Tool For Functional Genomics Research Articles

CRISPR Interference Efficiently Silences Latent and Lytic Viral Genes in Kaposi's Sarcoma-Associated Herpesvirus-Infected Cells.

Uncovering viral gene functions requires the modulation of gene expression through overexpression or loss-of-function. CRISPR interference (CRISPRi), a modification of the CRISPR-Cas9 gene editing technology, allows specific and efficient transcriptional silencing without genetic ablation. CRISPRi has been used to silence eukaryotic and prokaryotic genes at the single-gene and genome-wide levels. Here, we report the use of CRISPRi to silence latent and lytic viral genes, with an efficiency of ~80–90%, in epithelial and B-cells carrying multiple copies of the Kaposi’s sarcoma-associated herpesvirus (KSHV) genome. Our results validate CRISPRi for the analysis of KSHV viral elements, providing a functional genomics tool for studying virus–host interactions.

Three-in-One Simultaneous Extraction of Proteins, Metabolites and Lipids for Multi-Omics.

Elucidation of complex molecular networks requires integrative analysis of molecular features and changes at different levels of information flow and regulation. Accordingly, high throughput functional genomics tools such as transcriptomics, proteomics, metabolomics, and lipidomics have emerged to provide system-wide investigations. Unfortunately, analysis of different types of biomolecules requires specific sample extraction procedures in combination with specific analytical instrumentation. The most efficient extraction protocols often only cover a restricted type of biomolecules due to their different physicochemical properties. Therefore, several sets/aliquots of samples are needed for extracting different molecules. Here we adapted a biphasic fractionation method to extract proteins, metabolites, and lipids from the same sample (3-in-1) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-omics. To demonstrate utility of the improved method, we used bacteria-primed Arabidopsis leaves to generate multi-omics datasets from the same sample. In total, we were able to analyze 1849 proteins, 1967 metabolites, and 424 lipid species in single samples. The molecules cover a wide range of biological and molecular processes, and allow quantitative analyses of different molecules and pathways. Our results have shown the clear advantages of the multi-omics method, including sample conservation, high reproducibility, and tight correlation between different types of biomolecules.

Strongyloides RNA-seq Browser: a web-based software platform for on-demand bioinformatics analyses of Strongyloides species.

Soil-transmitted gastrointestinal parasitic nematodes infect approximately 1 billion people worldwide, predominantly in low-resource communities. Skin-penetrating gastrointestinal nematodes in the genus Strongyloides are emerging as model systems for mechanistic studies of soil-transmitted helminths due to the growing availability of functional genomics tools for these species. To facilitate future genomics studies of Strongyloides species, we have designed a web-based application, the Strongyloides RNA-seq Browser, that provides an open source, user-friendly portal for accessing and analyzing Strongyloides genomic expression data. Specifically, the Strongyloides RNA-seq Browser takes advantage of alignment-free read mapping tools and R-based transcriptomics tools to re-analyze publicly available RNA sequencing datasets from four Strongyloides species: Strongyloides stercoralis, Strongyloides ratti, Strongyloides papillosus, and Strongyloides venezuelensis. This application permits on-demand exploration and quantification of gene expression across life stages without requiring previous coding experience. Here, we describe this interactive application and demonstrate how it may be used by nematode researchers to conduct a standard set of bioinformatics queries.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing.

The corn planthopper, Peregrinus maidis, isa pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

BART3D: inferring transcriptional regulators associated with differential chromatin interactions from Hi-C data.

SummaryIdentification of functional transcriptional regulators (TRs) associated with chromatin interactions is an important problem in studies of 3-dimensional genome organization and gene regulation. Direct inference of TR binding has been limited by the resolution of Hi-C data. Here, we present BART3D, a computational method for inferring TRs associated with genome-wide differential chromatin interactions by comparing Hi-C maps from two states, leveraging public ChIP-seq data for human and mouse. We demonstrate that BART3D can detect relevant TRs from dynamic Hi-C profiles with TR perturbation or cell differentiation. BART3D can be a useful tool in 3D genome data analysis and functional genomics research.Availability and implementationBART3D is implemented in Python and the source code is available at https://github.com/zanglab/bart3d.Supplementary informationSupplementary data are available at Bioinformatics online.

