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Changes in human genetic diseases and agronomic traits of crops are usually caused by mutations in individual or small nucleotides in the genome.
single-base gene editing technology provides an important tool for the key nucleotide variation in the targeted editing genome. In rice
, Gao Caixia, a researcher at the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences, evaluated the specificity of two cytosine editors (CBE) BE3 and HF1-BE3, as well as a adenine editor (ABE), and for the first time comprehensively analyzed and compared the genome-wide targeting effect of the monobase editing system in the body using whole genome sequencing techniques.
research published online March 1 in the journal Science. The key nucleotide variation in the
identification and directional correction genome 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, and has been widely used in basic research, disease treatment and crop genetic improvement.
according to the different target base, the single base editor is divided into two main categories, cytosine monobase editor and adenine monobase editor, respectively, by the cytosine deaminase or modified adenine deaminase and nCas9 protein fusion, corresponding to the target site in the genome to achieve C and gt; T or A. G's base editing.
But it is not easy to accurately edit a target gene in a whole genome with vast amounts of data, and can sometimes "destroy" genes that control good traits or functions.
"Currently, CBE and ABE have been widely used in many species.
however, the detection of their off-target effect is still inadequate, because the data from previous studies are mainly derived from in vitro experimental studies, or the detection of finite target sequence similar sites predicted using bioinformatics software, and the off-target effect of CBE and ABE in the whole genome of the body has not been evaluated in detail.
" the first author of the paper, said Yu Shuai, a doctoral student at the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences.
the cloned plant samples by genome-wide sequencing analysis can overcome the above-mentioned data limitations, so as to objectively evaluate the specificity of single-base editing technology at the entire genome level.
researchers sequenced the genome-wide genome of 56 T0-generation rice plants converted by different monobase editing systems and 21 control plants.
further sequence statistical analysis found that after the single-base editing system, the number of base mutations inserted or removed in the genome did not change significantly compared with the control group.
but in the absence of sgRNA, BE3 and HF1-BE3 can cause a large number of additional single nucleotide variations in the rice genome, mostofy c. T-type base mutation.
plants treated with the BE3 and HF1-BE3 systems occur in the genome-wide scale compared to control plants that have been converted with aucobacteria but do not contain any single-base editing systems. T's single nucleotide variation increased by 94.5% and 231.9%, respectively.
, the current commonly used off-target prediction software Cas-OFFinder software is difficult to predict the addition of the above additional C. Off-target site of T single nucleotide variation.
in addition, the study found that these The variation of T is evenly distributed across chromosomes, but there is a tendency of enrichment in the transcriptional active region.
contrary 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 off-target effect within the genome was not detected, demonstrating very high specificity.
the researchers concluded that the ABE editor was able to accurately achieve single-base editing, but the cytosine editors of BE3 and HF1-BE3 had off-target editing throughout the genome, most likely due to random mutations in the genome caused by the use of cytosine deaminase or UGI.
this study is of great significance to the application and next transformation of single-base editing tools.
the future, how to reduce or eliminate the target of cytosine monobase editing tools will be an important direction for gene editing technology optimization.
Source: Science.com.