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Chinese scientists have found an all-genome-wide miss effect on cytosine single-base editors

pubdate:2019/03/02 source:

Chinese scientists have found an all-genome-wide miss effect on cytosine single-base editors

Human genetic diseases and changes in agronomic traits of crops are usually caused by mutations in individual or a few nucleotides in the genome. Single-base gene editing technology provides an important tool for key nucleoside variation in directional editing genomes. Kaoaaka team, Researcher, Institute of Genetic and Developmental Biology, Chinese Academy of Sciences on two cytosine editors (CBE) in riceThe BE3 and Hf1-be3, as well as the specificity of a adenine editor (ABE), were evaluated throughout the genome level, and for the first time, the Miss effect of single-base editing system on genomic level was comprehensively analyzed and compared by using whole genome sequencing technique in vivo.

The research results were published online in the journal Science on March 1.Identification and directional correction of key nucleotide variations in genomes is an important research direction in the treatment of human genetic diseases and animal and plant breeding.

Single-base gene editing technology based on CRISPR system is one of the revolutionary technologies obtained in recent years, which has been widely used in basic research, disease treatment and crop genetic improvement.According to the different target bases, the single base editor is mainly divided into two types, the cytosine single base editor and the Adenine single base editor, respectively, by cytosine deaminase or modified adenine deaminase and NCas9 protein fusion, corresponding to the target sites in the genome to achieve c>t or a>g base editing at the point.But it is not easy to accurately edit a target gene throughout the genome with large amounts of data information, and sometimes it can "destroy" genes that control good traits or functions."At present, CBE and Abe have been widely used in a number of species. However, the detection of their off-target effect is not sufficient, because the previous research data mainly from in vitro experimental research, or the use of bioinformatics software to predict the detection of similar sites of limited target sequences, and CBE and Abe in vivo genome-wide range of the Miss effect has not yet beenTo a detailed assessment. "first author of the paper, 靳帅, PhD student, Institute of Genetic and Developmental Biology, Chinese Academy of Sciences.

Through genome-wide sequencing analysis of cloned plant samples, the above data limitations can be overcome, so as to objectively evaluate the specificity of single-base editing technology at the entire genome level.The researchers sequenced the whole genome of 56 T0-generation rice plants and 21 control plants transformed by different single-base editing systems. Further sequence statistical analysis showed that the number of base mutations inserted or removed in the genome did not change significantly compared with the control group after the single base editing system was processed. However, whether in the absence of Sgrna, BE3 and Hf1-be3 areA large number of additional single nucleotide variants can be caused in the rice genome, and most of them are base mutations of the c>t type. Compared with the control plants transformed by Agrobacterium but not containing any single base editing system, the single nucleotide variation of c>t in the whole genome range of plants treated by BE3 system and HF1-BE3 system increased by 94.5% and 231, respectively.9%.

Moreover, the current use of off-target prediction software Cas-offinder software is difficult to predict the above additional c>t single nucleotide variation of the target point.In addition, the study also found that these c>t variations were evenly distributed among chromosomes, but showed a tendency to be enriched in transcription active areas.

In contrast to the CBE system, there was no significant difference between the number of single nucleotide variants in the Abe system and the control group, and the Miss effect in the genome was not detected, showing a very high specificity.The researchers concluded that the Abe editor was able to accurately implement single-base editing, but that BE3 and Hf1-be3 's cytosine editors had off-target editing throughout the genome-wide range, The reason for the miss is likely to be the random mutation of the genome caused by the cytosine deaminase or UGI used. This research has important guiding significance for the application of single base editing tool and the next transformation. In the future, how to reduce or eliminate the target of cytosine single base editing tool will be an important direction of gene editing technology optimization.