The sea anemone Nematostella vectensis, an emerging model for biomedical research: Mechano-sensitivity, extreme regeneration and longevity

Nematostella has fascinating features such as whole-body regeneration, the absence of signs of aging and importantly, the absence of age-related diseases. Easy to culture and spawn, this little sea anemone in spite of its "simple" aspect, displays interesting morphological characteristics similar to vertebrates and an unexpected similarity in gene content/genome organization. Importantly, the scientific community working on Nematostella is developing a variety of functional genomics tools that enable scientists to use this anemone in the field of regenerative medicine, longevity and mecano-sensory diseases. As a complementary research model to vertebrates, this marine invertebrate is emerging and promising to dig deeper into those fields of research in an integrative manner (entire organism) and provides new opportunities for scientists to lift specific barriers that can be encountered with other commonly used animal models.

Development of EMS-induced Mutagenized Groundnut Population and Discovery of Point Mutations in the <i>ahFAD2</i> and <i>Ara h 1</i> Genes by TILLING

Reducing allergenicity and increasing oleic content are important goals in groundnut breeding studies. Ara h 1 is a major allergen gene and Delta(12)-fatty-acid desaturase (FAD2) is responsible for converting oleic into linoleic acid. These genes have homoeologues with one copy in each subgenome, identified as Ara h 1.01, Ara h 1.02, ahFAD2A and ahFAD2B in tetraploid groundnut. To alter functional properties of these genes we have generated an Ethyl Methane Sulfonate (EMS) induced mutant population to be used in Targeting Induced Local Lesions in Genomes (TILLING) approach. Seeds were exposed to two EMS concentrations and the germination rates were calculated as 90.1% (1353 plants) for 0.4% and 60.4% (906 plants) for 1.2% EMS concentrations in the M1 generation. Among the 1541 M2 mutants, 768 were analyzed by TILLING using four homoeologous genes. Two heterozygous mutations were identified in the ahFAD2B and ahFAD2A gene regions from 1.2% and 0.4% EMS-treated populations, respectively. The mutation in ahFAD2B resulted in an amino acid change, which was serine to threonine predicted to be tolerated according to SIFT analysis. The other mutation causing amino acid change, glycine to aspartic acid was predicted to affect protein function in ahFAD2A. No mutations were detected in Ara h 1.01 and Ara h 1.02 for both EMS-treatments after sequencing. We estimated the overall mutation rate to be 1 mutation every 2139 kb. The mutation frequencies were also 1/317 kb for ahFAD2A in 0.4% EMS and 1/466 kb for ahFAD2B in 1.2% EMS treatments. The results demonstrated that TILLING is a powerful tool to interfere with gene function in crops and the mutagenized population developed in this study can be used as an efficient reverse genetics tool for groundnut improvement and functional genomics.

Synthesis of GalNAc-Oligonucleotide Conjugates Using GalNAc Phosphoramidite and Triple-GalNAc CPG Solid Support.

GalNAc oligonucleotide conjugates demonstrate improved potency in vivo due to selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis. GalNAc-siRNA and GalNAc-antisense oligonucleotides are at various stages of clinical trials, while the first two drugs were already approved by FDA. Also, GalNAc conjugates are excellent tools for functional genomics and target validation in vivo. The number of GalNAc residues in a conjugate is crucial for delivery as cooperative interaction of several GalNAc residues with asialoglycoprotein receptor enhances delivery in vitro and in vivo. Here we provide a robust protocol for the synthesis of triple GalNAc CPG solid support and GalNAc phosphoramidite, synthesis and purification of RNA conjugates with multiple GalNAc residues either to 5'-end or 3'-end and siRNA duplex formation.

Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells

The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34+ HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo.

RNAi and CRISPR/Cas9 as Functional Genomics Tools in the Neotropical Stink Bug, Euschistus heros.

