Establishing chromosomal design-build-test-learn through a synthetic chromosome and its combinatorial reconfiguration

Jee Loon Foo; Shohei Kitano; Adelia Vicanatalita Susanto; Zhu Jin; Yicong Lin; Zhouqing Luo; Linsen Huang; Zhenzhen Liang; Leslie A Mitchell; Kun Yang; Adison Wong; Yizhi Cai; Jitong Cai; Giovanni Stracquadanio; Joel S Bader; Jef D Boeke; Junbiao Dai; Matthew Wook Chang

doi:10.1016/j.xgen.2023.100435

Establishing chromosomal design-build-test-learn through a synthetic chromosome and its combinatorial reconfiguration

Cell Genom. 2023 Nov 9;3(11):100435. doi: 10.1016/j.xgen.2023.100435. eCollection 2023 Nov 8.

Authors

Jee Loon Foo^{1

2

3

4}, Shohei Kitano^{1

2

3

4}, Adelia Vicanatalita Susanto^{1

2

3

4}, Zhu Jin^{1

2

3

4}, Yicong Lin^{5

6}, Zhouqing Luo⁵, Linsen Huang^{5

6}, Zhenzhen Liang^{5

6}, Leslie A Mitchell⁷, Kun Yang⁸, Adison Wong^{1

9}, Yizhi Cai¹⁰, Jitong Cai¹¹, Giovanni Stracquadanio^{11

12}, Joel S Bader¹¹, Jef D Boeke^{13

7

8}, Junbiao Dai^{5

6}, Matthew Wook Chang^{1

2

3

4}

Affiliations

¹ NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), National University of Singapore, Singapore 117456, Singapore.
² Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117456, Singapore.
³ Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore.
⁴ Wilmar-NUS Corporate Laboratory (WIL@NUS), National University of Singapore, Singapore 117599, Singapore.
⁵ CAS Key Laboratory of Quantitative Engineering Biology, Guangdong Provincial Key Laboratory of Synthetic Genomics and Shenzhen Key Laboratory of Synthetic Genomics, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
⁶ Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China.
⁷ Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA.
⁸ Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn, NY 11201, USA.
⁹ Singapore Institute of Technology, 10 Dover Drive, Singapore 138683, Singapore.
¹⁰ Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
¹¹ High-Throughput Biological Center and Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
¹² School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3BF, UK.
¹³ Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016, USA.

Abstract

Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a redesigned synthetic Saccharomyces cerevisiae chromosome XV, synXV. We designed and built synXV to harbor strategically inserted features, modified elements, and synonymously recoded genes throughout the chromosome. Based on the recoded chromosome, we developed a method to enable chrDBTL: CRISPR-Cas9-mediated mitotic recombination with endoreduplication (CRIMiRE). CRIMiRE allowed the creation of customized wild-type/synthetic combinations, accelerating genotype-phenotype mapping and synthetic chromosome redesign. We also leveraged synXV as a "build-to-learn" model organism for translation studies by ribosome profiling. We conducted a locus-to-locus comparison of ribosome occupancy between synXV and the wild-type chromosome, providing insight into the effects of codon changes and redesigned features on translation dynamics in vivo. Overall, we established synXV as a versatile reconfigurable system that advances chrDBTL for understanding biological mechanisms and engineering strains.

Keywords: CRISPR-Cas9; Saccharomyces cerevisiae; chromosome reconfiguration; design-build-test-learn; mitotic recombination; synthetic biology; synthetic chromosome; synthetic genomics; synthetic yeast.