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The project we want to accomplish is a chromosomal stabilization element group centered on centromeres, which transfer large DNA fragments by transferring chromosomes. The difference between the source host and the destination host of the chromosome may be very large, so we need to enhance the stability of the chromosome.
Chromosomal stabilization element group
In the application of our chromosome-based gene transfer technology to the actual process, it is highly probable that the objective chromosome and the chromosome of Saccharomyces cerevisiae are far apart. At this time, in order to ensure the stable existence and stable inheritance of our large fragments after introduction into host cells, we attempt to introduce chromosomal stabilization element group on chromosomes with large fragments.
Figure. 1 Elements that can make chromosome stable
Through the investigation of the centromere, we found that the centromere can be roughly divided into three kinds: point centromere, regional centromere and intermediate transitional centromere. In order to study the difference in the effects of different kinds of centromeres, we selected the representative strains of these three centromeres, S. cerevisiae with point centromere, S. pombe with regional centromere and Y. lipolytica with intermediate transition state centromere to experiment.
Figure.2 Phylogenetic tree of centromere
First we put our sights on the point centromere and the regional centromere. Our first target chromosome is chromosome III of S. pombe. The literature indicates that the chromosome III of S. pombe has the ability to replicate autonomously in mouse cells, and it has the potential to become a shuttle vector for mammalian cells, and our chromosomal stabilization element group can increase its stability in S. cerevisiae and help us reform it better.
1. Centromere introduction
In order to explore the effect of centromere on chromosomes, we first select S. cerevisiae (point centromere) and S. pombe (regional centromere), which differ greatly in centromere evolution. We introduce the centromere from chromosome XV of S. cerevisiae into the chromosome III of S. pombe.
Figure. 3 Centromere design site schematic diagram of S.pombe
The reform site we choose is far from the original centromere of S. pombe, so as not to affect its normal division (S. pombe has a peripheral heterochromatin region on the centromere. Because of this, it will affect the binding of dynein to the centromere of S. pombe itself if the site is too close, and it will affecting its division).
Figure 4. Schematic diagram of the difference in the structure of S. cerevisiae & S. pombe centromere
Then we choose S. cerevisiae (point centromere) and Y. lipolytica (transitional centromere) which are not significantly different in centromere evolution. We added centromeres on the E chromosome of Y. lipolytica in the synthetic chromosome V of S. cerevisiae with lycopene pathway and explore the role of the centromere, in order to achieve the purpose of transferring the chromosome of Saccharomyces cerevisiae.
Figure 5. Centromere design site schematic diagram of Y.lipolytica
In this period, we found some interesting phenomena in the experiment. If the synthetic chromosome of Saccharomyces cerevisiae not only has its own centromere, but also has the centromere of Y. lipolytica, the growth of S. cerevisiae is very poor.
Therefore, we speculate that both the two centromeres (i.e., point centromere and transitional centromere) can play roles in S. cerevisiae, and the reason for the growth potential is that both centromeres can be identified, so the spindle may have two directions of traction for the two centromeres. When the two directions are opposite, S. cerevisiae breaks the synthetic chromosome V of S. cerevisiae. We then confirmed this conjecture in the model section.
So we adjusted the experiment: using the centromere of the E chromosome of Y. lipolytica to replace the centromere of synthetic chromosome V of S. cerevisiae, and then the strain we obtained grows well.Click here to learn more.
Figure. 6 Centromere replacement of S. cerevisiae
In addition, we will transfer the synthetic chromosome V with lycopene pathway on this strain to Y. lipolytica. Because Y. lipolytica's naturally occurring liposomes are suitable for enrichment of hydrophobic products, and their ability to utilize cheap raw materials can help us build high-yield Y. lipolytica strains faster, and then optimize yields with the help of a genomic rearrangement system which are unique to synthetic chromosomes.
2. Chromosome transfer
On the one hand, we have integrated the S. cerevisiae centromere into S. pombe and then we expected to achieve the goal that chromosome III of S. pombe can be modified by S. cerevisiae easily when the chromosome transferred into it by cell fusion. In subsequent studies, we will add the telomeres of tetrahymena to the chromosome of S. pombe, thereby further enhancing the stability of the S. pombe chromosome in S. cerevisiae and thus it has the potential of using it as a shuttle vector between S. cerevisiae and mammalian cells.
Figure 7. Schematic diagram of fusion between S.cerevisiae and S.pombe
On the other hand, we replaced the centromere of S.cerevisiae with the centromere of Y. lipolytica, so that when we transfer this chromosome into Y. lipolytica, it not only has the characteristics of being able to use cheap raw materials, but also has all genes that needed in the lycopene peoduction. Appart from this, it can store these lycopene with its abundant liposomes which can achieve a higher production than S. cerevisiae. After that, we will perform SCRaMbLE on the synthetic chromosome V in Y. lipolytica to achieve high-throughput gene replication, deletion, inversion, and translocation, thereby further increasing the yield.
Figure 8. Schematic diagram of SCRaMbLE
Characterization of chromosome transfer
In order to detect whether the large fragment transfer is successful more conveniently, we added the mRFP fluorescent protein gene to the synthetic chromosome V of S. cerevisiae, which can make it easier to confirm whether the chromosome has transferred.
Figure 9. Schematic diagram of fluorescence before and after chromosome transfer occurs
2. CRISPR-dCas9+ Mutant fusion protein
We creatively use a new type of technology to characterize the occurrence of fusion events. This component is divided into three parts:
1) Fusion protein: dCas9+A→G mutant enzyme
2) The DNA sequence（three repeats） to which gRNA binds and the PAM site to which dCas9 binds
3) gRNA sequence
When the fusion protein and the gRNA are simultaneously present in the same cell, the gRNA binds to the corresponding sequences of the chromosome, which designed by us and have been tested that different from other fragments on the chromosome and it have low off-target rate. Then the fusion protein on it will mutate adenine, which is not far from it, to guanine, thereby forming a new PAM sequence for dCas9 binding in the next time.
Figure. 9 CRISPR-dCas9+ Mutant fusion protein schematic diagram
Our highlight is that the specific site of gRNA binding has three repeat regions, and three sequential mutations can be made because of it. Therefore our design has many advantages: for example, it can reduce the bad influences because of off-target in gRNA and dCas9 on the detection. By using our detection system, the form of second PAM sequence(where dCas9 binding) can occurs only when the first site is mutated(similarly, the n+1th dCas9-binding PAM sequence can be formed only after the nth point mutation is generated for the next mutation). In conslusion, our detection method is very reliable because we can ensure that a positive result is detected and used in subsequent experiments only when the sequencing results show that more than one mutation has occurred.
Video 1. Fusion protein and gRNA demonstration
In addition, we hope that other teams, we can use the system we developed in more directions in the future researches: for example, designing the promoter of gRNA as a constant expression and designing the promoter of dCas9 as a induced promoter by special cell events(for example, it is changed to the promoter of DNA polymerase), and it is expressed at a specific time, thereby using sequential mutations to record cell events (such as the number of cell passages) and something more than that.
We put 1)&2) into the chromosome that needs to be transferred, and put the gRNA into the host strain’s chromosome. After the transfer of chromosomes occurs, we can tell the occurrence of this event through sequencing.
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