Simple SummaryUnderstanding the biology of insect pests is an important step towards developing appropriate control strategies. In this study, a CRISPR/Cas9 gene knockout work flow was established for the first time and was together with RNAi used as tools to study gene functions in the Neotropical stink bug, Euschistus heros. RNAi was first employed to study the function of three genes, abnormal wing disc (awd), tyrosine hydroxylase (th) and yellow (yel). Targeting awd and th resulted in distinct malformed phenotypes such as a deformed wing or a lighter cuticle pigmentation/defects in cuticle sclerotization, respectively. However, no distinct phenotype was observed for yel. To further investigate the function of yel, a CRISPR/Cas9 gene editing protocol was developed for E. heros. A total of 719 eggs were microinjected with single-guide (sgRNA) and Cas9 and total of six insects hatched. Out of these six nymphs, one insect showed mutation in yel but no clear phenotype was visible. Although, we were unable to generate insects with a distinct phenotype for yel, a successful gene editing workflow was established to complement RNAi for future functional gene studies in E. heros. Additionally, we provided recommendations to improve the established gene editing workflow.The Neotropical brown stink bug, Euschistus heros, is one of the most important stink bug pests in leguminous plants in South America. RNAi and CRISPR/Cas9 are important and useful tools in functional genomics, as well as in the future development of new integrated pest management strategies. Here, we explore the use of these technologies as complementing functional genomic tools in E. heros. Three genes, abnormal wing disc (awd), tyrosine hydroxylase (th) and yellow (yel), known to be involved in wing development (awd) and the melanin pathway (th and yel) in other insects, were chosen to be evaluated using RNAi and CRISPR/Cas9 as tools. First, the genes were functionally characterized using RNAi knockdown technology. The expected phenotype of either deformed wing or lighter cuticle pigmentation/defects in cuticle sclerotization was observed for awd and th, respectively. However, for yel, no obvious phenotype was observed. Based on this, yel was selected as a target for the development of a CRISPR/Cas9 workflow to study gene knockout in E. heros. A total of 719 eggs were injected with the Cas9 nuclease (300 ng/µL) together with the sgRNA (300 ng/µL) targeting yel. A total of six insects successfully hatched from the injected eggs and one of the insects showed mutation in the target region, however, the phenotype was still not obvious. Overall, this study for the first time provides a useful CRISPR/Cas9 gene editing methodology to complement RNAi for functional genomic studies in one of the most important and economically relevant stink bug species.

Breeding, Genetics, and Genomics Approaches for Improving Fusarium Wilt Resistance in Major Grain Legumes.

Fusarium wilt (FW) disease is the key constraint to grain legume production worldwide. The projected climate change is likely to exacerbate the current scenario. Of the various plant protection measures, genetic improvement of the disease resistance of crop cultivars remains the most economic, straightforward and environmental-friendly option to mitigate the risk. We begin with a brief recap of the classical genetic efforts that provided first insights into the genetic determinants controlling plant response to different races of FW pathogen in grain legumes. Subsequent technological breakthroughs like sequencing technologies have enhanced our understanding of the genetic basis of both plant resistance and pathogenicity. We present noteworthy examples of targeted improvement of plant resistance using genomics-assisted approaches. In parallel, modern functional genomic tools like RNA-seq are playing a greater role in illuminating the various aspects of plant-pathogen interaction. Further, proteomics and metabolomics have also been leveraged in recent years to reveal molecular players and various signaling pathways and complex networks participating in host-pathogen interaction. Finally, we present a perspective on the challenges and limitations of high-throughput phenotyping and emerging breeding approaches to expeditiously develop FW-resistant cultivars under the changing climate.

A CRISPR/dCas9 toolkit for functional analysis of maize genes

BackgroundThe Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9).ResultsIn this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression.ConclusionsWe anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.

Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research.

Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.

RNAi-Based Functional Genomics in Hemiptera.

Simple SummaryRNA interference (RNAi) is a powerful strategy to understand the function of novel and critical insect genes. In this review, we highlight the pros and cons of using RNAi as a functional genomics tool, the range of applications and explore RNAi delivery approaches such as topical and carrier/nano-particle-mediated RNAi for silencing insect genes in Hemiptera. We explore factors contributing to observed variations in RNAi efficiency and possible solutions to improve RNAi based investigations. We briefly summarise and provide experimental insight on the key RNAi studies in agricultural hemipteran pests that will be applicable to lesser-studied hemipteran’s, as well as informing functional genomics studies across diverse insect pests.RNA interference (RNAi) is a powerful approach for sequence-specific gene silencing, displaying tremendous potential for functional genomics studies in hemipteran insects. Exploiting RNAi allows the biological roles of critical genes to be defined and aids the development of RNAi-based biopesticides. In this review, we provide context to the rapidly expanding field of RNAi-based functional genomics studies in hemipteran insects. We highlight the most widely used RNAi delivery strategies, including microinjection, oral ingestion and topical application. Additionally, we discuss the key variables affecting RNAi efficacy in hemipteran insects, including insect life-stage, gene selection, the presence of nucleases, and the role of core RNAi machinery. In conclusion, we summarise the application of RNAi in functional genomics studies in Hemiptera, focusing on genes involved in reproduction, behaviour, metabolism, immunity and chemical resistance across 33 species belonging to 14 families.

