Crop breeding is an important global strategy to meet sustainable food demand. CRISPR/Cas is a most promising gene-editing technology for rapid and precise generation of novel germplasm and promoting the development of a series of new breeding techniques, which will certainly lead to the transformation of agricultural innovation. In this review, we summarize recent advances of CRISPR/Cas technology in gene function analyses and the generation of new germplasms with increased yield, improved product quality, and enhanced resistance to biotic and abiotic stress. We highlight their applications and breakthroughs in agriculture, including crop de novo domestication, decoupling the gene pleiotropy tradeoff, crop hybrid seed conventional production, hybrid rice asexual reproduction, and double haploid breeding; the continuous development and application of these technologies will undoubtedly usher in a new era for crop breeding. Moreover, the challenges and development of CRISPR/Cas technology in crops are also discussed.

Nowadays, with the rapid development of biotechnology, the CRISPR/Cas technology in particular has produced many new traits and products. Therefore, rapid and high-resolution detection methods for biotechnology products are urgently needed, which is extremely important for safety regulation. Recently, in addition to being gene editing tools, CRISPR/Cas systems have also been used in detection of various targets. CRISPR/Cas systems can be successfully used to detect nucleic acids, proteins, metal ions and others in combination with a variety of technologies, with great application prospects in the future. However, there are still some challenges need to be addressed. In this review, we will list some detection methods of genetically modified (GM) crops, gene-edited crops and single-nucleotide polymorphisms (SNPs) based on CRISPR/Cas systems, hoping to bring some inspiration or ideas to readers.

Molecular characterization is a key step in the risk assessment of genetically modified organisms (GMOs) for regulatory approval. Herein, we describe a method for analyzing copy number, insertion loci, and flanking sequences through whole-genome sequencing (WGS) and bioinformatics. Comprehensive molecular characterization of G2-6 transgenic rice was performed using this pipeline. The results showed that one copy of the foreign gene was inserted into rice chromosome 8. There was no vector backbone insertion but an unexpected insertion and DNA rearrangement at the 3′ end of the T-DNA. We also obtained the 5′ and 3′ flanking sequences of the T-DNA. Our results suggested that the use of a combination of WGS and bioinformatics is an effective strategy for the molecular characterization of GMOs.

Background: Soybean downy mildew (SDM), caused by Peronospora manshurica (Pm), is a major fungal disease in soybean. To date, little is known regarding the defense mechanism at molecular level and how soybean plants response to Pm infection. In this study, differential gene expression in SDM-resistant (HR) and SDM-susceptible (HS) genotype was analyzed by RNA-seq to identify differentially expressed genes (DEGs) following Pm infection. Results: Of a total of 55,017 genes mapped to the soybean reference genome sequences, 2581 DEGs were identified. Clustering analysis of DEGs revealed that these genes could be grouped into 8 clusters with distinct expression patterns. Functional annotation based on gene ontology (GO) and KEGG analysis indicated they involved in diverse metabolism pathways. Of particular interest were the detected DEGs participating in SA/ROS and JA signalling transduction and plant/pathogen interaction. Conclusion: Totally, 52 DEGs with P value < 0.001 and log(2) fold change > 2 or < - 2 upon fungal inoculation were identified, suggesting they were SDM defense responsive genes. These findings have paved way in further functional characterization of candidate genes and subsequently can be used in breeding of elite soybean varieties with better SDM-resistance.

The safety assessment and control of stacked transgenic crops is increasingly important due to continuous crop development and is urgently needed in China. The genetic stability of foreign genes and unintended effects are the primary problems encountered in safety assessment. Omics techniques are useful for addressing these problems. The stacked transgenic maize variety 12–5 × IE034, which has insect-resistant and glyphosate-tolerant traits, was developed via a breeding stack using 12–5 and IE034 as parents. Using 12–5 × IE034, its parents (12–5 and IE034), and different maize varieties as materials, we performed proteomic profiling, molecular characterization and a genetic stability analysis. Our results showed that the copy number of foreign genes in 12–5 × IE034 is identical to that of its parents 12–5 and IE034. Foreign genes can be stably inherited over different generations. Proteomic profiling analysis found no newly expressed proteins in 12–5 × IE034, and the differences in protein expression between 12 and 5 × IE034 and its parents were within the range of variation of conventional maize varieties. The expression levels of key enzymes participating in the shikimic acid pathway which is related to glyphosate tolerance of 12–5 × IE034 were not significantly different from those of its parents or five conventional maize varieties, which indicated that without selective pressure by glyphosate, the introduced EPSPS synthase is not has a pronounced impact on the synthesis of aromatic amino acids in maize. Stacked-trait development via conventional breeding did not have an impact on the genetic stability of T-DNA, and the impact of stacked breeding on the maize proteome was less significant than that of genotypic differences. The results of this study provide a theoretical basis for the development of a safety assessment approach for stacked-trait transgenic crops in China.

Maize (Zea mays L.) is a food crop with the largest planting area and the highest yield in the world, and it plays a vital role in ensuring global food security. Conventional breeding methods are costly, time-consuming, and ineffective in maize breeding. In recent years, CRISPR-Cas editing technology has been used to quickly generate new varieties with high yield and improved grain quality and stress resistance by precisely modifying key genes involved in specific traits, thus becoming a new engine for promoting crop breeding and the competitiveness of seed industries. Using CRISPR-Cas, a range of new maize materials with high yield, improved grain quality, ideal plant type and flowering period, male sterility, and stress resistance have been created. Moreover, many patents have been filed worldwide, reflecting the huge practical application prospects and commercial value. Based on the existing patent data, we analyzed the development process, current status, and prospects of CRISPR-Cas technology in dissecting gene function and creating new germplasm in maize, providing information for future basic research and commercial production.