Construction of a Genome-Wide Mutant Library in Rice Using CRISPR/Cas9

Abstract

Rice (Oryza sativa L.) is one of the world's most important staple crops and a powerful model system for studying monocot species because of its relatively small genome, rich genomic resources, and a highly efficient transformation system. With the completion of rice genome sequencing, the challenge of the post-genomic era is to systematically analyze the functions of all rice genes. Gene knockout is a frequently used and effective strategy for achieving this goal. Thus, generation of large-scale mutants at the whole-genome level is of great value for both functional genomics and genetic improvement of rice. Traditionally, large numbers of mutants are produced by physical, chemical, or biological mutagenesis. Mutants created by these methods have made enormous contributions to basic plant research and crop improvement. T-DNA insertion (Jeon et al., 2000), TILLING (targeting-induced local lesions in genomes) (Till et al., 2007) and RNAi (RNA interference) (Wang et al., 2013) are the three most common methods of performing genetic studies. T-DNA insertion and TILLING are time-consuming and labor-intensive in generating genome-wide mutant libraries, because large mutagenized populations must be generated to ensure sufficient genome-wide coverage. In addition, the T-DNA insertions occur randomly and often in intergenic and noncoding regions, for TILLING mutants it is difficult to identify the targeted mutations for the observed phenotypes, and the RNAi method only reduces the expression of targeted genes rather than generating the knockout mutants. Recently, a simple and highly efficient genomic engineering tool, the CRISPR (Clustered Regularly Interspaced Palindromic Repeats)/Cas9 system, has been developed; this technology can create small insertions and deletions (indels) in specific target genes and has been applied to many organisms. Because it is an easy and convenient technique, some CRISPR/Cas9 mutant libraries have been developed for genomewide mutation screens in cultured eukaryotic cells (Shalem et al., 2015). However, no large-scale CRISPR/Cas9 mutant libraries have yet been generated in higher plants. Here, we report the construction of a high-throughput CRISPR/Cas9 mutant library in rice and demonstrate its application for identifying gene functions and its potential use for genetic improvement.

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

“…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).…”

“…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). …”

“…For example, Meng et al (2017) discovered that 11 T 1 plants that were offspring of a single T 0 plant had altered tiller number and reduced height. They were able to show that the phenotypes were caused by loss-of-function mutations in the TAD1 gene (Meng et al, 2017).…”

“…For example, Meng et al (2017) discovered that 11 T 1 plants that were offspring of a single T 0 plant had altered tiller number and reduced height. They were able to show that the phenotypes were caused by loss-of-function mutations in the TAD1 gene (Meng et al, 2017). Large-scale barcoded/NGS analysis will establish sequence-indexed CRISPR/Cas9 mutant collections similar to sequence-indexed Arabidopsis mutant collections (Alonso et al, 2003), which have greatly benefited the Arabidopsis community.…”

“…Although still limited in scale, the studies in rice and tomato discussed here demonstrated that the CRISPR/Cas9-mediated targeted mutagenesis is reliable, efficient, and high-throughput for generating mutant collections that cover most genes in a genome (Jacobs et al, 2017; Lu et al, 2017; Meng et al, 2017). The CRISPR technology has revolutionized genetic studies in crops because of its unprecedented scale, versatility, and speed.…”