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Design and synthesis of the minimal S. cerevisiae mitochondrial chassis——MitoCRAFT

What is MitoCRAFT?

MitoCRAFT is a sequence-simplified Saccharomyces cerevisiae mitochondrial chassis (approximately 39Kbp) consisting of two parts. One is a pure chassis that provides a basic transcriptional translation system and maintains aerobic respiration, which we call MitoZero; the other is an extension that extends the functionality to create a diversified toolbox, which we call CRAFT Plus.
MitoCRAFT, derived from the mitochondrial genome (about 86 Kbp) of the wild-type S. cerevisiae S288C strain, does not contain intergenic redundant regions of the wild-type genome, thanks to which the possible metabolic burden is therefore reduced. Conducive to the direct regulation, the complex intron regulatory system of the original genome is not contained either. Apparently, it is less than half the size of the original genome and can be more easily extended by any combination and function.

The theoretical basis of MitoCRAFT:

Redundancy is an important feature of genome, which was thought to be related to the resistance of organisms and complex gene regulation. However, under controlled experimental conditions, these “Plan Bs” may be redundant and the preliminary complex gene regulation also retards engineered design. The wild-type S. cerevisiae strain S288C has a mitochondrial genome of 85,779 bp in length and contains a large number of "redundant" intergenic regions with the potential of being simplified.
The crucial regulatory role of mitochondria in eukaryotes has been confirmed by more and more studies, pushing forward the exploration of mitochondria-based cell regulation that scientists have been buckling down to. SynBio's rapid development has ignited new ideas to the development of a scalable and easy-to-use mitochondrial regulation toolbox.

Design of MitoCRAFT：

Mito Zero：

We analyzed and simplified the wild-type S. cerevisiae mitochondrial genome based on the necessity of gene function and the conservation of gene sequences. First, the entire genome was divided into seven syntenic orthologous blocks, the sequences between which were not conserved and took up a large proportion in the genome. With consideration of simplification, these sequences were deleted. The next step was to analyze its eight functional genes related to aerobic respiration, the results revealing that these genes exerted vitally essential biological functions and were highly conserved; therefore, they were all retained. The introns of these functional genes are nonetheless virtually non-conservative and thus could be deleted to ameliorate the maneuverability of MitoCRAFT. In view of functional integrity, we also deleted one remaining conserved intron. After the above three optimizations, we have ultimately established the basic chassis of MitoCRAFT --- MitoCRAFT One.

CRAFT Plus:

We designed a GFP reporter gene module particularly optimized for MitoCRAFT One, which contains a specific optimized GFP codon sequence, along with the supporting promoters and terminators. In addition to being able to verify if MitoCRAFT One's transcription translation system is working properly, this module can also serve as an example of the simplest extension module.

Synthesis of MitoCRAFT:

At present, there are still many deficiencies in mitochondrial gene editing technology, which cannot meet our needs. Hence, we chose a strategy of de novo synthesis in vitro. The extremely low GC content of the mitochondrial genomes and the presence of local GC clusters have caused great difficulties in synthesis and splicing in vitro. We adopted a hierarchical assembly strategy that combines in vitro synthesis and in vivo assembly. The first step was to specifically divide the MitoCRAFT into 21 fragments of less than 3 kbp for initial synthesis. The 40K genome was then constructed employing a homologous recombination system in yeast.

Subsequent testing and optimization:

We planned to use the gene gun method to deliver the MitoCRAFT genome to S. cerevisiae rho0 cells that have lost the mitochondrial genomes. The function of MitoCRAFT was tested by the GFP reporter module and specific biochemical detection methods. Meanwhile, we also designed two control test genomes that separately verified intergenic region deletion and intron region deletion, which were used to control the test of the function of MitoCRAFT after synthesis.