Genome-wide Targeted Mutagenesis in Rice Using the CRISPR/Cas9 System

Lu, Yuming; Xiao, Yunhua; Guo, Ruru; Jing, Huang; Wang, Wei; Tang, Jiuyou; Tan, Liwen; Zhu, Jian‐Kang; Chu, Chengcai; Qian, Yangwen

doi:10.1016/j.molp.2017.06.007

Cited by 242 publications

(179 citation statements)

References 11 publications

Supporting

Mentioning

178

Contrasting

Order By: Relevance

“…Two recent studies in rice demonstrated that it is technically and logistically feasible to generate large-scale CRISPR/Cas9 mutant libraries that cover most of the genes in rice for the purpose of defining gene functions and improving crops (Lu et al, 2017; Meng et al, 2017). Designing and constructing genome-scale CRISPR/Cas9 plasmids are straightforward and relatively inexpensive because the targeting specificity of CRISPR/Cas9 is determined by the first 20-bp sequence in a gRNA molecule, which can be easily synthesized using array-based oligonucleotide synthesis (Figure 1).…”

mentioning

confidence: 99%

“…Both studies reported efficient editing of the target sites and very few off-target events. Together the two studies generated almost 100,000 independent CRISPR/Cas9 T 0 rice lines, providing tremendous resources for the plant biology community (Lu et al, 2017; Meng et al, 2017). …”

mentioning

confidence: 99%

“…Lu et al (2017) creatively used barcodes and next-generation sequencing (NGS) to identify the gRNAs for each transgenic plant in a CRISPR/Cas9 mutant collection that they generated, enabling high-throughput analysis of the relationship between genotypes and phenotypes. As a pilot analysis, Lu et al (2017) conducted 96 × 96 PCR reactions with a unique pair of barcoded PCR primers for each sample to amplify gRNAs from 9216 transgenic plants, resulting in the identification of gRNAs in more than 5500 plants (some of the PCR or sequencing reactions did not work) (Figure 1). This study demonstrates that genotyping CRISPR/Cas9 mutant library has become efficient and inexpensive, greatly accelerating the identification of the causal mutations for a given phenotype (Lu et al, 2017).…”

mentioning

confidence: 99%

“…As a pilot analysis, Lu et al (2017) conducted 96 × 96 PCR reactions with a unique pair of barcoded PCR primers for each sample to amplify gRNAs from 9216 transgenic plants, resulting in the identification of gRNAs in more than 5500 plants (some of the PCR or sequencing reactions did not work) (Figure 1). This study demonstrates that genotyping CRISPR/Cas9 mutant library has become efficient and inexpensive, greatly accelerating the identification of the causal mutations for a given phenotype (Lu et al, 2017). …”

mentioning

confidence: 99%

“…Indeed, Lu et al (2017) reported that multiple T 0 plants had obvious developmental phenotypes, including spotted leaves, altered tiller number, altered tiller angle, dwarf, and sterility. The genes responsible for the phenotypes at T 0 generation potentially can be inferred from the gRNA sequences, which can be obtained through barcoded/NGS analysis or simple PCR/sequencing (Figure 1).…”

mentioning

confidence: 99%

See 4 more Smart Citations

Revolutionize Genetic Studies and Crop Improvement with High-Throughput and Genome-Scale CRISPR/Cas9 Gene Editing Technology

2017

View full text Add to dashboard Cite

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Revolutionize Genetic Studies and Crop Improvement with High-Throughput and Genome-Scale CRISPR/Cas9 Gene Editing Technology