RNA interference dynamics in juvenile Fasciola hepatica are altered during in vitro growth and development

For over a decade RNA interference (RNAi) has been an important molecular tool for functional genomics studies in parasitic flatworms. Despite this, our understanding of RNAi dynamics in many flatworm parasites, such as the temperate liver fluke (Fasciola hepatica), remains rudimentary. The ability to maintain developing juvenile fluke in vitro provides the opportunity to perform functional studies during development of the key pathogenic life stage. Here, we investigate the RNAi competence of developing juvenile liver fluke. Firstly, all life stages examined possess, and express, core candidate RNAi effectors encouraging the hypothesis that all life stages of F. hepatica are RNAi competent. RNAi effector analyses supported growing evidence that parasitic flatworms have evolved a separate clade of RNAi effectors with unknown function. Secondly, we assessed the impact of growth/development during in vitro culture on RNAi in F. hepatica juveniles and found that during the first week post-excystment liver fluke juveniles exhibit quantitatively lower RNAi mediated transcript knockdown when maintained in growth inducing media. This did not appear to occur in older in vitro juveniles, suggesting that rapidly shifting transcript dynamics over the first week following excystment alters RNAi efficacy after a single 24 h exposure to double stranded (ds)RNA. Finally, RNAi efficiency was found to be improved through use of a repeated dsRNA exposure methodology that has facilitated silencing of genes in a range of tissues, thereby increasing the utility of RNAi as a functional genomics tool in F. hepatica.

Large-scale identification and characterization of phenolic compounds and their marker–trait association in wheat

Phenolic compounds (PCs) are important biomolecules as they affect processing (color, aroma, and taste quality of bread, and grain hardness) and nutrition quality (antioxidant and other health benefits). This study identified a set of 115 PCs by screening 100 Indian bread wheat varieties that showed wide variation in PCs content. These PCs were classified into 29 phenolic acid and their derivative, 71 flavonoids, 2 chalcones, 8 stilbenoids 1 theaflavin, 1 eugenol, and 3 coumarins. Of the 115, standards for 25 PCs were validated by matching their RT (retention time), MS and MS/MS fragmentation pattern on UPLC-QTOF-MS/MS. The range of PC content was from 0.01 µg/100 g for luteolin in ‘GW 503’ to 547.63 µg/100 g for vanillin in ‘Durgapur 65.’ The marker–trait association analysis identified 81 SSR markers which were associated with twelve PCs. Of 81, 53 were significantly associated on 5% FDR at true value (p < q). After multiple test correction (adjusted p value and 5% FDR), eight SSR markers were significantly associated with five PCs, namely ‘rutin,’ ‘hesperidin,’ ‘2,4-dihydroxybenzoic acid,’ ‘vanillin,’ and ‘salicylic acid.’ A substantial number of markers showed coefficient of variation (R2) ranged from ~ 5 to 45% for these PCs. The variations present in the set of wheat varieties and linked markers can be exploited for the improvement of PCs affecting processing and nutrition quality of wheat through molecular breeding and functional genomics tools.

Integration of genomics, metagenomics, and metabolomics to identify interplay between susceptibility alleles and microbiota in adenoma initiation

