. This article reviews the recent progress of HDR-mediated editing and discusses novel strategies such as cell cycle synchronization, modulation of DNA damage repair factors, engineering of Cas protein favoring HDR and CRISPR-Cas reagents delivery methods to improve efficiency for generating knock-in events in both plants and animals. Further, multiplexing of described methods may be promising towards achieving higher donor template-assisted homologous recombination efficiency at the target locus.

Genome-wide identification and expression profiling of WUSCHEL-related homeobox (WOX) genes confer their roles in somatic embryogenesis, growth and abiotic stresses in banana.

Plant-specific WUSCHEL-related homeobox (WOX) transcription factors are known to be involved in plant developmental processes, especially in embryogenesis. In this study, a total of thirteen WOX members were identified in the banana (Musa acuminata) genome (MaWOX) and characterized for in-silico analysis. Phylogenetic analysis revealed that these genes were divided into three clades (ancient, intermediate and modern) which reflected the evolutionary history of WOX families. Furthermore, modern clade members have shown higher variations in gene structural features and carried unique conserved motifs (motif 3 and motif 4) when compared to the members of other clades. The differential expression of all 13 MaWOX was observed in early (embryogenic cell suspension (ECS), multiplying ECS, germinating embryos, young leaflet and node of germinated plantlets) and late (unripe fruit peel and pulp, ripe fruit peel and pulp) developmental stages of banana cultivar Grand Naine. The maximum expression of MaWOX6 (18 fold) and MaWOX13 (120 fold) was found during somatic embryogenesis and in unripe fruit pulp, respectively. Moreover, numerous cis-elements responsive to drought, cold, ethylene, methyl jasmonate (MeJA), abscisic acid (ABA) and gibberellic acid (GA) were observed in all MaWOX promoter regions. The subsequent expression analysis under various abiotic stresses (cold, drought and salt) revealed maximum expression of the MaWOX3 (830 fold), MaWOX8a (30 fold) and MaWOX11b (105 fold) in salt stress. It gives evidence about their possible role in salt stress tolerance in banana. Hence, the present study provides precise information on the MaWOX gene family and their expression in various tissues and stressful environmental conditions that may help to develop climate-resilient banana plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03387-w.