中国生物工程杂志

Objective: In order to improve the on-target effect of CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9).At the same time, it can prove the application value of the yeast hybridization system in the study of off-target effect. Methods: Based on the activase gene-edited rice strains previously constructed in our laboratory, the T7 endonuclease I assay was firstly used to predict off-target rates in 30 gene-edited rice strains. Subsequently, the yeast hybrid system was further used to investigate the off-target rate. During this period, at first, standard sgRNA and truncated sgRNA of on-target gene were cloned into the CRISPR/Cas9 expression vector pDW3769 to be recombinant vectors pHZ2 and pHZ4, and then those vectors were transfered into YPH499 yeast haploids to be the recombinant yeast YpHZ2 and YpHZ4. Seven off-target sequences A, B, C, D, E, F, G derived from off-target site prediction and on-target sequence were selected and cloned into the high copy vector pDW3133 and the low copy vector pDW3134, both of which contained the reporter genemCherry. The corresponding high copy recombination vectors were pHZ5, pHZ7, pHZ9, pHZ11, pHZ13, pHZ15, pHZ17 and pHZ19 and the corresponding low copy recombination vectors were pHZ6, pHZ8, pHZ10, pHZ12, pHZ14, pHZ16, pHZ18 and pHZ20, and all of them were respectively transformed into YPH500 yeast haploids to be recombinant yeast YpHZ5-20. Subsequently, recombinant yeast YpHZ2 and YpHZ4 individually hybridized with recombinant yeast YpHZ5-20, colonies of diploid yeast were picked and the fluorescence values were examined at various time periods, and at last off-target rate on the basis of fluorescence values was predicted. Results: The significant and stable fluorescence value was at 144-192 h, and off-target sgRNA sequences which have higher homology to the targeted sgRNA became more likely to be edited by CRIPSR/Cas9, too. However, truncated sgRNA can reduce the off-target rates compared with standard sgRNA. The result of off-target detection according to rice plants showed about 20%, and the off-target detection based on yeast hybridization was about 20%-28%. Conclusions: The fluorescence values are most significant and stable during the stationary phase in batch culture of yeast and directly proportional to the copies of the vector, and sgRNA sequences and structure can affect and targeting of CRISPR/Cas9. The prediction results of off-target rate were similar between the two methods so that the yeast hybrid platform can be proved to be a highly useful method in evaluating the off-target rate of the CRISPR/Cas9 system and investigating factors affecting the off-target rate.