Carl Jonas Jorge Spetz
Research Scientist
Authors
Xiaoyu Yuan Keya Xu Fang Yan Zhiyuan Liu Carl Jonas Jorge Spetz Huanbin Zhou Xiaojie Wang Huaibing Jin Xifeng Wang Yan LiuAbstract
Wheat dwarf virus (WDV, genus Mastrevirus, family Geminiviridae) is one of the causal agents of wheat viral disease, which severely impacts wheat production in most wheat-growing regions in the world. Currently, there is little information about natural resistance against WDV in common wheat germplasms. CRISPR/Cas9 technology is being utilized to manufacture transgenic plants resistant to different diseases. In the present study, we used the CRISPR/Cas9 system targeting overlapping regions of coat protein (CP) and movement protein (MP) (referred to as CP/MP) or large intergenic region (LIR) in the wheat variety ‘Fielder’ to develop resistance against WDV. WDV-inoculated T1 progenies expressing Cas9 and sgRNA for CP/MP and LIR showed complete resistance against WDV and no accumulation of viral DNA compared with control plants. Mutation analysis revealed that the CP/MP and LIR targeting sites have small indels in the corresponding Cas9-positive plants. Additionally, virus inhibition and indel mutations occurred in T2 homozygous lines. Together, our work gives efficient results of the engineering of CRISPR/Cas9-mediated WDV resistance in common wheat plants, and the specific sgRNAs identified in this study can be extended to utilize the CRISPR/Cas9 system to confer resistance to WDV in other cereal crops such as barley, oats, and rye.
Abstract
Weeds are one of the biggest problems that modern agriculture is facing worldwide due to the impact they have on crop productivity. Thus, there is a necessity to develop crop varieties with herbicide resistance or tolerance, which would provide cost-effective tools for helping farmers control weeds in the field. Development of herbicide-tolerant crops was initially based on conventional plant breeding and transgenic technology. In recent years, the emerging genome technologies, including ZFNs (zinc-finger nucleases), TALENs (transcription activator-like effector nucleases), and CRISPR (clustered regularly interspaced short palindromic repeat), provide us a new way for crop improvement through precise manipulation of endogenous genes in the plant genomes. Among these, CRISPR technologies, including nuclease systems, base editors, and prime editors, are really promising in creating novel crop germplasms with herbicide tolerance as they are simple, easy to use, and highly efficient. In this review, we briefly summarize the latest development and breakthroughs of CRISPR technologies in creating herbicide-tolerant crops. Finally, we discuss the future applications of CRISPR technologies in developing herbicide-tolerant crops.
Authors
Ziyan Xu Yongjie Kuang Bin Ren Daqi Yan Fang Yan Carl Jonas Jorge Spetz Wenxian Sun Guirong Wang Xueping Zhou Huanbin ZhouAbstract
Background Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome. Results In this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines. Conclusions The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.