Utilizing CRISPR-Cas in Tropical Crop Improvement: A Decision Process for Fitting Genome Engineering to Your Species

Adopting modern gene-editing technologies for trait improvement in agriculture requires important workflow developments, yet these developments are not often discussed. Using tropical crop systems as a case study, we describe a workflow broken down into discrete processes with specific steps and decision points that allow for the practical application of the CRISPR-Cas gene editing platform in a crop of interest. While we present the steps of developing genome-edited plants as sequential, in practice parts can be done in parallel, which are discussed in this perspective. The main processes include 1) understanding the genetic basis of the trait along with having the crop’s genome sequence, 2) testing and optimization of the editing reagents, development of efficient 3) tissue culture and 4) transformation methods, and 5) screening methods to identify edited events with commercial potential. Our goal in this perspective is to help any lab that wishes to implement this powerful, easy-to-use tool in their pipeline, thus aiming to democratize the technology.

CicerSpTEdb: A web-based database for high-resolution genome-wide identification of transposable elements in Cicer species

Recently, Cicer species have experienced increased research interest due to their economic importance, especially in genetics, genomics, and crop improvement. The Cicer arietinum, Cicer reticulatum, and Cicer echinospermum genomes have been sequenced and provide valuable resources for trait improvement. Since the publication of the chickpea draft genome, progress has been made in genome assembly, functional annotation, and identification of polymorphic markers. However, work is still needed to identify transposable elements (TEs) and make them available for researchers. In this paper, we present CicerSpTEdb, a comprehensive TE database for Cicer species that aims to improve our understanding of the organization and structural variations of the chickpea genome. Using structure and homology-based methods, 3942 C. echinospermum, 3579 C. reticulatum, and 2240 C. arietinum TEs were identified. Comparisons between Cicer species indicate that C. echinospermum has the highest number of LTR-RT and hAT TEs. C. reticulatum has more Mutator, PIF Harbinger, Tc1 Mariner, and CACTA TEs, while C. arietinum has the highest number of Helitron. CicerSpTEdb enables users to search and visualize TEs by location and download their results. The database will provide a powerful resource that can assist in developing TE target markers for molecular breeding and answer related biological questions. Database URL: http://cicersptedb.easyomics.org/index.php

Toward Integrated Multi-Omics Intervention: Rice Trait Improvement and Stress Management

Rice (Oryza sativa) is an imperative staple crop for nearly half of the world’s population. Challenging environmental conditions encompassing abiotic and biotic stresses negatively impact the quality and yield of rice. To assure food supply for the unprecedented ever-growing world population, the improvement of rice as a crop is of utmost importance. In this era, “omics” techniques have been comprehensively utilized to decipher the regulatory mechanisms and cellular intricacies in rice. Advancements in omics technologies have provided a strong platform for the reliable exploration of genetic resources involved in rice trait development. Omics disciplines like genomics, transcriptomics, proteomics, and metabolomics have significantly contributed toward the achievement of desired improvements in rice under optimal and stressful environments. The present review recapitulates the basic and applied multi-omics technologies in providing new orchestration toward the improvement of rice desirable traits. The article also provides a catalog of current scenario of omics applications in comprehending this imperative crop in relation to yield enhancement and various environmental stresses. Further, the appropriate databases in the field of data science to analyze big data, and retrieve relevant information vis-à-vis rice trait improvement and stress management are described.

Von Genen zu Chromosomen: Pflanzenzüchtung mit CRISPR-CAS

Using the CRISPR-Cas system, it has been possible to introduce different kinds of mutations in single or multiple genes for trait improvement in crops. Last year, for the first time, the CRISPR-Cas-mediated induction of different kinds of targeted heritable chromosomal rearrangements has been achieved in plants. This novel application has the potential to revolutionize plant breeding as genetic exchange and linkage drag are now becoming controllable in a targeted manner.

