Liyun Yang , Lijiao Chen , Yang Zheng , Li Deng , Raoxian Bai , Ting Zhang , Zhengbo Wang , Shangang Li
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引用次数: 0
Abstract
The CRISPR/Cas9 system can induce off-target effects in programmed gene editing, but there have been few reports on cleavage detection and their affection in embryo development. To study these events, sgRNAs with different off-target rates were designed and compared after micro-injected into mouse zygotes, and γH2AX was used for DNA cleavage sites analysis by immunostaining and CUT&Tag. Although the low off-target sgRNA were usually selected for production gene editing animals, γH2AX immunofluorescence indicated that there was a relative DSB peak at 15 h after Cas9 system injection, and the number of γH2AX foci at the peak was significantly higher in the low off-target sgRNA-injected group than in the control group. Further, the result of CUT&Tag sequencing analysis showed more double-strand breaks (DSBs) related sequences were detected in low off-target sgRNA-injected group than control and the distribution of DSB related sequences had no chromosome specificity. Gene Ontology (GO) annotation analysis of the DSB related sequences showed that these sequences were mainly concentrated at genes associated with some important biological processes, molecular functions, and cell components. In a conclusion, there are many sgRNA-sequence-independent DSBs in early mouse embryos when the Cas9 system is used for gene editing and the DSB related sequence could be detected and characterized in the genome. These results and method should also be considered in using or optimizing the Cas9 system.
期刊介绍:
Genomics is a forum for describing the development of genome-scale technologies and their application to all areas of biological investigation.
As a journal that has evolved with the field that carries its name, Genomics focuses on the development and application of cutting-edge methods, addressing fundamental questions with potential interest to a wide audience. Our aim is to publish the highest quality research and to provide authors with rapid, fair and accurate review and publication of manuscripts falling within our scope.