Development And Optimization Of Adenine Transversion Base Editors

Base editors(BEs),a class of promising gene editing technology,have been widely utilized to modify single nucleotide,which were developed through combination of CRISPR/Cas system with distinct deaminases.Compared with canonical nuclease(CRISPR/Cas)-induced homology-directed repair(HDR),base editing is capable of installing efficient base substitutions without dependence of DNA double-strand break(DSB)process and the requirement for donor templates,refraining from potential risks such as P53 activation、chromosomal abnormalities in the course of HDR technique.Currently,three main types of base editing tools constitute BEs family,including cytosine base editors(CBEs)that mediate C-to-T mutations,adenine base editors(ABEs)catalyzing A-to-G conversions as well as cytosine transversion editors(CGBEs)which predominantly execute C-to-G transversion in mammals but C-to-A edits as major products in bacteria.However,no base editors that achieve effective A to C/T mutations have been described,and adenine transversion editing not only plays a pivotal role in basic biological research(e.g.,lineage tracing,molecular evolution),but also has profound implications for gene therapy of 25% pathogenic single nucleotide polymorphisms(SNPs).This study hopes to develop a class of base editors that efficiently mediate A·T-to-C·G/T·A transversions to fill in the gap,providing technical support for a myriad of potential applications.C-to-G/A transversion events normally are observed in editing products of CBE in the absence of uracil glycosylase inhibitor(UGI),influenced by activated base excision repair followed by cytosine deamination.Conversely,A to non-G transversion products are demanding to observe in endogenous systems(product purity of canonical ABEmediated A-to-G editing is typically ≥99%)probably due to the devoid of natural adenosine deaminases that acts directly on DNA and active DNA glycosylases to perform inosine(adenine deamination product)excision repair.In this study,through screening nine enzymes that potentially mediate inosine excision,we found that the fusion of mouse alkyladenine/3-methyladnine DNA glycosylase with highly-active adenosine deaminase Tad A-8e and catalytically impaired Cas9 nickase(Cas9n)can induce efficiently programmable A-to-Y(Y=C/T)products,resulting in adenine transversion editing agent we named AXBE.Data of in vitro and mammalian cells revealed that AXBE-induced adenine transversion edits rely on specific sequence context(mainly YAR-motif,R=A/G).To increase the efficiency of transversion and expand the targeting range,two key mutations(m AAG-EF)in m AAG were identified based on structure-guided molecular evolution,which significantly improved the resection activity of its substrate inosine and the editing efficiency of A-to-Y(A-to-C as the main product),especially transversion mutations at non-YAR-motif sites were significantly enhanced,thus effectively expanding the sequence selectivity of transversion editing.In addition,this study demonstrates that the m AAG-EF variant can also better adapt to PAM-relaxed Cas9 variants such as sp NG and sp RY,indicating that adenine transversion editing is expected to achieve a wider coverage of genomic region.Then this study developed a series of highly efficient A-to-C base editors(ACBEs)by use of reengineered adenine deaminases and Cas9 embedding strategy,among which the high-accuracy ACBE-Q version can precisely install the desired A-to-C transversion with background levels of Cas9-independent off-target events.More importantly,even high-efficiency A-to-C edits can be observed in ACBE-Q-treated mice,F0 mice exhibited average editing efficiencies of 45-53% with allelic frequencies of up to 100%.We also demonstrated that ACBEs can generate stop codons(e.g.,TAG and TGA)based on adenines in mammalian cell lines and mouse embryos,which terminates gene expression to obtain desired cell/animal disease models while canonical BEs only depend on cytosines to introduction of premature stop codons.Finally,disease-associated mutations were efficiently corrected or created in human cells with employment of ACBEs,showing that ACBEs have the potential to correct17% of C·G to A·T mutation-causing SNPs(the second most common category).In summary,this study aimed at inosine excision protein screening/evolution to create AXBEs and ACBEs series with combination of deaminase modification and protein embedding strategies,expectedly providing powerful novel technical support for applications such as molecular evolution,genetic screening,and lineage tracing due to available capacity of a wider range of codon and amino acid changes.Introducing premature stop codons in AT-rich regions will further expand the scope of gene regulation.Due to the high compatibility of m AAGs with PAM-relax Cas9 variants,the combination of ACBEs with different CRISPR/Cas systems will further expand the targeting range,indicating the therapeutic prospect of creating A-to-C correction in a broader genome range.In addition,ACBEs can serve as a unique tool to probe the DNA repair mechanisms of specific abasic sites opposite thymidine.Therefore,the development of AXBEs and ACBEs has significantly advanced the application capacity of base editing in basic research and potential therapeutic scenarios.