Complete assembly of a eukaryotic chromosome in vitro using programmable DNA-guided nicking endonucleases

Recent advances in DNA synthesis technologies have made it possible to construct small genomes, such as those of viruses or bacteria. However, synthesizing larger genomes, especially linear eukaryotic chromosomes, remains a substantial challenge. Here, we report an efficient assembly method for constructing linear DNA molecules in vitro using small fragments with extra-long overhangs at both ends. These overhangs are user-designed without length or sequence preference, and generated using a programmable DNA-guided strand cleavage (DSC) technology. DSC is driven by a nicking endonuclease and a guide DNA (gDNA), which tether the enzyme to the target sequence via gDNA and precisely cleave the targeted single strand DNA. By combining multiple DSCs onto two strands of a duplex DNA, designer overhangs can be generated at both ends of DNA fragments destined for assembly of much larger DNA molecules. As a proof-of-concept, the 30-kb T7 phage genome was assembled using 15 DSC-treated PCR amplicons and infectious T7 virions were rescued from transfected bacterial host cells. Finally, a synthetic version of yeast chromosome I (180kb) was constructed in vitro using 62 designed synthetic fragments. The assembled linear chromosomal DNA was purified and successfully transformed into yeast, where it successfully established itself as a new chromosome. The technology described here is generally useful for seamless construction of large DNA molecules and can accelerate future genome synthesis projects.