Development Of A Novel Adenine Base Editor And Application Of In Vivo Base Editing Technology Mediated By LNP

Single base editing is a gene editing strategy for single base substitution,which is divided into two working systems according to the difference of single-cut Cas9(Cas9n)deaminase.Among them,Adenine Base Editors(ABEs)convert adenine(A)to guanine(G)in the editing window to correct C·G to T·A genetic mutations.Compared with the traditional CRISPR/Cas9 gene editing strategy,ABEs have improved the efficacy,accuracy and safety of gene therapy,and have a wide range of application potential and high development prospects.However,as a powerful gene editing tool,base editing system also has many technical limitations,such as narrow target range,low precision,low editing activity and off-target effects.In this study,a novel adenosine deaminase was obtained by metagenomic analysis.The mutation and modification of the novel base editor can further enhance the editing activity and expand the editing window,which is a beneficial attempt for the development and optimization of base editing tools.First,we replaced TadA8e deaminase with a new adenosine deaminase 5V1 to constructed a new 5V1-ABE base editor.We tested its editing characteristics in mammalian cell lines and compared it with ABE8e base editor.We found that5V1-ABE had lower editing activity and a smaller editing window range than ABE8e.Therefore,based on the homology modeling results of 5V1 and TadA8e,we selected three residues Q150/Q151/P152 for single point mutation and combination mutation of 5V1-ABE to obtain a better performing base editor 5V1-GMR-ABE.The editing window of 5V1-GMR-ABE was A3-A8,the same as that of 5V1-ABE,but the average editing efficiency in the window was increased by 1.5～3.4 times,and no sgRNA-dependent DNA off-target was detected.The editing range of5V1-GMR-ABE was smaller than ABE8e,and the editing activity at A5,A6 and A8 positions was comparable to ABE8e.Secondly,we inserted the novel deoxyadenosine deaminase or the control deaminase TadA8e into the relevant permissive sites within Cas9 n protein to construct new fusion base editors.Among the 11 permissive sites tested,1249 showed the best results.Compared with adenosine deaminase at the N-terminus,the editors with same deaminase at 1249 site had altered editing windows and improved editing activities.ABE8e-1249 variant showed the most significant improvement in editing activity,while 5V1-1249-ABE and 5V1-GMR-1249-ABE variants showed less improvement.In addition,we studied the efficiency of ABE8e in vivo editing in mouse liver and established a feasible method to deliver base editor and sgRNA in vivo using Lipid Nanoparticle(LNP),which can be used to verify the performance of novel base editors in vivo.In conclusion,this study successfully developed new ABE tools,achieved efficient editing in mammalian cells,and further enriched the base editing toolbox.This study also confirmed that LNP-mediated ABE8e was highly efficient editing in mouse livers,providing a valuable therapeutic strategy for familial hypercholesterolemia.