Gene Editing And Information Storage In Escherichia Coli

Deoxyribonucleic acid(DNA)is the main information carrier of life on earth and has the characteristics of programmability,persistence,easy replication,high storage density and fidelity.It is also a foundational element in synthetic biology,more recently used as a template for gene editing and a medium for DNA information storage.Nevertheless,there are still many limitations in large-scale gene insertion and information storage in cells,such as introduction of exogenous gene-editing templates and low information storage capacity.To overcome these limitations,we took advantage of high-copy plasmids and engineered E.coli cells to develop an extensible and low-cost method,which allowed us to directly synthesize single-stranded DNA(ss DNA)to edit genes and store digital information in the form of DNA in living cells.The ss DNA synthesis system based on high-copy plasmids with a modular rolling circle replication structure allows us to synthesize circular ss DNA with arbitrary length and sequence in E.coli cells.Based on the ss DNA synthesis system and Cas9 nuclease,a tightly controlled RC-Cas system was designed to generate circular ss DNA,from which a desired fragment was cut out using the Spy Cas9 nuclease in a protospacer adjacent motif(PAM)-independent manner.Combined with ?Red recombinase,the desired linear ss DNA fragment could be used as donor for precise genome engineering.Donor ss DNA fragments from hundreds to thousands of nucleotides in length were synthesized in E.coli cells,allowing successive genome editing in growing cells and enabling the insertion of large sequences(>1kbps).At the same time,the stability,efficiency and programmability of high-copy plasmids during replication in E.coli cells allowed the use of mixed cultures of bacterial cells carrying a very large oligo pool as a stable material for large-scale data storage.Data storage in a mixed culture of living cells enables us to simply bridge the in vitro and in vivo storage systems while retaining the advantages of both storage capacity and economical information propagation.The method was used to successfully store a DNA library of 97.7 kbps and recover the complete data(100 %).Thereafter,over ten thousand distinct oligos,totalling 2304 kbps encoding 445 kbytes of digital data,were stored to expand the information storage capacity in cells,and the stored data could be recovered almost completely(84.15 %).In summary,we developed a new strategy based on high-copy-number plasmids for ss DNA synthesis,genome editing and DNA information storage in E.coli cells.The linear ss DNA templates(>1 kbps)could be synthesized in living cell and used as a template for large-scale sequence insertion in the bacterial chromosome.A DNA library could be stored in vivo in a mixed culture of E.coli cells quickly and cheaply,and the data could be recovered almost completely.This study provides new research ideas and models for genome engineering and synthetic biology.