KARYOTYPE ENGINEERING: SYNTHESIS AND FUSION OF YEAST CHROMOSOMES
Embargo until
2022-12-01
Date
2018-06-19
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Publisher
Johns Hopkins University
Abstract
As part of the Sc2.0 project, we designed and synthesized synthetic chromosome I. The total length of synI is ~21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype balance. We used a robust CRISPR-Cas9 method to fuse chromosome I to chromosome arms of varying length: chrIXR (84kb), chrIIIR (202kb) and chrIVR (1Mb). All fusion chromosome strains grew as well as wild type so we decided to attach synI to synIII. Through investigation of three-dimensional structures of fusion chromosome strains, a strange loop and a twisted complex structure were formed in chrIII-I and chrIX-III-I fusion chromosomes, which are dependent on the presence of silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. In meiosis, we used these fusion chromosomes to show that axial element Red1 binding is not strictly chromosome size dependent even though Red1 binding is enriched on the three small chromosomes in wild-type yeast and we discovered an unexpected role for centromeres in Red1 binding.
Using this CRISPR-Cas9 method, we have successfully fused yeast chromosomes, generating a near-isogenic series of strains with progressively fewer chromosomes ranging from n=16 to n=2. A strain carrying only two ~6 Mb long chromosomes exhibited only modest transcriptomic changes and strikingly, grows without major defects compared to the n=16 strain. In heterotypic crosses (n<16 X n=16), two trends were noted. As n dropped below 16, spore viability decreased dramatically, reaching <10% at n=12 X n=16. As n decreased further, yeast sporulation was arrested, with drastically reduced full tetrad formation detected in the n=8 X n=16 cross, with <1% spore viability, from which no viable spores could be recovered. However, homotypic crosses between pairs of n=8, 4 and 2 strains gave excellent sporulation and spore viability. These results indicate that as few as 8 chromosome-chromosome fusion events suffice to isolate strains reproductively. Overall, budding yeast tolerates reduction in chromosome number surprisingly well, providing a dramatic new example of the remarkable robustness of the yeast genome to change.
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Keywords
synthetic biology, yeast fusion chromosomes, CRISPR/Cas9, synthetic yeast, Sc2.0