Rationally Engineered Base Editors For Efficient And High-Fidelity C To G Transversion Editing

Background and ObjectiveA large-scale analysis of human genetic diseases finds that the largest category of pathogenic mutations in the human genome is point mutations,also known as single nucleotide polymorphisms(SNPs).The genetic diseases caused by SNPs account for about 58% of the total.Therefore,precise base editing on the single-base level plays a vital role in the research of genetic diseases.The single-base editing technology is a new gene editing technology based on CRISPR/Cas system modification.It can accurately replace one base with another base in the DNA without causing the DNA double-strand breaks(DSBs).Currently,the developed single base editors include the cytosine base editor(CBE)that can convert cytosine(C)to thymine(T),the adenine base editor(ABE)that can convert adenine(A)to guanine(G)and the recently developed CGBE that can perform C to G base transversion.Two recent articles have shown that replacing CBE's Uracil DNA Glucoamylase Inhibitor(UGI)with Uracil DNA Glucoamylase(UNG)can achieve C to G conversion.This study aims to obtainOPTI-CGBE by optimizing CGBE,improving C to G conversion efficiency and reducing the risk of missing the target.It then systematically analyzes the motif preference ofOPTI-CGBE and of other CGBE variants in order to provide guidance for the selection of sgRNA.Finally,theOPTI-CGBEs is uesd to perform efficient base editing in mouse embryos so as to verify the practicability of the system.Methods1.Construct the CGBE system by replacing the UGI of BE3 with UNG from different sources,and improve the CGBE system by optimizing r Apobec1,adding the nuclear localization sequence,optimizing the codon of SpCas9 n,and changing the position of UNG.Construct sgRNA system plasmid.CGBEs and sgRNA are co-transfected into HEK293 T cells.Positive cells are sorted by flow cytometry,and the target sequence is amplified by PCR.After deep sequencing of amplicons,the editing status of the target position is obtained.Then,analyze the editing efficiency of different versions of CGBEs C to G,and screen out the bestOPTI-CGBEs.The random off-target risk ofOPTI-CGBEs is further analyzed by whole-genome sequencing and transcriptome sequencing,and the sgRNA-dependent off-target risk is analyzed by Cas-OFFinder.2.Compare the influence of the upstream and downstream bases of the edited C site on its editing byOPTI-CGBEs,analyze the motif preference ofOPTI-CGBEs in the target sequence,and construct specific sgRNA plasmid according to the analysis results.Construct CGBE variants that contain different cytosine deaminases and then analyze their motif preferences.The variant of CGBE is constructed to identify “NG” PAM and the best version is screened out.3.The mouse embryos is edited withOPTI-CGBEs to test its editing efficiency on the embryos.After the edited embryos are further transferred to the surrogate mother mice,the young mice are delivered.And the edited mice are identified.Results1.The CGBE which is composed of c UNG from C.elegan and e UNG from E.coli can achieve efficient C to G conversion(29.6% and 22.7% respectively)through the deaminase optimization,the relative position optimization of fusion protein domain and the codon optimization.We call it cOPTI-CGBE(FNLS-c UNG-YE1-CGBE)and cOPTI-CGBE(FNLS-c UNG-YE1-CGBE).And the system has low off-target risk.2.It is found that both cOPTI-CGBE and eOPTI-CGBE have a significant preference for “QCW” motif through comparing the editing efficiency of cOPTI-CGBE and eOPTI-CGBE in the “QCW” motif site and the non QCW motif site(30.2% vs 9.5%;34.0% vs 12.1%).Besides,further analysis shows that e A3A-eOPTI-CGBE and e A3 AcOPTI-CGBE have a significant preference for “TCW,” while h A3G-eOPTI-CGBE,h A3G-CTD-eOPTI-CGBE,A3G-cOPTI-CGBE and h A3G-CTD-cOPTI-CGBE have a significant preference for “CCN” motif.The editing efficiency of Cas9n-NG and spGn is higher than that of x Cas9 n.3.The editing efficiency ofOPTI-CGBE in embryos can reach 33.2%,and 60% of the mice born after embryo transfer are edited,which shows thatOPTI-CGBE can effectively edit mouse embryos and produce mice with edited genes.ConclusionBy optimizing CGBE,we get a new variant namedOPTI-CGBE,which has the advantages of high C to G editing efficiency and low off-target risk.It is shown thatOPTI-CGBE and CBE have different motif preference,and CGBE of different deaminase types prefer different types of motifs.We have successfully usedOPTICGBE to edit mouse embryos and produced mice with edited genes.OPTI-CGBE will play an important role in applications that need the C to G editing.