Construction And Application Of Novel Cytosine Base Editors Based On MS2 And Suntag System

Background and ObjectiveGene editing is a technology to achieve programmable modification in genome by the deletion,insertion,or replacement of a fragment or specific bases of the genome,which allows the precise removal,addition,or alteration of genetic information.Due to its efficiency and simplicity,CRISPR/Cas9 is widely used in genome manipulation.Guided by sgRNA to the target sequence,Cas9 can introduce a DNA double-strand break（DSB）at the binding site.The DSB can be repaired by either non-homologous end joining（NHEJ）or homology-directed repair（HDR）.HDR generates precise modification,while NHEJ generates indels and deletions（indels）at the DSB site,which eventually resulting in frameshift mutations,translocations or other DNA rearrangements causing gene disruption.In addition,the DSBs are preferentially repaired by the alternative pathway of NHEJ,which causes many non-targeted stochastic changes in genome.To avoid DSB formation,a strategy based on CRISPR/Cas9 was developed,named base editing.Cytosine base editors（CBEs）were developed by fusing a nickase Cas9（nCas9）or a catalytically deficient Cas9（dCas9）with cytosine deaminases to generate C-to-T（G-to-A on the complementary DNA strand）base conversion at the targeting site under the guidance of sgRNA.The most used cytosine deaminases in CBEs mainly are rat APOBEC1（rAPOBEC1）and activation induced cytidine deaminase（AID）.The editing windows of most current CBEs are approximately 5nt.In addition,BEs based on rAPOBEC1（BE3 and BE-PLUS）has sequence preference which shows low editing efficiency in GC dinucleotides.Due to the narrow editing window and the context sequence preference of these BEs,there are many potential target sites in genome that cannot be edited because of lots of conditional restrictions.In order to increase genomic scope of base editing,reduce the preference of base sequences and improve editing efficiency,we try to build base editing tools with a wide editing window,no sequence preference and high editing efficiency.We constructed two new CBEs based on the MS2 system and the Suntag system,respectively.MS2-BE and BE-PLUS-AID broadened the range of application of base editing in the genome,and provide new tools for studying gene regulatory regions.Methods1.Construction of a base editor based on the MS2 system1.1 Based on the MS2 system,two copies of MS2 aptamers were ligated to the 3’end of sgRNA,and the MCP was fused to the N terminal of rAPOBEC1 or AID with nCas9（D10A）to construct a new base editor,MS2-BE.1.2 Verify the base editing efficiency of MS2-BE using the pSP189 blue-white reporting system.1.3 Verify the editing efficiency of the MS2-BE consisting of different versions of Cas9proteins with sgRNA-2X MS2 and MCP-AID in the pSP189 reporter system.1.4 Replacing different deaminases and exploring the editing efficiency of MS2-BE consisting of MCP-rAPOBEC1/MCP-AID,sgRNA-2X MS2 and dCas9/n Cas9（D10A）/n Cas9（H840A）in the pSP189 reporting system.1.5 The sgRNA-12X MS2 plasmid was constructed by fusing 12X MS2 to the 3’end of sgRNA.Verify the editing efficiency of different copies of MS2 with MCP-rAPOBEC1/MCP-AID and dCas9 in the pSP189 reporter system.2.Construction of a base editor based on the Suntag system2.1 Design sgRNA targeting the"GC"-rich sequences（VEGF Grich sg1,C9ORF72Grich sg1and sg2）,and verify the sequence preference of the base editors based on rAPOBEC1（BE-P and BE3）.2.2 Analyze the proportion of―GC‖in the promoter and 5’UTR of human reference genome using a custom python script.2.3 Based on the Suntag system,the cytidine deaminase rAPOBEC1 in BE-PLUS,was replaced with the truncated hAID^Δ182-198（K10E/E156G/T82I）to construct a new base editing tool,BE-PLUS-AID（BE-PA）,BE-PA3A,BE-P,BE-A3A and BE-A were constructed using the same method.2.4 Edit the"GC"-rich sequences and verify the base editing efficiency of BE-PA in"GC"or non-"GC"sequences.2.5 Target endogenous genes and verify the editing window and the sequence preference of BE-PA.2.6 Evaluate the precision of BE-PA by analyzing