Mengyan Bai, Juehui Yuan, Huaqin Kuang, Pingping Gong, Suning Li, Zhihui Zhang, Bo Liu, Jiafeng Sun, Maoxiang Yang, Lan Yang, Dong Wang, Shikui Song, Yuefeng Guan.

Plant Biotechnol J. 2020 Mar; 18(3):721-731.  doi: 10.1111/pbi.13239. Epub 2019 Sep 9.

Abstract

The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR-Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR-Cas9 as a mutant screening tool. Here, we report a pooled CRISPR-Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR-Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1-1/1-2/1-3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops.

See: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004907/pdf/PBI-18-721.pdf

Figure 1 A pooled CRISPR-Cas9 mutagenesis procedure in soya bean. (a) sgRNAs are designed to target multiple loci or specific single loci, and CRISPR vectors are cloned individually. In the optimized procedure, the gene editing efficiency of each sgRNA is assessed in hairy roots. Highly efficient vectors are pooled into a sublibrary in A. tumefaciens, which is then transformed into soya bean. The T0 and T1 plants are genotyped by SSP (the coloured bars indicate sgRNA positive), and gene editing is verified by PCR and Sanger sequencing. Desired homozygous mutants will be obtained in the T1 or T2 generation and then subjected to phenotyping. (b) Architecture of the pGES201 vector. Type IIS BsaI restriction sites were used for sgRNA cloning between the U6 promoter and sgRNA scaffold. In between two BsaI restriction sites is a toxin gene, CcdB.pM4 was used to drive spCas9. 35S-driven BASTA is the selection marker.