2017

View full text Add to dashboard Cite

Thirty Years of Genome Engineering in Rice: From Gene Addition to Gene Editing

Meynard

Vernet

Meunier

et al. 2020

Annual Plant Reviews Online

View full text Add to dashboard Cite

Rice, the staple food for more than half of humankind and the model monocot crop, was the first cereal in which efficient gene transfer procedures were implemented: 30 years ago, the first transgenic rice plant was regenerated following direct gene transfer to cell suspension‐derived protoplasts. Shortly thereafter, transgenic plants were regenerated from zygotic embryo‐derived cells following their subjection to micro‐projectile acceleration and Agrobacterium ‐mediated transfection treatments. The high efficiency of transfer deoxyribonucleic acid (T‐DNA) integration in seed embryo‐derived cells of rice has allowed the transfer of genes of agronomical relevance and the generation of large collections of insertion lines and has provided a key contribution to deciphering the function of more than 2000 rice genes. The high efficiency of T‐DNA integration in seed embryo‐derived cells of rice also permitted the first implementation of gene targeting and knock‐in (KI) events, relying on the albeit very low natural frequency of homology‐directed repair (HDR) in the rice genome. In the late 2000s, the advent of site‐directed nucleases (SDNs) that induce either single or double‐strand breaks at a high frequency and their rapid application to rice permitted routine targeted mutagenesis, which can be multiplexed to simultaneously alter several targets or create deletions, and base and gene editing (e.g. correction of amino acids). Currently, the challenge remains to attain a high frequency of KI and replacement of long stretches of DNA for protein domain or coding sequence swapping. We present herein a historical perspective of the advances that have been readily implemented to determine the function of rice genes and to manipulate traits of agronomical relevance. Two main bottlenecks remain to be alleviated in rice genomic engineering: the low frequency of HDR and the genotype dependence of gene transfer efficiency. Alleviation of these bottlenecks is needed to reach the potential of intra‐ and interspecific gene replacement and SDN‐mediated multiplex editing of alleles in elite materials, which will assist in the breeding and deployment of rice cultivars embedded in sustainable and climate‐smart agricultural practices.

show abstract

The Promise of eQTL Studies in Dissecting Crop Genetic Basis and Evolution

Leng

Wang

et al. 2022

Annual Plant Reviews Online

View full text Add to dashboard Cite

Most traits important for crop production are affected by polymorphisms in the genome. The application of genome‐wide association studies (GWASs) has analysed numerous genetic loci controlling complex traits, highlighting the significant roles of non‐coding regulatory variants and networks. During the past decade, there have been continuous advancements in high‐throughput RNA sequencing technology applied to crop genomes, along with expression quantitative trait locus (eQTL) mapping based on GWASs with diverse lines. These developments have facilitated considerably superior resolution to uncover the detailed genetic basis of whole transcriptomic variation, which can connect DNA polymorphism to individual genes and provide an effective approach to elucidate the regulatory networks underlying phenotypes. In this article, we comprehensively summarise the technical and computational considerations of eQTL mapping, especially using RNA sequencing of association populations. Through integration with trans hotspots, response or dynamic eQTLs, and network analyses, eQTL studies on crops aid in understanding the regulatory networks and connections among genes in metabolic pathways, development, or response to stimuli. We outline the approaches to integrate eQTL results with GWASs, which can greatly improve the possibility of identifying causal genes and regulatory networks responsible for complex traits. Regulation information from eQTL mapping can also be used to elucidate the divergence and stabilisation of gene expression during crop evolution. Although facing several technical challenges and presently limited to a few sample points in crops, eQTL studies hold great potential for advancing our knowledge of the genomic base and evolution of complex traits, and supports molecular breeding of crops.

show abstract

Genome-wide Targeted Mutagenesis in Rice Using the CRISPR/Cas9 System

Cited by 242 publications

References 11 publications

Revolutionize Genetic Studies and Crop Improvement with High-Throughput and Genome-Scale CRISPR/Cas9 Gene Editing Technology

Revolutionize Genetic Studies and Crop Improvement with High-Throughput and Genome-Scale CRISPR/Cas9 Gene Editing Technology

Thirty Years of Genome Engineering in Rice: From Gene Addition to Gene Editing

The Promise of eQTL Studies in Dissecting Crop Genetic Basis and Evolution

Contact Info

Product

Resources

About