Revolutionize Genetic Studies and Crop Improvement with High-Throughput and Genome-Scale CRISPR/Cas9 Gene Editing Technology  Ning Yang, Rongchen Wang, Yunde Zhao  Molecular Plant  Volume 10, Issue 9, Pages 1141-1143 (September 2017) DOI: 10.1016/j.molp.2017.08.001 Copyright © 2017 The Author Terms and Conditions

Figure 1 Define Gene Functions in a Single Generation using Genome-Scale and High-Throughput CRISPR/Cas9-Mediated Mutagenesis. A pooled CRISPR/Cas9 plasmid library is generated using array-based oligonucleotide synthesis. Transformation of the CRISPR/Cas9 plasmid library into rice by Agrobacterium-mediated transformation generates T0 plants, which can be phenotypically scored. If the T0 plant population is large enough, allelic mutants with similar phenotypes can be obtained. Identification of the gRNA sequences followed by sequencing the targeted genes can lead to the identification of the causal mutations for the phenotypes. Note that in order to define a gene function at T0 generation, editing efficiency and the rate of finding homozygous/bi-allelic mutants must be sufficiently high, which is the case in rice. Tissues or seeds from T0 plants can be stored in plate formats, allowing PCR amplification of gRNA sequences using barcoded primers and next-generation sequencing (NGS) of the mixed PCR products to identify the corresponding gRNAs for each T0 plant. T1 plants can be further screened for desired phenotypes. Molecular Plant 2017 10, 1141-1143DOI: (10.1016/j.molp.2017.08.001) Copyright © 2017 The Author Terms and Conditions