Human genetic diseases and crop agronomic traits are usually caused by mutations in single or minority nucleotides in the genome. Single-base gene editing technology provides an important tool for directed editing of key nucleotide variations in the genome. Gao Caixia, a researcher at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, conducted a genome-wide assessment of the specificity of two cytosine editors (CBE) BE3 and HF1-BE3, and an adenine editor (ABE) in rice. For the first time, in vivo, whole-genome sequencing technology was used to comprehensively analyze and compare the off-target effects of single base editing systems at the genome level. Related research results were published online in the journal Science on March 1.
Identifying and directionalally correcting key nucleotide variations in the genome is an important research direction for the treatment of human genetic diseases and animal and plant breeding. The single-base gene editing technology based on CRISPR system is one of the revolutionary technologies acquired in recent years, and has been widely used in basic research, disease treatment and crop genetic improvement.
According to the different bases, single base editors are mainly divided into two types, cytosine single base editor and adenine single base editor, respectively, by cytosine deaminase or modified adenine deaminase. Fusion with the nCas9 protein, correspondingly to achieve base editing of C>T or A>G at the targeting site in the genome.
But accurately editing a target gene in a whole genome with vast amounts of data is not an easy task, and sometimes it can “break down” genes that control good traits or functions.
“CBE and ABE have been widely used in many species at present. However, the detection of their off-target effects is still insufficient, because the data of previous studies mainly originated from in vitro experimental studies or the use of bioinformatics. The software predicted the detection of similar sites in the limited target sequence, and the off-target effects of CBE and ABE in the genome-wide range have not been evaluated in detail." The first author of the paper, the doctoral student of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences said . By analyzing whole-genome sequencing for clonal plant samples, the above data limitations can be overcome to objectively evaluate the specificity of single-base editing techniques at the entire genome level.
The researchers performed genome-wide sequencing of 56 T0 rice plants and 21 control plants transformed by different single-base editing systems. Further sequence statistical analysis revealed that the number of base mutations inserted or deleted in the genome was not significantly changed compared with the control group after treatment by the single base editing system. However, both in the presence or absence of sgRNA, both BE3 and HF1-BE3 caused a large number of additional single nucleotide variations in the rice genome, and most of them were C>T type base mutations.
Compared to control plants transformed with Agrobacterium but without any single-base editing system, plants treated with the BE3 system and the HF1-BE3 system increased the single nucleotide variation of C>T by 94.5 in the genome-wide range, respectively. % and 231.9%. Moreover, the currently used off-target prediction software Cas-OFFinder software is difficult to predict the above-mentioned additional target site of C>T single nucleotide variation.
In addition, the study also found that these C>T variations were evenly distributed between chromosomes, but showed a tendency to enrich in the active regions of transcription. In contrast to the CBE system, the number of single nucleotide variations in the ABE system was not significantly different from that in the control group, and no off-target effects in the genome were detected, showing very high specificity.
The researchers concluded that the ABE editor is capable of accurate single-base editing, but the cytosine editors for BE3 and HF1-BE3 have off-target editing across the genome. The reason for off-targeting is probably the cytosine deaminase used or UGI causes random variation in the genome. This study has important guiding significance for the application of single base editing tools and the next step. In the future, how to reduce or eliminate off-targeting of cytosine single-base editing tools will be an important direction for optimization of gene editing technology.
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