Pei Ni

Prime editing is limited by low efficiency in plants. Here, we develop an upgraded engineered plant prime editor in hexaploid wheat, ePPEplus, by introducing a V223A substitution into reverse transcriptase in the ePPEmax* architecture. ePPEplus enhances the efficiency by an average 33.0-fold and 6.4-fold compared to the original PPE and ePPE, respectively. Importantly, a robust multiplex prime editing platform is established for simultaneous editing of four to ten genes in protoplasts and up to eight genes in regenerated wheat plants at frequencies up to 74.5%, thus expanding the applicability of prime editors for stacking of multiple agronomic traits.

Common wheat (Triticum aestivum) is one of the most widely cultivated and consumed crops globally. In the face of limited arable land and climate changes, it is a great challenge to maintain current and increase future wheat production. Enhancing agronomic traits in wheat by introducing mutations across all three homoeologous copies of each gene has proven to be a difficult task due to its large genome with high repetition. However, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) genome editing technologies offer a powerful means of precisely manipulating the genomes of crop species, thereby opening up new possibilities for biotechnology and breeding. In this review, we first focus on the development and optimization of the current CRISPR-based genome editing tools in wheat, emphasizing recent breakthroughs in precise and multiplex genome editing. We then describe the general procedure of wheat genome editing and highlight different methods to deliver the genome editing reagents into wheat cells. Furthermore, we summarize the recent applications and advancements of CRISPR/Cas technologies for wheat improvement. Lastly, we discuss the remaining challenges specific to wheat genome editing and its future prospects.

Two methods of synthesis, namely, using a polymerization catalyst versus a non-catalytic route, were investigated to produce lignin-based polyurethanes. The films were characterized with respect to crosslink density, ultimate tensile behavior and glass transition temperature. The results indicated that use of the catalyst for polymerization is an effective way for producing films with consistent properties, even at lignin contents as high as 45 to 50 wt%. To illustrate the catalyst effectiveness, crosslink densities of catalyzed films with 20 wt% of lignin content increased drastically from 0.2-0.3 to 1.7-2.7 mmol/cm 3 when the NCO/OH molar ratio increased from about 1.3 to 3.0, without much increase in the corresponding crosslink densities of the non-catalyzed films. Also, when the NCO/OH molar ratio increased from 1.2 to 3.2, the tensile strength increased from 1.9 MPa to a maximum of 55 MPa (NCO/OH=2.6) before decreasing. Also, for same NCO/OH ratios, ultimate strain decreased drastically from 174.4% to 4.3%, with a corresponding increase in Young's Modulus from 0.03 GPa to 2.8 GPa. The glass transition temperatures of the catalyzed films also increased from 35°C to 89°C. Without the catalyst, only polyurethanes with low NCO/OH ratios, low lignin contents, and inferior mechanical properties, could be synthesized.

Grain filling, a crucial process that determines grain weight, is regulated by the efficiency of sugar transport to the caryopsis. However, the regulation of sugar transport during this process in wheat remains largely unknown. In this study, we conducted genetic and transcriptomic analyses to investigate the role of TaSWEET11 in grain filling and its contribution to grain weight. TaSWEET11 encodes a membrane-localized protein and is primarily expressed in developing grains, specifically in the vascular bundle and nucellar projection. Knocking out TaSWEET11 disrupted starch synthesis in developing grains, resulting in shrunken and empty-pericarp grains. Further investigation revealed that TaSWEET11 is involved in sucrose transport, as knockout lines exhibited significantly reduced sucrose content. Transcriptomic analysis showed significant downregulation of genes related to starch synthesis and sucrose metabolism in knockout lines, shedding light on the mechanism behind grain shrinkage. Notably, overexpressing TaSWEET11 had a positive impact on effective tiller number, spike length, grain number per spike, and ultimately grain yield in CB037. In addition, TaSWEET11, as a key factor for grain filling, underwent strong selection during wheat domestication and breeding programs. Overall, these findings highlight the crucial role of TaSWEET11 in sucrose transport during grain filling and suggest its potential as a target for increasing wheat yield.

Additional file 2: Table S1. pegRNA target sites, RT templates and PBS sequences. Table S2. Summary of genotypes of individual plants induced by CMPE-ePPEplus in wheat plants in T0 generation. Table S3. Summary of mutations in each targeted gene induced by CMPE-ePPEplus in wheat plants in T0 generation. Table S4. Summary of simultaneous editing of multiple genes induced by CMPE-ePPEplus in wheat plants in T0 generation. Table S5. Analysis of potential off-target effects in regenerated wheat plants. Table S6. Segregation and transegene-free analysis of six T0 lines drived from CMPE-ePPEplus [63]. Table S7. PCR primers used in this study.