| More than half of all human genetic diseases are known to be caused by point mutations,so the development of tools that can accurately "rewrite" a single base is crucial to the study and treatment of such genetic diseases.The development of base editors bring hope to it.The base editor is composed of a nuclease-impaired Cas9 and a deaminase module,which do not directly cause DNA double-stranded breaks(DSB).The repair process does not rely on the delivery of exogenous DNA template,and is more efficient and precise than the strategy of homologous recombination to achieve base conversions in specific regions of genomic DNA.By fusing the activation-induced cytidine deaminase/apolipoprotein B m RNAediting enzyme catalytic polypeptide-like(AID/APOBEC)family deaminase with nCas9 to form the initial version of CBE,large amounts of C-to-G or C-to-A byproducts are usually produced.This is mainly because cytosine deaminase can deaminate C to form U,and U:G mismatch will be recognized by uracil DNA N-glycoylase(UDG).UDG excises U to form AP site,and induces unpredictable base conversions through TLS polymerase and DNA replication process.C-to-G base transversions will be mainly induced in some genomic DNA sites,a new class of base editor was constructed—CGBE.CGBEs developed based on natural cytosine deaminase have a wide range of applications,but at the same time they produce a high rate of DSB leading to small fragment insertion-deletion(indels)products,bystander editing,and Cas9-independent off-target editing at the DNA and RNA levels.Instead,ABE uses an unnatural adenine deaminase evolved from TadA in Escherichia coli to catalyse adenine deaminase in single-stranded DNA,achieving efficient and high purity(over 99.9%)A-to-G base transitions that does not cause indels to be produced.Recent studies have shown that ABEs can also induce cytosine editing in TC*N motifs.ABE8 e,a superactive ABE variant containing the TadA-8e deaminase showed enhanced cytosine deaminase activity,suggesting that evolved TadA can catalyse the deaminization of unicyclic cytosine as well as double-cycled adenine.Therefore,we imagine that it is possible to construct a novel base editor derived from non-natural cytosine deaminase for C-to-G transposition by modifying TadA to narrow the pockets of catalytic activity to allow only smaller volumes of cytosine to enter.This project is mainly aimed at the highest activity of TadA-8e modification.First,14 potential amino acid sites were selected near pockets that may affect the interaction of TadA-8e with substrate DNA,and 31 candidate variants were constructed for testing.The introduction of an N46 L variant in TadA-8e eliminated its adenine deaminase activity and led to the TadA-8e-derived C-to-G base editor(Td-CGBE)catalyzing the C·G-to-G·C transversions.Through large number of endogenous target evaluation,the catalytic activity of Td-CGBE was comparable to that of classical CGBE1,with a maximum efficiency of 72.8%,but the frequency of the generated indels was reduced by two times while accurately targeting C5/C6(window narrowing to 1-2nt).Then,based on the comprehensive off-target evaluation of Cas9-dependent/independent and RNA-seq,compared with representative CGBEs,we found that the off-target of Td-CGBE was reduced to the background level,providing a strong technical platform support for precision gene therapy.Finally,Td-CGBE was used to produce early termination codon at S184 of mouse Tyr gene.The albino phenotype was observed in 21 F0-generation mice,14 of which produced C-to-G transversions at the target site,with an average efficiency of 36% and a maximum efficiency of 85%.In addition,we have demonstrated the feasibility of Td-CGBE for precision therapy and other applications by generating or correcting pathogenic single nucleotide variants(SNVs)in homopolymeric cytosine sites.In summary,this study provides a breakthrough concept of base editing tool modification.The key variant N46 L affecting TadA deaminase substrate selection was found,which destroyed the inherent adenine deaminase activity and significantly improved cytosine editing,overcoming the defects of natural deaminase to a certain extent.The first new C-to-G base editor that does not rely on the AID/APOBEC protein family deaminase— Td-CGBE,has been innovatively developed.Compared with classical CGBEs,Td-CGBE produce the least amount of indels,have accurate editing window,greatly reduce genomic and transcriptomic off-target editing.The development of Td-CGBE enrich the toolbox of base editing technology,and provide a new idea for protein evolution engineering. |
