CRISPR-Cas9 is a common tool for gene editing, and appropriate sgRNAs are the key factor for successful editing. In this study, the effect of sgRNA length and number on editing efficiency was analyzed in rice using CYP81A6 as the target gene. A series of CRISPR-Cas9 plant expression vectors containing single sgRNAs with different lengths (17, 18, 19, 20, 21, 22, 23 nt) or two sgRNAs 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 sgRNAs was higher than that with a single sgRNA. Editing efficiency and sgRNA length showed a normal distribution with 20 nt sgRNA (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 sgRNA. These results provide data for successful gene editing or rice by CRISPR-Cas9.

With the widespread cultivation of transgenic crops, there is increasing concern about unintended effects of these crops on soil environmental quality. In this study, we used the Biolog method and ELISA to evaluate the possible effects of OsrHSA transgenic rice on soil microbial utilization of carbon substrates under field conditions. There were no significant differences in average well-color development (AWCD) values, Shannon–Wiener diversity index (H), Simpson dominance indices (D) and Shannon–Wiener evenness indices (E) of microbial communities in rhizosphere soils at eight samplings between OsrHSA transgenic rice and its non-transgenic counterpart. The main carbon sources utilized by soil microbes were carbohydrates, carboxylic acids, amino acids and polymers. The types, capacities and patterns of carbon source utilization by microbial communities in rhizosphere soils were similar throughout the detection period. We detected no OsrHSA protein in the roots of OsrHSA transgenic rice. We concluded that OsrHSA transgenic rice and the rHSA protein it produced did not alter the functional diversity of microbial communities in the rhizosphere.