In cereal crops, grain starches are composed of different proportions of amylose and amylopectin, which determine the cooking and eating qualities. The amylose synthesis is controlled by the Waxy (Wx ) gene encoding a granulebound NDP‐glucose‐starch glucosyltransferase (Shure et al ., 1983). In rice (Oryza sativa L.), the varied activities of natural Wx alleles regulate different amylose contents (AC), gel consistency (GC), and pasting viscosity of grain starches; these factors together influence the grain appearance, cooking/eating quality, and starch physical characters (Zhang et al ., 2019).

Nutrient deficiencies in crops are a serious threat to human health, especially for populations in poor areas. To overcome this problem, the development of crops with nutrient-enhanced traits is imperative. Biofortification of crops to improve nutritional quality helps combat nutrient deficiencies by increasing the levels of specific nutrient components. Compared with agronomic practices and conventional plant breeding, plant metabolic engineering and synthetic biology strategies are more effective and accurate in synthesizing specific micronutrients, phytonutrients, and/or bioactive components in crops. In this review, we discuss recent progress in the field of plant synthetic metabolic engineering, specifically in terms of research strategies of multigene stacking tools and engineering complex metabolic pathways, with a focus on improving traits related to micronutrients, phytonutrients, and bioactive components. Advances and innovations in plant synthetic metabolic engineering would facilitate the development of nutrient-enriched crops to meet the nutritional needs of humans.

Summary: Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site‐specific A‐to‐G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer‐adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high‐efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single‐stranded DNA‐binding domain (DBD), based on PAM‐less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n‐NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base‐editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e‐DBD‐SpRYn (hyABE8e‐SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)‐dependent off‐target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base‐editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.

Recently, some newly engineered SpCas9 variants, such as xCas9 3.7 and Cas9‐NG, expand the PAM recognition site to NG and show function efficiency in mammalian cells. However, there is still not enough comparative data for their efficiencies on the genome and base editing in plants. In addition, there is also a lack of enhanced specific Cas9 variant with NG PAM. Here, we developed a serial of genome editing tools using different Cas9 variants containing the xCas9, the Cas9‐NG and the enhanced specific Cas9‐NG (eCas9‐NG), and of cytosine base editors (CBE 4 editors, xCas9n‐CBE, Cas9n‐NG‐CBE and eCas9n‐NG‐CBE) and adenine base editors (ABE 7.10 editors, xCas9n‐ABE, Cas9n‐NG‐ABE and eCas9n‐NG‐ABE). We systematically investigated the editing activities of these tools with expending target scope in transgenic rice. Our results indicated that the Cas9‐NG and eCas9‐NG variants enable more efficient genome editing and targeted C‐T single nucleotide substitutions with extended non‐canonical PAMs than those of xCas9. But their ABE editors worked inefficiently in rice. The Cas9‐NG variant is more suitable for genome engineering in rice. Taken together, our work has engineered and yielded Cas9 variant tools with a broadened PAM compatibility, which will facilitate plant functional genome and crop genetic improvement.