SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes

Area of Science:

• Synthetic biology
• Genomics
• Molecular biology

Background:

• Combinatorial genomic diversity is crucial for evolutionary studies.
• Precisely controlling genomic rearrangements is a challenge in synthetic biology.

Purpose of the Study:

• To apply SCRaMbLE to a synthetic yeast chromosome arm (synIXR).
• To computationally infer the sequence of recombination events.
• To assess the capability of SCRaMbLE for generating genomic diversity and identifying fitness constraints.

Main Methods:

• Application of SCRaMbLE to yeast synthetic chromosome arm synIXR with 43 recombinase sites.
• Deep sequencing of resulting SCRaMbLE strains.
• Computational pipeline for inferring recombination sequences.

Main Results:

• Generated unique genomes with 156 deletions, 89 inversions, 94 duplications, and 55 complex rearrangements.
• Rearrangements occurred exclusively at designed loxPsym sites.
• Identified genes essential for viability and fast growth based on deletion frequencies.
• Observed minimal fitness impact from 3' UTR replacement.

Conclusions:

• SCRaMbLE effectively generates extensive and precise genomic diversity within designated regions.
• The method can identify genes critical for cellular fitness.
• SCRaMbLE is scalable for simultaneous evolution of multiple synthetic chromosomes.