Programmable Base-editing In Bombyx Mori Using BE3

In recent years,CRISPR/Cas9 genome-editing technology has been rapidly and widely adapted for cutting-edge applications in stem cell biology,genomic biology,developmental biology,and cancer research because of its high efficiency and simplicity.Under the direction of a guide RNA(gRNA),the Cas9 nuclease binds to an opened DNA strand paired with the gRNA and induces a double-strand break(DSB).At this point,the intrinsic cellular DNA-repair mechanism will generally repair the DSB via non-homologous end joining(NHEJ),resulting in deletions or insertions(indels)in most cases.When present,the cell can use a homologous DNA fragment as a template to repair the DSB by another mechanism,known as homology-directed repair(HDR).The sequence shifts caused by NHEJ may disrupt the function of the target genes.The DNA surrounding the cleavage site could be replaced with a designed homologous DNA template through HDR.Although the traditional CRISPR/Cas9 system is powerful,its DSB-dependent genome editing still has some defects.The key for Cas9 editing gene is the response to the DSB.The repair method of NHEJ is mainly used for gene knockout.The NHEJ is so random that target genes may not be knocked out sometimes for the number of inserted or deleted bases is difficult to control.The HDR is inefficient and difficult to achieve,although it is very accurate.DSBs generated by the over-cutting of Cas9 might lead to excessive DNA damage that could cause targeted chromosome structural changes,such as deletions of fragments,translocations and eli mination and cell death when targeting several genomic loci simultaneously.When the damage exceeds the tolerance of the cell,it may even lead to cell death.In order to solve the above problems,many scientists are committed to researching new technology systems and implementing safe and efficient genome editing with DSB-free.In 2016,the David R.Liu's group developed the base editor(BE)which base on the CRISPR/Cas9 system by combining cytosine dea minase(APOBEC1)and uracil glycosylase inhibitor(UGI).This system can generate site-specific base conversions from C:G base pairs to T:A base pairs without inducing DSBs,while effectively avoiding indels,avoiding a series of negative effects brought by DSB.And it didn't take long for other base-editing systems(Target-AID,TAM,and CRISPR-X)to appear.The development of base-editing systems has both improved the scope and effectiveness of genome editing.To date,several organisms have been subjected to base editing,mostly using BE,including mammals,rice,mice and zebrafish.However,no reports have described the performance of any base-editing system in invertebrates.Here,we demonstrated that CRISPR/Cas9-dependent base editor(BE3)converts C:G to T:A with a high frequency up to 66.2% in the invertebrate Bombyx mori.Using BE3 as a knock-out tool,we efficiently inactivated exogenous and endogenous genes.Notably,up to 14 bases were substituted simutltaneously in a single DNA molecule,with a low indel frequency of 0.6%,when 32 gRNAs were co-transfected.The main conclusions are as follows:1.Establishing a base-editing system for Bombyx moriTo confirm whether base editing could be achieved by BE3(rAPOBEC1-XTEN-nCas9-UGI)in B.mori,we co-transfected the codon-optimized BE3 vectors and gRNA vectors into B.mori embryo cell line(BmE).Genomic regions spanning the target site were amplified by PCR for sequencing.Sanger sequencing for both the Blos2 and Yellow-e PCR products showed a site-specifical substituted C:G with T:A base pair.The efficiency of C-T substitutions for gRNA-Blos2 and gRNA-Yellow-e reached 40% and 51.2%,respectively.Notably,with the guidance of gRNA-Yellow-e,BE3 could edit up to 4 bases simultaneously.2.A comprehensive research of the editing window of BE3 in Bombyx moriRecent reports showed that the BE3 could induce C-to-T substitutions with dea mination windows covering 9(C1–C9)and 5(C4-C8)bases in plants and mammalian cells respectively,whereas we detected a broader editing window with effective C substitutions spreading from C1 to C13.The editing window,ranging from C1 to C13,is wider than that in plants and mammalian cells,which provides greater potential and feasibility for modifying the genome at target sites with a larger scope.Differing efficiencies probably reflected the different positions of C:G base pairs within gRNAs.The most efficient sites for base editing are mainly concentrated in C4-C7.3.Establishing a effective BE3-mediated knockout-system for Bombyx moriHere,we also used BE3 to efficiently inactivate exogenous and endogenous genes through substitution-induced nonsense mutations in B.mori.The frequency of introducing a stop codon was up to 66.2%,which was much more efficient than that found with SpCas9,SaCas9,and Cpf1 in B.mori cells.From this point of view,BE3 is more suitable for knock-out studies in invertebrates.To deter mine the targetable sites for knock-out by BE3 at the genome scale,we identified all candidate codons(CAA,CAG,CGA and TGG)that can potentially be converted to stop codons(TAA,TAG,TGA)by BE3.This genome-scale analysis revealed a pool of 151,551 targetable knockout sites in 14,106 genes,with a median of 11 sites per gene and 96.5% targetable genes(among 14,623 total genes).Furthermore,the distributions of these codons were well-distributed within the CDSs,suggesting that these targetable sites could effectively stop mRNA translation.BE3 provides an alternative for functional-genomics studies and other knock-out experiments and represents an improvement over Cas9 in terms of its ability to perform base substitutions,without generating DSBs.4.With 32 gRNAs together,14 C:G-to-T:A conversions occurred in EGFPGenome editing without generating DSBs is the biggest advantage of the base-editing system over Cas9.Such a capacity will guarantee the integrity of the genome to a much larger extent.In this study,we only detected a few indels generated by 32 gRNAs individually targeting EGFP with an average indel frequency of 2.2%,compared with a 1.2% indel frequency in control samples(which might have been induced by PCR or sequencing).However,when we co-transfected all 32 gRNAs together with the BE3 vector,the indel frequency in the experimental group was almost the same as that in control cells.This result indicated that multiple bases can be edited simultaneously with almost no indels occurring around the target sites.Using the C:G base editor BE3 and the recently described A:T base editor ABE,all four transition mutations can be programed with a library of gRNAs to enable mapping of thousands of parallel a mino acids and promoter mutations simultaneously.