SummaryThe RNA‐guided Cas9 system is a versatile tool for genome editing. Here, we established a RNA‐guided endonuclease (RGEN) system as an in vivo desired‐target mutator (DTM) in maize to reduce the linkage drag during breeding procedure, using the LIGULELESS1 (LG1) locus as a proof‐of‐concept. Our system showed 51.5%–91.2% mutation frequency in T0 transgenic plants. We then crossed the T1 plants stably expressing DTM with six diverse recipient maize lines and found that 11.79%–28.71% of the plants tested were mutants induced by the DTM effect. Analysis of successive F2 plants indicated that the mutations induced by the DTM effect were largely heritable. Moreover, DTM‐generated hybrids had significantly smaller leaf angles that were reduced more than 50% when compared with that of the wild type. Planting experiments showed that DTM‐generated maize plants can be grown with significantly higher density and hence greater yield potential. Our work demonstrate that stably expressed RGEN could be implemented as an in vivo DTM to rapidly generate and spread desired mutations in maize through hybridization and subsequent backcrossing, and hence bypassing the linkage drag effect in convention introgression methodology. This proof‐of‐concept experiment can be a potentially much more efficient breeding strategy in crops employing the RNA‐guided Cas9 genome editing.

This study evaluated the effects of different dietary levels and sources of zinc (Zn) on performance and carbonic anhydrase (CA) activity in eggshell formation and quality in aged laying hens. A total of 504 Hy-line Grey layers aged 59 wk were fed a basal diet (Zn, 28.4 mg/kg) for 4 wks, then randomly allocated to 7 groups that were fed a basal diet or a basal diet supplemented with inorganic (ZnSO4·H2O) or organic (amino acid metals, 9.58%) Zn at 35, 70, or 140 mg Zn per kg of feed for 6 weeks. Each group had 6 replicates of 12 hens. Results showed that egg weight decreased linearly with the supplemental level of organic Zn (P < 0.05). Dietary Zn supplementation had linear and quadratic effects on the CA activity in plasma (P < 0.05), and it was higher in the organic Zn-added groups at wks 2 and 4 (P < 0.05). Dietary Zn supplementation had a quadratic effect on the CA activity in the eggshell gland (P < 0.05). Shell thickness was greater in the organic Zn-added groups (P < 0.05), and its relationship with the supplemental level of Zn showed linearly and quadratically, increasing with the organic Zn and with the inorganic Zn at wk 4, while linearly increasing with the inorganic Zn at wk 6 (P < 0.05). At wk 4, the supplemental level of inorganic Zn had a linear effect on shell weight, and linear and quadratic effects on shell index and ratio (P < 0.05), while shell weight, the index, and ratio increased linearly and quadratically with the organic Zn level in the diet (P < 0.05), with more obvious effects in the organic Zn-added groups (P < 0.05). Overall, dietary Zn supplementation, up to 140 mg/kg feed, could increase eggshell thickness by enhancing CA activity in the plasma and eggshell gland of aged layers; thicker eggshells were found in the organic Zn-added groups, but the breaking strength did not increase despite the eggshell thickness increasing.

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The study aimed to determine the effects of methionine hydroxyl analog chelated zinc (MHA-Zn) on laying performance, eggshell quality and mineral deposits, and the activities of Zn-containing enzymes on aged laying hens. A total of 960 layers (Hy-Line Grey, 57 wk old) were fed a basal diet (Zn: 35.08 mg/kg) without extra zinc for 2 wk. During the ensuing 14 wk, birds were randomly divided into 4 groups according to body weight and laying rate, with 8 replicates per treatment, and each group had 8 replicates of 30 hens. Four levels of Zn (ZnSO4: 80 mg/kg; MHA-Zn: 20, 40, 80 mg/kg) were added to the diet, respectively. The results shown that dietary Zn did not affect laying rate, average egg weight, average daily feed intake, or feed conversion ratio (P > 0.05); however, compared to the inorganic group, dietary supplementation with 40 or 80 mg/kg MHA-Zn decreased broken egg rate significantly in the whole period (P < 0.05), while significantly increased eggshell weight in week 62 to 72, eggshell thickness and eggshell strength in wk 66 to 72, eggshell weight percent and eggshell density in week 62 to 72 (P < 0.05). Besides, dietary supplementation with different sources and levels of Zn did not affect ash concentration of eggshell (P > 0.05), whereas dietary supplementation with 80 mg/kg MHA-Zn improved the Zn and Ca concentrations of eggshells and carbonic anhydrase (CA) activity of liver, and 40 mg/kg MHA-Zn increased Zn concentration of liver (P < 0.05). Moreover, no significant differences in alkaline phosphatase activity were observed among the treatment groups (P > 0.05). Therefore, dietary supplementation with 40 mg/kg MHA-Zn can improve eggshell quality by promoting Ca deposition and CA activity.

The study aimed to determine the effects of methionine hydroxy analog chelate zinc on the tibia quality, mineral deposit, apparent retention of nutrients, and liver metallothionein (MT) expression level of aged laying hens. A total of 960 layers (Hy-Line Grey, 57 wk old) were randomly assigned into 4 groups, and each group had 8 replicates of 30 hens. During the first 2 wk, groups were fed a basal diet without extra zinc (Zn: 35.08 mg/kg). During the ensuing 14 wk, 4 levels of Zn (inorganic Zn: 80 mg/kg; organic Zn: 20, 40, 80 mg/kg) were added to the diet. The results indicated that both the Zn source and level did influence tibia strength and calcium (Ca) and Zn concentrations of tibia (P < 0.05), whereas there were no differences in the copper (Cu) and phosphorus (P) concentrations of the tibia and the tibia length (P > 0.05). Moreover, dietary supplementation with 40 or 80 mg/kg of organic Zn showed higher Zn and Ca concentrations in the tibia and higher tibia strength. The Cu concentration in the liver showed no difference among the 4 treatments, whereas the Zn concentration in the liver increased with the increasing Zn level. The apparent retention of P, iron (Fe), and manganese (Mn) was not affected by the Zn level or source (P > 0.05). However, the organic Zn group increased the apparent retention of Cu, Zn, Ca, crude protein (CP), and energy, and the group supplemented with 40 or 80 mg/kg of organic Zn obtained significant effects (P< 0.05). Moreover, dietary supplementation with 40 or 80 mg/kg organic Zn increased the MT mRNA expression of the liver at week 72, whereas 20 mg/kg of organic Zn decreased it (P < 0.05). In conclusion, this study suggested that an optimum dietary (40 mg/kg) organic Zn level plays a key role in promoting the apparent retention of minerals and nutrients, trace element deposit, and MT mRNA expression.

For some Cas nucleases, trans -cleavage activity triggered by CRISPR/Cas-mediated cis -cleavage upon target nucleic acid recognition has been explored for diagnostic detection. Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture. Here, we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool. We systematically characterized AsCas12a, LbCas12a, LwaCas13a, and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection. Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input. Using this tool, we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field. Our method, from sample preparation to result readout, could be rapidly and easily deployed in the field. This system could be extended to other crop pathogens, including those that currently lack a detection method and have metabolite profiles that make detection challenging. This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping, transgene detection, and qualitative detection of gene expression in the field. Supporting Information The supporting information is available online at 10.1007/s11427-021-2028-x. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.