Manipulation of large-scale genetic information provides a powerful approach to deciphering and engineering complex biological functions. However, the manipulation of large DNA, such as assembly and delivery, remains complex and difficult. Here we describe the experimental design strategy and protocol for a chromosome elimination-mediated large DNA assembly and delivery method (HAnDy), which enables efficient Mb-scale DNA assembly and delivery in yeast conveniently. This protocol is divided into three parts: (1) CRISPR-Cas9-mediated elimination of chromosome, which includes design and integration of a synthetic single-guide RNA (sgRNA) site near the centromere, activation of chromosome elimination by mating, and verification of the chromosome elimination. It can be used to eliminate multiple chromosomes, achieving haploidization in yeast. (2) Haploidization-mediated DNA assembly, which includes the design and construction of initial assembly strains, DNA assembly by programmed haploidization and verification of the assembled clones. (3) Haploidization-mediated DNA delivery, which includes the design and construction of inducible haploidization strains, DNA delivery by programmed haploidization and verification of the delivered clones. Users can utilize this protocol entirely or selectively depending on their needs. With the use of this protocol, it takes 10 d to achieve chromosome elimination and 7-11 d to achieve a standard cycle of haploidization-mediated DNA assembly or delivery. This protocol provides an efficient approach that is useful for the elimination, assembly and delivery of large DNA in yeast, requiring basic molecular biology skills.

• PMID: 40640356
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Journal Article | Review

Authors

Ma Y, Xiong Y, Xu J, Xu H, Fu Z, Zhao GR, Wu Y, Yuan YJ

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