The new single base editing tool is efficient and safe

a classic single base editing tool deaminase with ssDNA binding region BD and catalytic active region AD (left). The deaminase of the YE1-BE3-FNLS single base editing tool loses the ssDNA binding region BD, preserving the catalytic active region AD (right).
May 18, Nature Methods published the latest results of collaborations between the Shenzhen Genomics Research Institute of the Chinese Academy of Agricultural Sciences, the
Institute of Neuroscience and the
Map Institute of Computational Biology.
Based on the protein structure, they predicted the important amino acids that determine the off-target effect during gene editing, and mutated the corresponding amino acids without affecting catalytic activity, and finally obtained a single base editing tool that significantly reduced the off-target effect of gene editing.
March 2019, the team published GOTI technology in the journal Science to detect the off-target rate of gene editing technology. The latest results are based on GOTI's in-depth exploration of ways to address the off-target effect of single-base editors, Zuo Erwei, co-author of the paper and a researcher at the Shenzhen Genomics Research Institute of the Chinese Academy of Agricultural Sciences, told China Science Daily.is a typical genetic disease caused by mutations in genetic points, with more than 3 million patients worldwide and more than 40 million people with sickle-type anemia. As of 2013, the disease has killed 176,000 people worldwide.
about 300 million people worldwide with rare diseases. Eighty per cent of the more than 7,000 known rare diseases are single-gene genetic diseases and 50 per cent occur in childhood. It is expected that 40 new gene therapy drugs will be available in 2022.
gene editing technology is one of the most important disruptive technologies in the field of biological research, and gene editing tools with CRISPR/Cas9 system as the core are widely used in medical research fields such as hematopoietic stem cells and nerve cells.
" traditional CRISPR/Cas9 system to cut off the DNA double strand, which brings some uncertainty to the results of gene editing. "The derivative, single-base editing technology, enables targeted mutations of single nucleotides without cutting off the DNA double strands," mr. Zuo said. This offers hope for the treatment of genetic diseases caused by single-base mutations.
Based on the CRISPR/Cas9 system, in 2016, Harvard University professor David Liu's team first reported on a new gene editing tool that works on a single base, cytosine deaminase mono-base editing technology (CBE), which received immediate attention.
the system uses catalytic cytosine deaminase, guided by sgRNA, to target binding to specific genomic locations and convert cytosine C into thymus T. In the same way, the conversion from Ostrich G to Adenine A can also be achieved.
2017, the David Liu team developed the Adenine single base editing tool, the ABE system, which enables efficient T-to-C and A-to-G conversion.
, however, the safety of single-base editing tools will be called into question in 2019. Several research teams at home and abroad, including Yang Hui, Gao Caixia, Keith Joung and David Liu, have reported serious DNA and RNA off-target effects in single base editors.
previous studies have significantly reduced RNA off-targeting by introducing mutations, DNA off-targeting in cytosine mono-base editors remains unresolved.GOTI technology is a new generation of gene editing off-target detection technology, which is far more sensitive, accurate and applicable than the previous off-target detection technology.
" with GOTI technology can accurately evaluate the safety of gene editing technology. Yang Hui, co-author of the paper and a researcher at the
Shanghai Institute of Neurology, told China Science daily that on this basis, there is a purpose to improve existing single-base editing tools or to develop new ones.
single-base editing tool is a protein molecule designed by humans to sequence amino acids, and the system consists of components such as deaminase and nCas9. Using GOTI technology, they found that CBE's off-targeting was caused by cytosine deaminase.
" deaminase plays a catalytic role in editing tools. It should have been led by sgRNA, according to the law of base pairing, led the Cas9 protein to bind to the corresponding position of the genome, the transformation of a single base. But deaminase itself has the ability to bind ssDNA to RNA, and it carries the Cas9 protein to trigger base mutations at any 'like' bit. This type of base mutation, which does not occur under the guidance of sgRNA and does not occur at the designed location, is a completely random and unpredictable off-target effect, Zuo said.
found out why CBE was off target, and the team set out to improve the single base editing tool.In off-target effect detection, computational biology can provide detailed calculation methods and patterns to quickly draw conclusions and analyze the extent and harm of off-targeting, said Li Yisso, co-author of the
paper and a researcher at the
Magnup Institute of Computational Biology, in an interview with China Science Daily. in improving single-base editing tools, computational biology has a powerful computational power: using high-volume bioinsynamics to screen the purpose points of deaminase modification. This has a great enlightenment effect on future research.
"We used ion sequence matching to predict areas where structurally unknown deaminase binds to ssDNA or RNA, and introduced specific mutations in important amino acids in this region. By changing the protein composition, the ability to bind deaminase to ssDNA or RNA is eliminated. Sun Yidi, co-first author
Center for Excellence and Innovation in Molecular Cells, told China Science Daily.
they constructed a total of 23 CBE mutants and fully detected their presence as off-target across the gene range. Four of these mutants did not affect gene editing efficiency, but also reduced random off-target effects.
"We optimized the above CBE mutants, increased labeling and nuclear positioning sequences, and significantly improved gene editing efficiency in high fidelity, making it a safe and efficient new gene editing tool, YE1-BE3-FNLS. Yang Said the new tool is expected to be used in gene therapy for genetic diseases to promote the clinical application of gene editing. The findings are consistent with those of David Liu's team, published in Nature Biotechnology on February 10 this year. Both articles reported that YE1 reduced off-targeting of DNA and RNA while maintaining high editing efficiency.
but Yang Hui's GOTI-based approach is unrestricted and can be used not only to detect single-base editors, but also to detect and improve the safety of other gene editing tools based on fusion proteins.
"We hope that GOTI technology will become the gold standard for detecting gene editing off-targeting." Yang Hui said that both scientific research on gene editing techniques and gene therapy for genetic diseases should use efficient and accurate testing methods to assess their safety, that is, off-target effects.
" is still relatively easy to implement in the laboratory phase. But if applied clinically, there is still a lot of work to be done to optimize and reduce costs. There is still a long way to go from proposing ideas and directions to truly commercializing. "Lee also the theology.
new single-base editing tool is "a bit bulky" for vector adeno-related viruses commonly used in gene therapy, according to Mr. Zuo. This is where they need to improve in the future.
", the technique requires the direct digestion of well-developed embryos from entire mice, a practice that is difficult to use in human beings. Sun said it's worth thinking about how the new gene editing technology can achieve risk-free trauma detection.
Yang Hui stressed that the current detection of off-target effects, improvements to single-base editing tools, etc. are done in the laboratory, "we have solved the safety and effectiveness of gene editing technology." "The safety and effectiveness of gene therapy has yet to be further validated in animal and clinical trials.
, Chinese and foreign scientists have launched clinical applications for gene therapy for sickle anemia based on traditional gene editing techniques. "There is no gene therapy for single base editing tools. We're still working on it. Yang Hui said.