Engineering A Highly Precise Adenine Base Editor With Infinitesimal Bystander Effects

DNA base editors,mainly CBEs and ABEs,are novel gene editing tools derived from CRISPR/Cas9 systems.By fusing the nickase Cas9 with cytosine(APOBEC/AID family)or adenine deaminase(evolved Tad A proteins),cytosine or adenine at specific sites in the genome can be converted into thymine or guanine with the guidance of sg RNA,respectively.Base editors can efficiently catalyze base substitutions without introducing DNA double-strand breaks and adding additional DNA donor templates,showing a wide range of application prospects.ABE can convert A·T base pair to G·C base pair,but if there are multiple adenines in the editing window(editing window represents bases deaminated by the deaminase within the target site),the non-target bases will be edited due to bystander effects.In addition,ABE’s DNA and RNA offtarget editing has aroused widespread concern about its safety.In addition,it has been reported recently that ABE can convert cytosines in the TCN motif in genomic DNA,resulting in undesired C-to-T/A/G conversions in addition to the A-to-G bystander effects.Further,with the improvement of ABE and its increased activity(such as ultrahigh activity ABE8e),ABE also induces more severe adenine and cytosine bystander effects and higher off-target editing at DNA and RNA levels.Therefore,developing safer ABE with high efficiency is an urgent need to solve the safety problems when applying ABE in clinical research.In this study,based on the cryo-electron microscopic structure of Tad A-8e adenine deaminase,we tried to modify ten key residues(E27,V28,P29,H57,F84,P86,N108,L145,F148 and Y149)in deaminase that could affect the interaction with DNA substrate.Twenty-one mutants with different single amino acid replacements were designed and constructed.Through preliminary screening,we found that compared with ABE8 e,Tad A-8e-V28 F,V28N,N108 Q,L145C,L145 T and L145 Q showed a tendency to narrow the editing window and reduce cytosine base conversions without reducing on-target activity.ABE8e-N108 Q had the highest activity and the lowest bystander editing effects.By testing 12 and 9 endogenous targets containing multiple adenines or cytosines in cells,we demonstrated that compared with ABE8 e,ABE8eN108Q could narrow the main activity window to A4-A7 while maintaining the main editing activity,and reduce the average bystander editing efficiency of cytosine from18.02% to 5.79%.However,the performance of this variant in reducing adenine and cytosine bystander editing was not perfect enough to achieve true single-base editing.Therefore,we wanted to further add potential point mutations that could improve deaminase specificity to develop a narrowed window ABE.In the preliminary screening,we constructed 14 combination mutants,and found that the combination of ABE8eN108 Q with an additional single mutation in E27,P29,F84 and L145 residues presented a condensed editing window,even to a single adenine at position 5.ABE8eN108Q/L145 T displayed the narrowest editing window while maintaining high ontarget activity,so we named it ABE9.The results at 12 endogenous targets containing multiple adenines showed that the overall editing activity of ABE9 was slightly decreased compared with ABE8 e,but its editing activity was much higher than ABE7.10 and ABE9 could significantly reduce adenine bystander editing and narrow the activity window to 1-2nt.Compared with ABE8e-N108 Q,the editing accuracy of ABE9 was improved by up to 8-fold(4.3-fold on average).The indels rate of ABE9 was comparable to ABE8 e and ABE8e-N108 Q or slightly decreased.In addition,at all11 tested targets containing multiple cytosine,ABE8 e and ABE8e-N108 Q induced severe cytosine bystander editing,while ABE9 had no cytosine editing at 10 out of 11targets(efficiency lower than 1% considered as no editing),reducing cytosine conversion rates by an average of 56.2-and 10.2-fold compared to ABE8 e and Abe8eN108 Q,respectively.These results indicated that ABE9 almost completely eliminates unnecessary cytosine editing.In addition,using the unbiased evaluation strategy of the target library containing 9120 sg RNAs,we found that ABE9 confined the editing window to 1-2nt with the highest editing activity and preference at A5.Through the profiling of the motif preferences of ABE9,we found that it was suitable for a wide range of accurate A-to-G editing without strict motif requirements.All these data suggested that the accuracy of ABE9 was dependent on the position relative to the protospacer but not on sequence context.In the off-target analysis,we designed 44 potential Cas9-dependent DNA off-target sites,and found that ABE8 e induced mild off-target editing at 11 tested sites(mean4.15%),while ABE9 only had mild off-target editing at 2 sites with high on-target activity.In the modified orthogonal R-loop experiment,compared with ABE8 e,ABE9greatly reduced the Cas9-independent DNA off-target editing,which was close to the background level(mean<0.3%).In genome-wide RNA-seq analysis,the RNA offtarget effect of ABE9 was also reduced to the background level,by reducing 726.1-and117.1-fold compared with ABE8 e and ABE8e-N108 Q,respectively.These results suggested that ABE9 was highly specific with only a minimal rate of DNA and RNA off-target editing.Due to minimized editing window,ABE9 could further broaden the scope for accurate correction of pathogenic point mutations when fusing to Cas9 variants with expanded PAM compatibility.In summary,by molecular evolution of adenine deaminase Tad A-8e,this study developed a new precise and safe ABE9.ABE9 had a narrowed editing window of 1-2nt and can accurately achieve A-to-G conversions in mammalian cells with minimal unpredictable DNA and RNA off-target editing rates while eliminating severe cytosine bystander effects of traditional ABE.Developing novel precise editing tools like ABE9 is crucial for both basic research and gene therapy.