A Novel Banana Mutant “RF 1” (Musa spp. ABB, Pisang Awak Subgroup) for Improved Agronomic Traits and Enhanced Cold Tolerance and Disease Resistance

Banana is a major fruit crop grown in tropical and subtropical regions worldwide. Among cultivars, “FenJiao, FJ” (Musa spp. ABB, Pisang Awak subgroup) is a popular variety of bananas, due to its better sugar-acid blend and relatively small fruit shape. However, because the traditional FJ variety grows relatively high in height, it is vulnerable to lodging and unsuitable for harvesting. In this study, we sought desirable banana mutants by carrying out ethyl methanesulfonate (EMS) mutagenesis with the FJ cultivar. After the FJ shoot tips had been treated with 0.8% (v/v) EMS for 4 h, we obtained a stably inherited mutant, here called “ReFen 1” (RF1), and also observed a semi-dwarfing phenotype. Compared with the wild type (FJ), this RF1 mutant featured consistently improved agronomic traits during 5-year field experiments conducted in three distinct locations in China. Notably, the RF1 plants showed significantly enhanced cold tolerance and Sigatoka disease resistance, mainly due to a substantially increased soluble content of sugar and greater starch accumulation along with reduced cellulose deposition. Therefore, this study not only demonstrated how a powerful genetic strategy can be used in fruit crop breeding but also provided insight into the identification of novel genes for agronomic trait improvement in bananas and beyond.

Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing

Background A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing. Results To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected. Conclusions With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.

Protoplast Regeneration and Its Use in New Plant Breeding Technologies

The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration.

Employing CRISPR/Cas9 for de-repression of specific miRNA-target genes in rice

MicroRNAs (miRNAs) target specific mRNA molecules based on sequence complementarity for their degradation or translation repression, thereby regulating various development and physiological processes in eukaryotic orgasms. Expressing the target mimicry (MIM) and short tandem target mimicry (STTM), can block endogenous mature miRNAs activity and eliminate the inhibition to their target genes, resulting in phenotypic changes due to higher expression of the target genes. Here, we report a strategy to achieve de-repression of interested miRNA-target genes through CRISPR/Cas9-based generation of in-frame mutants within the miRNA-complementary sequence of the target gene. We show that two rice genes, OsGRF4 and OsGRF8 carrying in-frame mutants with disrupting the miR396 recognition sites, escape from miR396-mediated post-transcriptional silence, resulting in enlarged grain size and increased the brown planthopper (BPH) resistance in their respective rice transgenic lines. These results demonstrate that CRISPR/Cas9-mediated disruption of miRNA target sites can be effectively employed to precisely de-repress particular target genes of functional importance for trait improvement in plants.

Targeted Sequencing Suggests Wild-Crop Gene Flow Is Central to Different Genetic Consequences of Two Independent Pumpkin Domestications

Studies of domestication genetics enrich our understanding of how domestication shapes genetic and morphological diversity. We characterized patterns of genetic variation in two independently domesticated pumpkins and their wild progenitors to assess and compare genetic consequences of domestication. To compare genetic diversity pre- and post-domestication and to identify genes targeted by selection during domestication, we analyzed ∼15,000 SNPs of 48 unrelated accessions, including wild, landrace, and improved lines for each of two pumpkin species, Cucurbita argyrosperma and Cucurbita maxima. Genetic diversity relative to its wild progenitor was reduced in only one domesticated subspecies, C. argyrosperma ssp. argyrosperma. The two species have different patterns of genetic structure across domestication status. Only 1.5% of the domestication features identified for both species were shared between species. These findings suggest that ancestral genetic diversity, wild-crop gene flow, and domestication practices shaped the genetic diversity of two similar Cucurbita crops in different ways, adding to our understanding of how genetic diversity changes during the processes of domestication and how trait improvement impacts the breeding potential of modern crops.

Tissue Specific Transcriptome Profiling and Genetic Region Marker Discovery of Indian Sesame (Sisemum indicum L.)

Sesame (Sesamum indicum L.), is rich source of oil, protein and potent antioxidants with wide applications. Molecular approach can be of great use for trait improvement as well more availability for this crop. The present work aims at tissue specific transcriptome profiling along with biochemical pathway analysis and genic region putative marker discovery. We report 14389, 9465 and 5490 DEGs in root, leaf and flower-bud tissues, respectively. 135, 113 and 120 cellular metabolic or signaling pathways having common 118 pathways were found in root-leaf (RL), leaf-flower (LF) and flower-root (FR), respectively. 218, 170 and 180 transcription factors were identified in root, leaf and flower transcriptome, respectively. Among DEGs, microRNA targets predicted were 534, 376 and 173 in root, leaf and flower, respectively. Genic region repeat analysis revealed 379 SSR. Further variant analysis revealed 3371, 5439 and 4975 SNPs and 2257, 2403 and 2411 INDELs in root, leaf and flower, respectively. The present study will aid in understanding the major biochemical pathways operating in different tissues. Genic region putative marker discovery can be a valuable genomic resource for future crop improvement program.