| TALE is a powerful tool for gene regulation and genome editing,which has been restricted due to its cumbersome and time-consuming construction process;at the same time,because of the emergence of CRISPR and the construction process of CRISPR/Cas9 is relatively simple and the construction period is short,so TALE was abandoned.However,TALE’s high targeting is the biggest advantage.For this reason,this study has developed a new method for efficiently and quickly constructing TALE-TFs and TALENs,and they are used in the field of tumor induction differentiation therapy and gene editing and chromatin immunoprecipitation,respectively.and has achieved fruitful results.1.New method for efficiently constructing TALE-TF for tumor-induced differentiationTALE has always been a promising tool for gene editing and regulation.However,with the advent of CRISPR/Cas9,TALE was quickly abandoned due to its time-consuming and inefficient construction process.However,the off-target effect of CRISPR/Cas9 has limited its in vivo application.In contrast,because TALE is highly targeted,it has been allowed to perform gene editing and treatment in vivo.In order to overcome the key limitations of TALE technology,this study developed a new method to efficiently construct any customized TALE-TFs.This study created 62 new monomers and a new construction process for quickly assembling custom TALE-TFs within two days.This study validated the new method by assembling 9 TALE-TFs targeting the promoters of two transcription factor genes,HNF4α and E47.In two cancer cells,Hep G2 and PANC1,TALE-TFs activated the expression of two endogenous genes HNF4α and E47,respectively,thereby promoting the differentiation of the two cancer cells into normal cells.Using this new method,customized TALE-TFs can be constructed as easily and quickly as CRISPR,which can promote the wide application of TALE technology.2.Highly constructed TALEN vector precisely edits NF-κB family genesTALEN has been used for gene editing and therapy in vivo because of its high targeting ability.To this end,TALEN’s construction program was further optimized.This protocol can prepare TALENs that target any 18 bp target within a day(about 12 hours),which is faster than CRISPR/Cas9 construction.This scheme uses a set of linearized monomers,TALE-Fok I backbone plasmids and a new process to assemble the ready-to-use TALEN expression plasmid.By using a pair of universal primers to perform high-fidelity PCR amplification in 96-well plates,it can be easily produced and Copy these linear monomers.Most importantly,the new TALEN construction process can easily and efficiently obtain many positive colonies(over80%).This study was conducted by preparing 5 pairs of TALENs targeting 5 NF-κB family genes(RELA,RELB,CREL,NFKB1,NFKB2)in different cell lines(293T,Hep G2,and PANC1).It shows that the new method has high efficiency and applicability.Moreover,the constructed TALEN has higher editing efficiency than CRISPR,In the past,the detection of editing efficiency required the use of fluorescent quantitative PCR or DNA sequencing of selected monoclonal cells,and the detection cycle was long and complicated.Therefore,this study developed a labeling system for labeling target proteins,and successfully repaired the NF-κB family genes with the homology of green fluorescent protein(Zs Green),which can realize the visualization of edited genes and the simplification for detection of editing efficiency.The three positive cells(293T,Hep G2,PANC1)successfully screened by flow cytometry can be widely used in gene regulation and immunotherapy,and are also ideal cell models.3.Cell and protein double labeling system based on TALEN and homologous repairScreening with antibiotics after gene editing is still the traditional method of enriching edited cells.However,using antibiotics to screen for positive cells is highly toxic and timeconsuming,and it is difficult to obtain cells that have been successfully edited.To this end,we developed a dual labeling system for simultaneously labeling target proteins and cells based on the TALEN rapid preparation protocol developed earlier.Through homology-directed repair,streptavidin-binding peptide(SBP)or Avi Tag successfully edited proteins and cells,which not only realized the visualization of edited genes,but also successfully displayed peptide tags on the surface of cell membranes,and realized the visualization of edited cells.And the peptide tag can specifically bind to the immunostreptavidin magnetic beads,which is beneficial to the screening of positive cells in the later stage.At the same time,the editing efficiency of TALEN and CRISPR was also compared,and it was found that TALEN has higher editing efficiency than CRISPR.The system can be widely used for protein(antigen)preparation,immunoprecipitation and transcription factor chromatin immune-precipitation determination.4.The double labeling system based on TALEN was used for ChIP-seqChromatin immunoprecipitation followed by next-generation DNA sequencing(ChIP-seq)is a widely applied technique for identifying transcription factor(TF)binding events throughout an entire human genome.However,ChIP-seq is restricted by the availability of suitable ChIPseq grade antibodies,futher the vast majority of transcription factors do not have corresponding antibodies.To ameliorate these technical obstacles,in this study,a dual labeling system based on TALEN is proposed for ChIP-seq.Targeted genomic editing results in an epitope-tagged transcription factor NF-κB,which is recognized by a well streptavidin immunomagnetic beads.We assessed the feasibility of DPL system by tagging NF-κB homologous proteins(RELA,RELB,CREL,NF-κB1,NF-κB2)in the Hep G2 cells.We can use this method to obtain the classic motif of RELA-DNA binding,including motif of RELB,CREL,NF-κB1,NF-κB2.Our data exhibit strong correlations between both replicate types as well as with standard ChIP-seq approaches that use TF antibodies in RELA protein.Collectively,these data highlight the applicability of the new method to accurately define the genome-wide binding profiles of DNAbinding proteins,allowing for a straightforward methodology to potentially assay the complete repertoire of TFs,including the large fraction for which ChIP-quality antibodies are not available.In addition,the dual labeling system can also be used for the study of protein interactions.In short,the new method can quickly build TALE-TFs and TALENs,And TALE-TFs have successfully transformed cancer cells into normal cells in tumor differentiation therapy.The constructed TALENs also successfully edited NF-κB,and applied the protein double labeling system to chromatin immunoprecipitation technology to obtain the DNA binding spectrum of NF-κB. |
