Design And Synthesis Of T-cell Acute Lymphoblastic Leukemia SIL-TAL1 Deletion

“Research on biological macromolecules and genes has entered the stage of precise regulation,the study of the rules of life operation developing from understanding life and transforming life to synthesizing life and designing life”.New ideas,new technologies to meet the study of multicellular eukaryotic genome complex sequence synthesis are required to be established.The main challenge is how to accurately design the genome sequence synthesis and transfer the genome to the target host for functional validation.In this study,we designed and synthesized the chromosomal DNA of 1p32 SIL-TAL1 deletion,combined with various techniques to explore new methods of synthetic large-segment DNA transfer,and proposed a strategy for in situ repair of 1p32 SIL-TAL1 deletion in acute T lymphoblastic leukemia.First,we designed the target DNA sequence to be synthesized.To ensure the functional activity of the synthesized SIL-TAL1 deletion gene fragments,the exons,introns,gene expression regulatory regions and unknown functional areas were precisely retained.To distinguish the synthetic fragments from natural fragments,the recombination sites,fluorescent signals and resistance labels were added,and the watermarking tags that did not affect the overall genome function were added by synonymous codon replacement.Based on the above design idea,different versions of the missing fragments to be synthesized were designed according to the different histone modifications of the TAL1 and STIL intergenic regions.Each sequence of all designed versions was divided into 5 kb unit sequences.Secondly,the de novo synthesis and assembly of the target segment DNA were performed.Starting from DNA unit fragments about 5 kb in length,the syn SILTAL1＿01 of 1p32 SIL-TAL1 fusion deletion with full-length 96,340 bp was assembled based on the developed "bottom-up" one-step synthetic assembly strategy.Further,syn SIL-TAL1＿02,syn SIL-TAL1＿03,syn SIL-TAL1＿04,syn SIL-TAL1＿05 with respective lengths of 7,767 bp,11,626 bp,16,767 bp,20,767 bp were assembled based on the prediction of the histone modification map.The technology of PCRTag,pulsedfield gel electrophoresis and next-generation sequencing are used to confirm the identity of the designed and synthetic sequences.Thirdly,to establish the flexible transfer process of synthetic genes,we developed multiple methods for heterogeneous cell contact fusion to mediate the transfer of synthetic DNA.Combined with UHF,femtosecond laser and microfluidic chip,this paper explored and developed various heterogeneous cell fusion to assist DNA transcell transfer technology.Especially,we established asymmetric double claw microfluidic chip structure to realize accurate co-localization and close contact in cell structure,proposed the one-to-many packaging strategy of yeast protoplast and mammalian cells and achieved fusion of heterogeneous cells in droplets,which opened a new direction for the development of large segment DNA transfer technology.Further,whether functional genes of fusion-deletion fragments can be normally expressed,and whether regulatory regions are functional,are crucial for mammalian genome synthesis studies.In this study,a stably transfected 293 T cell line with 1p32SIL-TAL1 deletion gene(108kb)was constructed.The functional characterization of the synthesized sections in m RNA,protein expression and transcriptional regulation,respectively,proved that synthetic genes could be normally expressed and affect the transcriptional regulation of cells.Finally,this research has formulated principles of the design for mammalian genomes,optimized the assembly method,developed new technology of large DNA transfer by combining UHF,femtosecond laser and microfluidic chip and evaluated the synthetic gene function in mammalian cells.Based on the synthetic gene,we proposed a strategy for loss of in situ repair of 1p32 SIL-TAL1 acute T lymphoblastic leukemia fusion,that provides new research ideas for the pathogenesis and potential treatment of chromosomal aberration-related diseases.This pipeline provides a framework for design and synthesis any mammalian genes for function evalution in cells.