BackgroundColorectal cancer (CRC) is a multifactorial disease resulting from both genetic predisposition and environmental factors including the gut microbiota (GM), but deciphering the influence of genetic variants, environmental variables, and interactions with the GM is exceedingly difficult. We previously observed significant differences in intestinal adenoma multiplicity between C57BL/6 J-ApcMin (B6-Min/J) from The Jackson Laboratory (JAX), and original founder strain C57BL/6JD-ApcMin (B6-Min/D) from the University of Wisconsin.MethodsTo resolve genetic and environmental interactions and determine their contributions we utilized two genetically inbred, independently isolated ApcMin mouse colonies that have been separated for over 20 generations. Whole genome sequencing was used to identify genetic variants unique to the two substrains. To determine the influence of genetic variants and the impact of differences in the GM on phenotypic variability, we used complex microbiota targeted rederivation to generate two Apc mutant mouse colonies harboring complex GMs from two different sources (GMJAX originally from JAX or GMHSD originally from Envigo), creating four ApcMin groups. Untargeted metabolomics were used to characterize shifts in the fecal metabolite profile based on genetic variation and differences in the GM.ResultsWGS revealed several thousand high quality variants unique to the two substrains. No homozygous variants were present in coding regions, with the vast majority of variants residing in noncoding regions. Host genetic divergence between Min/J and Min/D and the complex GM additively determined differential adenoma susceptibility. Untargeted metabolomics revealed that both genetic lineage and the GM collectively determined the fecal metabolite profile, and that each differentially regulates bile acid (BA) metabolism. Metabolomics pathway analysis facilitated identification of a functionally relevant private noncoding variant associated with the bile acid transporter Fatty acid binding protein 6 (Fabp6). Expression studies demonstrated differential expression of Fabp6 between Min/J and Min/D, and the variant correlates with adenoma multiplicity in backcrossed mice.ConclusionsWe found that both genetic variation and differences in microbiota influences the quantitiative adenoma phenotype in ApcMin mice. These findings demonstrate how the use of metabolomics datasets can aid as a functional genomic tool, and furthermore illustrate the power of a multi-omics approach to dissect complex disease susceptibility of noncoding variants.

Chloroplast genome sequences of Carya illinoinensis from two distinct geographic populations

Pecan (Carya illinoinensis) is the most economically important member of the Carya genus and has been collected and evaluated across its broad geographic range in the process of crop improvement. In this study we obtained complete chloroplast genome sequences from two pecan genotypes, 87MX3-2.11 and the ‘Lakota’ cultivar (160,545 and 160,819 bp in length, respectively). The chloroplast genome of C. illinoinensis maintains the conserved structure typical of Juglandaceae and other land plants and is a circular molecule that includes a large single-copy (LSC) and a small single-copy (SSC) region, separated by a pair of inverted repeats (IRa and IRb). There were 124 genes found on the 87MX3-2.11 chloroplast genome and 123 on ‘Lakota’ (including multiple copies of the same gene), with 108 and 107 unique genes, respectively (counting only one copy of each gene). Different genes are found among C. illinoinensis, C. sinensis, and Juglans chloroplast genomes. C. illinoinensis is missing rps16 gene and has fewer copies of some tRNA genes, with ‘Lakota’ lacking a start codon of rps12 gene, compared with other related species. The nucleotide divergence between the two pecan chloroplast genomes reflects the genetic diversity of geographically separated populations of the species. Genomic divergence was also confirmed by the phylogenetic relationship of 19 whole chloroplast genome sequences representing Juglandaceae taxa. The complete chloroplast genome sequences in this study provide a foundation for understanding the influences of geographical adaptation, gene flow, and horticultural trait inheritance, in order to develop functional genomic tools for regional selection and pecan breeding.

Performance evaluation of induced mutant lines of black gram (Vigna mungo (L.) Hepper)

Performance evaluation of induced mutant lines of black gram (Vigna mungo (L.) Hepper)

Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook.

In most crop breeding programs, the rate of yield increment is insufficient to cope with the increased food demand caused by a rapidly expanding global population. In plant breeding, the development of improved crop varieties is limited by the very long crop duration. Given the many phases of crossing, selection, and testing involved in the production of new plant varieties, it can take one or two decades to create a new cultivar. One possible way of alleviating food scarcity problems and increasing food security is to develop improved plant varieties rapidly. Traditional farming methods practiced since quite some time have decreased the genetic variability of crops. To improve agronomic traits associated with yield, quality, and resistance to biotic and abiotic stresses in crop plants, several conventional and molecular approaches have been used, including genetic selection, mutagenic breeding, somaclonal variations, whole-genome sequence-based approaches, physical maps, and functional genomic tools. However, recent advances in genome editing technology using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated (Cas) proteins have opened the door to a new plant breeding era. Therefore, to increase the efficiency of crop breeding, plant breeders and researchers around the world are using novel strategies such as speed breeding, genome editing tools, and high-throughput phenotyping. In this review, we summarize recent findings on several aspects of crop breeding to describe the evolution of plant breeding practices, from traditional to modern speed breeding combined with genome editing tools, which aim to produce crop generations with desired traits annually.