Recently, Shanghai BRL Medicine Inc. (hereinafter referred to as "BRL Medicine"), in collaboration with the team of Professor Li Dali and Professor Liu Mingyao of East China Normal University, and published an article in Nature Biotechnology on June 15, 2023, reporting the development of a series of novel adenine transversion editing tools (AXBEs and ACBEs),which provide new tools for diversified genetic manipulation and gene therapy for the second largest class of single-base mutations (SNVs) in humans.

Nature Biotechnology  publishes

AXBEs: Enabling Broad Genome Targeting, Expanding the scope of Applications

Base transversions rely on the creation of apurinic and apyrimidinic (AP) sites, followed by the base excision repair pathway (BER). In view of the low efficiency of inosine excision repair ability of endogenous glycosidases, the research team of BRL Medicine and East China Normal University searched for other enzymes that could potentially use inosine as a catalytic substrate, and compared it with adenine deaminase TadA-8e and nCas9 fusion. Surprisingly found that when fused with mouse-derived alkyladenosine DNA glycosidase (mAAG), 8.7% of A to Y (Y=C or T) base transversions were achieved, and it was named AXBE (X stands for any base). In order to increase the efficiency of adenine transversion editing and expand the targeting range, two key mutations (mAAG-EF) in mAAG were identified based on structure-guided rational design and screening, which greatly improved the resection activity of its substrate inosine. Based on this The resulting AXBEv2 mediated more efficient A to Y editing, and even transversion mutations at non-YAR-motif sites were significantly enhanced, thus effectively improving the sequence-background selectivity of transversion editing.

The ABE system can only generate 122 codons and 32 amino acid conversions, while AXBEs can induce the conversion of adenine to the other three bases. Based on the ABE system, it can generate unique 436 codons and 115 Amino acid mutations have shown that the genome can be broadly targeted, expanding the scope of application, and providing powerful gene editing tools for molecular evolution, genetic screening, lineage tracing and other applications.

ACBEs: Precise, Efficient, with Great Therapeutic Potential

In order to further reduce the serious non-target base A to G mutation, the researchers tried to transform adenine deaminase and adopt the strategy of Cas9 embedding, so that the editing by-products from A to G were greatly reduced, and this type of editor was named respectively for ACBE and ACBE-Q . ACBE can achieve up to 45% of A to C editing and 73% of base transversion editing, while ACBE-Q can more accurately edit the A4-A6 positions of sgRNA, the accuracy is increased by up to 171 times, and only the background level is generated. Cas9-independent off-target events (average off-target efficiency <0.3%), showing high application safety. By comparing the isotopes with the prime editing technology, it is found that ACBE can more efficiently realize the precise conversion of A-to-C, while the PE system needs to be screened and optimized through dozens of parameter combinations, but the efficiency is very low and it will Generates a high rate of deletion mutations. It proves that ACBE is simpler and easier to implement, and has significant advantages in efficiency.

In addition, studies have shown that ACBE-Q also exhibits extremely high editing efficiency and precision in mice. In the establishment of the mouse disease model of Duchenne muscular dystrophy, 70% of the mutant mice (21/30) realized the editing of the target site A6 to C, the average editing efficiency was 56%, and the highest editing purity of A>C could reach 99.8 %, showing its great potential for in vivo applications. Finally, in order to study the therapeutic potential of ACBEs, the researchers constructed a stable cell line carrying the mutation of STAT3 c.1145G>T (the hot spot mutation causes recurrent infectious disease), and ACBEs introduced the desired A to C correction editing at the target position . The results showed that the ability of ACBEs to introduce stop codons in AT-rich regions in advance expanded the scope of gene regulation, and the compatibility of mAAG with different Cas variants further expanded the targeting range from A to C, indicating great therapeutic promise for correcting the second largest class of pathogenic SNVs in humans . Great therapeutic promise for pathogenic SNVs.

ACBEs are precisely edited in mice and mimic or correct human pathogenic SNVs in cell lines

In this study, the development of novel adenine transversion editing tools provides a new strategy for diversified genetic manipulation and gene therapy of human genetic diseases. ACBEs can be expected to correct many genetic diseases caused by single point mutations from C G to A T, for example, phenylketonuria, liver metabolic diseases such as  hyperammonemia due to ornithine transaminase (OTC) deficiency, and hemophilia B. Compared with the PE system, ACBE has the characteristics of being more efficient and simple in establishing A-to-C base transversions. Overall, the development of ACBEs has added another pivotal member to the base editing toolbox, which can be said to be the last piece of the puzzle of base editing technology, which is expected to help people overcome a wider range of genetic diseases.