Effects of sgRNA length and number on gene editing efficiency and predicted mutations generated in rice

CRISPR-Cas9 is a common tool for gene editing, and appropriate sg RNAs are the key factor for successful editing. In this study, the effect of sg RNA length and number on editing efficiency was analyzed in rice using CYP81 A6 as the target gene. A series of CRISPR-Cas9 plant expression vectors containing single sg RNAs with different lengths(17, 18, 19, 20, 21, 22, 23 nt) or two sg RNAs were constructed and introduced into rice cultivar Zhonghua11 by Agrobacterium-mediated transformation. Analysis of the editing status of 1283 transgenic rice plants showed that 371 were successfully edited with base preference.Single A or T insertions were the most frequent among the six edited types. The editing efficiency of transgenic rice with two sg RNAs was higher than that with a single sg RNA. Editing efficiency and sg RNA length showed a normal distribution with 20 nt sg RNA(25%) being the most efficient. The editing efficiency decreased slightly with decreases of 1–2 bases(19 nt 20%, 18 nt 21%), but decreased significantly with a decrease of 3 bases(17 nt 4.5%). Editing efficiency was significantly reduced by adding 1 to 3 bases(21 nt 16.8%, 22 nt 13%, 23 nt 13%) to the sg RNA. These results provide data for successful gene editing or rice by CRISPR-Cas9.

Grain carbon emission transfer and its spatiotemporal shifts based on the increasing supply-demand separation in China over the past three decades

The food system is one of the major sources of anthropogenic greenhouse gas(GHG)emissions.The impact of emission transfer due to the separation between food production and consumption within the context of carbon neutrality remains unclear.In this study,we constructed an emission inventory for three types of grains at the production stage of their life cycle and then analysed the spatiotemporal evolution characteristics of the grain supply and demand.With the use of a spatial equilibrium model,we simulated the spatial distribution flow of the different types of grains from 1990 to 2018 and calculated the resulting GHG emission transfer efficiency.The main results include the following:(1)The imbalance between the grain supply and demand intensified,which was mainly reflected in the distance between the geographic centre of the grain supply and consumption increasing 3.2 times,and thus,the self-sufficiency decreased.(2)The total emission transfer TET of rice and wheat decreased because of the increase in the intra-regional supply,while that of maize gradually increased due to the increase in the inter-regional supply.(3)Overall,grain trade improved the carbon efficiency of grain production in China.The trade efficiency of crops varied,with wheat and maize leading to overall effective carbon reductions,while the carbon transfer efficiency of rice from trade was relatively low.(4)The carbon footprint of grain production in China's provinces exhibited a downward trend,but due to the intensified separation between the grain supply and demand,certain major grain-producing areas achieved inefficient carbon increases.Therefore,we suggest further optimization of the spatial structure of planting and breeding,strengthening of the grain supply in the region,and enhancement in the optimization of the low-carbon production structure and adjustment of cultivated land use combined with regional governance strategies.The application of these measures could contribute to achieving dual-carbon goals.