Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement

Abstract

Advances in molecular technologies over the past few decades, such as high-throughput DNA marker genotyping, have provided more powerful plant breeding approaches, including marker-assisted selection and genomic selection. At the same time, massive investments in plant genetics and genomics, led by whole genome sequencing, have led to greater knowledge of genes and genetic pathways across plant genomes. However, there remains a gap between approaches focused on forward genetics, which start with a phenotype to map a mutant locus or QTL with the goal of cloning the causal gene, and approaches using reverse genetics, which start with large-scale sequence data and work back to the gene function. The recent establishment of efficient CRISPR-Cas-based gene editing promises to bridge this gap and provide a rapid method to functionally validate genes and alleles identified through studies of natural variation. CRISPR-Cas techniques can be used to knock out single or multiple genes, precisely modify genes through base and prime editing, and replace alleles. Moreover, technologies such as protoplast isolation, in planta transformation, and the use of developmental regulatory genes promise to enable high-throughput gene editing to accelerate crop improvement.

Genetic factors underlying anaerobic germination in rice: Genome‐wide association study and transcriptomic analysis

“…The selected candidate genes identified in our study can be further confirmed using the two accessions and several other accessions with contrasting AG phenotypes via reverse transcription polymerase chain reaction and other genomic tools. Moreover, functional validation can be performed using transgenic studies including knocking out each candidate gene in the tolerant genotype with gene editing (Thomson et al., 2022). Furthermore, these findings will help in designing markers to be used in marker‐assisted breeding programs for rice improvement against flooding during germination stress (Gonzaga et al., 2015).…”

Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene

“…Protoplasts have been used for the testing of CRISPR/Cas9 constructs and sgRNA activity in a diverse range of species, including legumes such as peanut and chickpea, but a protocol has not yet been developed for cowpea [22][23][24]. Likewise, Agroinfiltration of leaves has also been used with the CRISPR-Cas9 system for sgRNA validation and gene expression assays [25][26][27]. The current study aims to knock out the cowpea phytoene desaturase (PDS) gene using a CRISPR-Cas9 construct designed with four multiplexed sgRNAs through PEG transformation in cowpea protoplasts and agroinfiltration in cowpea leaves.…”

Guidelines for Performing CRISPR/Cas9 Genome Editing for Gene Validation and Trait Improvement in Crops

“…Plants 2023, 12, 3564 2 of 14 Subsequently, a list of candidate genes is refined, and functional validation is performed through complementation and/or genome editing [12]. Recent advances in genome editing techniques have begun to greatly accelerate progress in the genetic dissection of key traits in plants but have not yet been thoroughly optimized for crop improvement [13].…”

“…The identification of causal genes controlling key traits in crop species often begins with quantitative trait locus (QTL) mapping and genome-wide association studies (GWASs). Subsequently, a list of candidate genes is refined, and functional validation is performed through complementation and/or genome editing [12]. Recent advances in genome editing techniques have begun to greatly accelerate progress in the genetic dissection of key traits in plants but have not yet been thoroughly optimized for crop improvement [13].…”