the indels,byproducts and off-target.2.7 Verify the knocking out efficiency of BE-PA in knocking out the target gene by introducing a premature stop codon using the GFP-iSTOP reporter system.2.8 Targeting the G-quadruplex sequence in the VEGFA promoter region to verify the utility of BE-PA in the functional regions of genome.Results1.Construction of a base editor based on the MS2 system1.1 The initial version of MS2-BE consists mainly of three parts:1)MCP-AID-UGI that MCP is ligated to the N terminal of AID-UGI;2)sgRNA-2X MS2 that 2X MS2 is ligated to the 3’end of sgRNA;3)nickase Cas9（D10A）.1.2.Targeting the supF region of pSP189 plasmid,MS2-BE induced C-to-T conversion which occurred in a wide window ranging from-32²8 bp around PAM sequence with an average base editing efficiency of 0.0519.1.3 Among the three Cas9 variants,the dCas9 group cotransfected with sgRNA and MCP-AID produced more white colonies（P<0.01）showing higher base editing efficiency in MS2-BE system.1.4 When using dCas9 or nCas9（D10A）,the editing efficiency of MCP-rAPOBEC1group is higher than that of MCP-AID group（dCas9 group:MCP-rAPOBEC1 vs.MCP-AID,P<0.0001;nCas9（D10A）group:MCP-rAPOBEC1 vs.MCP-AID,P<0.01）.When using nCas9（H840A）there was no significant difference in efficiency between MCP-rAPOBEC1group and MCP-AID group（MCP-rAPOBEC1 vs.MCP-AID,P>0.05）.1.5 When MCP-rAPOBEC1 was used,the editing efficiency of sgRNA-2X MS2 group was higher than that of sgRNA-12X MS2 cotransfected with dCas9.（P<0.01）While when MCP-AID was used,there was no significant difference in editing efficiency between sgRNA-2X MS2 and sgRNA-12X MS2 cotransfected with dCas9.（P>0.05）.1.6 When using sgRNA-2X MS2,the editing efficiency of MCP-rAPOBEC1 group was higher than MCP-AID group（P<0.001）.When using sgRNA-12X MS2,there was no significant difference between MCP-rAPOBEC1 and MCP-AID（P>0.05）.1.7 The hotspot region of MS2-BE system is from-32^-2 counting from PAM and the average efficiency in hotspot region of MS2-BE system with MCP-rAPOBEC1was 17.26%,which was higher than MS2-BE system with MCP-AID（P<0.05）.1.8 In hotspot region,MS2-BE with sgRNA-2x MS2 could simultaneously introduce more transitions of C-to-T than MS2-BE with sgRNA-12x MS2（P<0.05）.1.9 The optimal version of the MS2-BE system consists of the following three components:1)MCP-rAPOBEC1-UGI;2)sgRNA-2X MS2;3)dCas9.2.Construction of a base editor based on the Suntag system2.1 At the"GC"site,the base editing efficiencies of BE-P（BE-PLUS）and BE3 are low,which equipped with rAPOBEC1,and there was sequence preference for BE-P and BE3.2.2 The"GC"dinucleotide sequences are not uniformly distributed across the genome,and are enriched in regulatory region,promoters and the 5’-UTRs.2.3 Replace the rAPOBEC1 with the C-terminal truncated AID^Δ182-198（K10E/E156G/T82I）in BE-P to generate a new version of BE,BE-PLUS-AID（BE-PA）.2.4 In"GC"contents,BE-PA had higher base editing efficiency and broader editing window compared to BE-PA3A,BE-P,BE-A3A and BE-A.2.5 When targeting endogenous genes,BE-PA showed broader editing window maintaining high base editing efficiency with no sequence preference.2.6 BE-P and BE-PA produced fewer indels and editing byproducts than BE-A.Although the off-target of BE-PA was slightly higher,there was no statistical difference among BE-P,BE-PA and BE-A.2.7 BE-PA was more effective than BE-P in reducing the expression of GFP in the"GC"sequence by introducing stop codons and BE-PA increased the usage of BE in the coding regions of genome.2.8 Compared to BE-P,BE-PA successfully disrupted the G-quadruplex structure in the VEGFA promoter more efficiently and significantly reduced the expression of VEGFA mRNA and protein.Conclusions1.Based on the MS2 system,the base editor MS2-BE-rAPOBEC1 was constructed to simultaneously edit multiple target bases in a window of-32^-2 bp.2.Based on the Suntag system,the base editing editor BE-PLUS-AID was successfully constructed.It can edit the―GC‖rich sequences with higher editing efficiency,broader editing window and shows no sequences preference.It is a more suitable tool for exploring gene regulatory regions.