CAFRI‐Rice: CRISPR applicable functional redundancy inspector to accelerate functional genomics in rice

Abstract

Rice (Oryza sativa L.) is a staple crop with agricultural traits that have been intensively investigated. However, despite the variety of mutant population and multi-omics data that have been generated, rice functional genomic research has been bottlenecked due to the functional redundancy in the genome. This phenomenon has masked the phenotypes of knockout mutants by functional compensation and redundancy. Here, we present an intuitive tool, CRISPR applicable functional redundancy inspector to accelerate functional genomics in rice (CAFRI-Rice; cafri-rice.khu.ac.kr). To create this tool, we generated a phylogenetic heatmap that can estimate the similarity between protein sequences and expression patterns, based on 2,617 phylogenetic trees and eight tissue RNA-sequencing datasets. In this study, 33,483 genes were sorted into 2,617 families, and about 24,980 genes were tested for functional redundancy using a phylogenetic heatmap approach. It was predicted that 7,075 genes would have functional redundancy, according to the threshold value validated by an analysis of 111 known genes functionally characterized using knockout mutants and 5,170 duplicated genes. In addition, our analysis demonstrated that an anther/pollen-preferred gene cluster has more functional redundancy than other clusters. Finally, we showed the usefulness of the CAFRI-Rice-based approach by overcoming the functional redundancy between two root-preferred genes via loss-of-function analyses as well as confirming the functional dominancy of three genes through a literature search. This CAFRI-Rice-based target selection for CRISPR/Cas9-mediated mutagenesis will not only accelerate functional genomic studies in rice but can also be straightforwardly expanded to other plant species.

Global Identification of ANTH Genes Involved in Rice Pollen Germination and Functional Characterization of a Key Member, OsANTH3

“…The meta-expression data in the context of the phylogenetic tree were produced to check the function of OsANTH genes with respect to anther and pollen development compared with other tissues/organs (Figure 3A). In addition, we examined their expression profiles in RNAsequencing samples (Supplementary Figure 3; Hong et al, 2020).…”

A Revolution toward Gene-Editing Technology and Its Application to Crop Improvement

“…Therefore, 46.5% of rice genes with gene-family members might not be suitable targets for gene-editing applications using a single target. In addition, the 7075 genes with other family members in the genome having higher similarity in both sequence and expression patterns are more probable targets for multiple-gene editing ( Figure 6) [197]. For example, OsMADS63 genes, which share expression in mature pollen with OsMADS62, did not yield a defect in pollen development, whereas multiple mutations of OsMADS62 and OsMADS63 with a PCC of 0.977 caused a severe defect in late pollen development, and RUPO mutation (PCC, 0.335) over LOC_Os03g55210 in the same family led to a severe defect in late pollen development and did not require multiple-gene editing.…”

“…Therefore, accurate estimation of gene redundancy within a family will accelerate crop improvement through gene-editing systems. This web tool (CAFRI-Rice, ) is only available for rice, but we expect its expansion to other crop species [ 197 ].…”

Advantage of Nanotechnology-Based Genome Editing System and Its Application in Crop Improvement

“…Speed editing strategies have been proposed to address this challenge recently. A web tool has been developed by Hong et al (2020) to accelerate speed editing strategies to achieve 2% genetic gain in crop productivity (2050 food demand challenge). The powerful GE technology CRISPR/Cas has facilitated functional genomic studies of several crops with simplicity and accuracy.…”

“…The tool is based on a phylogenetic heatmap that can estimate the similarity between protein sequences and expression patterns. This CAFRI-Rice-based target selection for CRISPR/Cas9-mediated mutagenesis has accelerated functional genomic studies in rice; moreover, it can also be easily expanded to other plant species (Ahmar et al, 2020b;Hong et al, 2020).…”