Technology, Tianjin University, Tianjin 300072, orcid.org/0000-0003-0553-0089

Large DNA transfer technology has been challenged with the rapid development of large DNA assembly technology. The research and application of synthetic yeast chromosomes have been mostly limited in the assembled host itself. The mutant of KARL prevents nuclear fusion during yeast mating, and occasionally single chromosome can be transferred from one parental nucleus to another. Using the kar1 mutant method, four synthetic yeast chromosomes of Sc2.0 (synIII, synV, synX, synXII) were transferred to wild-type yeasts separately. SynIII was also transferred into an industrial strain Y12, resulting in an improvement of thermotolerance. Moreover, by combining abortive mating and chromosome elimination by CRISPR-Cas9, which has been reported in our previous study, we developed a strategy for consolidation of multiple synthetic yeast chromosomes. Compared to the previous pyramidal strategy using endoreduplication backcross, our method is a linear process independent of meiosis, providing a convenient path for accelerating consolidation of Sc2.0 chromosomes. Overall, the method of transfer and consolidation of synthetic yeast chromosomes by abortive mating and chromosome elimination enables a novel route that large DNA was assembled in donor yeast and then in vivo directly transferred to receptor yeasts, enriching the manipulation tools for synthetic genomics.