In-locus gene silencing in plants using genome editing.

Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5'untranslated region (5'UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG). Here, we report an efficient gene silencing method by introducing a tailor-designed uATG-containing element (ATGE) into the 5'UTR of genes in plants, occupying the original start site to act as a new pATG. Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding. Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement.

Targeted insertion of regulatory elements enables translational enhancement in rice.

In-locus editing of agronomically-important genes to optimize their spatiotemporal expression is becoming an important breeding approach. Compared to intensive studies on mRNA transcription, manipulating protein translation by genome editing has not been well exploited. Here, we found that precise knock-in of a regulating element into the 5'UTR of a target gene could efficiently increase its protein abundance in rice. We firstly screened a translational enhancer (AMVE) from alfalfa mosaic virus using protoplast-based luciferase assays with an 8.5-folds enhancement. Then the chemically modified donor of AMVE was synthesized and targeted inserted into the 5'UTRs of two genes (WRKY71 and SKC1) using CRISPR/Cas9. Following the in-locus AMVE knock-in, we observed up to a 2.8-fold increase in the amount of WRKY71 protein. Notably, editing of SKC1, a sodium transporter, significantly increased salt tolerance in T2 seedlings, indicating the expected regulation of AMVE knock-in. These data demonstrated the feasibility of such in-locus editing to enhance protein expression, providing a new approach to manipulating protein translation for crop breeding.

Large-scale genome editing in plants: approaches, applications, and future perspectives.

As a powerful genome editing technology, CRISPR/Cas is revolutionizing both fundamental research and crop breeding, and has now evolved into large-scale editing tools that are efficient, simple, and programmable. With such CRISPR screening technologies, the numbers of genome-edited crops are rapidly increasing. Here, we describe the general workflow of a CRISPR screen in plants, including the selection of appropriate editors, genome-wide guide RNA design, pooled library construction, massive transformation, and high-throughput genotyping. We also discuss applications for the screening of candidate genes, the optimization of spatiotemporal expression, the evolution of protein activities, and the establishment of genome-wide libraries of knockout mutant. After considering the current challenges and limitations, we finally envision a virus-mediated strategy to improve CRISPR screens.

High-throughput genome editing in rice with a virus-based surrogate system.

With the widespread use of clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate (WDV-surrogate), to facilitate high-throughput genome editing. WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene (HygM) for surrogate selection; the other is a WDV-based construct expressing two single guide RNAs (sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency (66.8% vs. 90.1%), plantlet regeneration efficiency (2.31-fold increase), and numbers of homozygous-edited plants (36.3% vs. 70.7%). Large-scale editing using pooled sgRNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDV-Gate on sequence knock-in for protein tagging. By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.

Application of γ-aminobutyric acid (GABA) and nitrogen regulates aroma biochemistry in fragrant rice.

The 2-acetyl-1-pyrroline (2AP) is a key aroma compound in fragrant rice. The present study assessed the γ-aminobutyric acid (GABA) and nitrogen (N) application induced regulations in the biochemical basis of rice aroma formation. Four N levels, that is, 0, 0.87, 1.75, and 2.61 g/pot, and two GABA treatments, that is, 0 mg/L (GABA0) and 250 mg/L (GABA250), were applied to three fragrant rice cultivars, that is, Yuxiangyouzhan, Yungengyou 14, and Basmati-385. Results showed that GABA250 increased 2AP, Na, Mn, Zn, and Fe contents by 8.44%, 10.95%, 25.70%, 11.14%, and 43.30%, respectively, under N treatments across cultivars. The GABA250 further enhanced the activities of proline dehydrogenase (PDH), ornithine aminotransferase (OAT) (both at 15 days after heading (d AH), and diamine oxidase (DAO) (at maturity) by 20.36%, 11.24%, and 17.71%, respectively. Significant interaction between GABA and N for Mn, Zn, and Fe contents in grains, proline content in leaves, GABA content in leaves at 15 d AH and maturity stage (MS), Δ1-pyrroline-5-carboxylic acid (P5C) contents in leaves at 15 d AH, and Δ1-pyrroline-5-carboxylate synthase (P5CS), PDH, and OAT activities in leaves at MS was noted. Moreover, the 2AP contents in grains at MS showed a significant and positive correlation with the proline contents in the leaves at 15d AH. In conclusion, GABA250 enhanced the 2AP, Na, Mn, Zn, and Fe contents, as well as the enzyme activities involved in 2AP biosynthesis. Exogenous GABA and N application improved the 2AP contents and nutrient uptake in fragrant rice.