Team:Tianjin/Measurement
mRFP(which can characterized chromosome transfer)
We initially use mRFP to represent chromosome transfer. We put mRFP on the chromosome which will be transfered, so when chromosome transfer occurs, the host cell will turn red under irradiation of excitation wavelength at 587nm and detect it at 610nm emission wavelength.
Figure 1. chromosome transfer detected by mRFP
dCas9-adenine base editor & gRNA(which can characterized chromosome transfer to specific cells)
We creatively proposed a new way to characterize chromosome tranfer: by using the fusion protein of dCas9- A to G mutant enzyme and gRNA to achieve this detection purpose.[1]
Firstly, we designed a sequence of gRNA and there are three main design standards:
1. There can’t be high similarity between gRNA sequence and genome sequence of the host, so as to reduce the missing target rate.
2. The mutation site of A-to-G mutant enzyme which exists on DNA sequence corresponding to gRNA cannot contain more than one adenine.
3. There should exist PAM sequence inside gRNA to realize sequential editing, which can enhance the credibility of detection results and reduce the impact of other factors.
Figure 2. Constructed gRNA sequence and its corresponding DNA sequence
According to the three standards above, we construct many sequences based on Saccharomyces cerevisiae, more over, we design a programme which can provided aided design of gRNA and corresponding DNA sequence.
Figure 3. Fusion protein and gRNA demonstration
We put the fusion protein (which contains dCas9 and A-to-G mutant protein) and the DNA sequence we designed into the chromosome that we want to transfer and put gRNA into the chromosome of the host cell. Because of that, when the chromosome transfer into the host cell, the fusion protein and gRNA will meet together and target to the corresponding DNA sequence to start the first mutation.
When the first mutation has done, a new PAM site will occured and gRNA will target to the second repeat of the corresponding sequence to mediate the next mutation.
After an interval, all of the mutation has done and we can get the DNA sequence by PCR and sequencing it for the time of mutation, so that we can detect the transfer of chromosome accurately.
Though our design, gRNA and the fusion protein can combine perfectly gRNA binds to the DNA sequence and dCas9 binds to PAM site. Moreover, the three repeat that we design in the DNA sequence ensure that only when more than one mutation occurs, we see the detection is successful, which can effectively reduce the impact caused by natural gene mutation.
Reference
[1]Gaudelli N M, Komor A C, Rees H A, et al. Publisher Correction: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage[J]. Nature, 2018, 559(7714):E8.