| Due to the high demand for genetic modification in medicine and bioengineering,the development of efficient and stable gene-editing technology has been a hot research field in basic and applied biology.Nowadays,the transfer of small fragments of foreign genes can be achieved in most species,but it is still fairly difficult to integrate large fragments.Considering that the generation of DNA double-strand breaks(DSBs)can greatly improve the efficiency of homologous recombination at target sites,this research investigated the possibility of improving recombination and integration of large gene fragments by inducting multipleDSBs in yeast.In addition,the impacts of spatial distance and the relative position between DSBs on DNA recombination efficiency and the molecular mechanisms when multiple DSBs exist at the same time were studied.In order to promote the stable transfer of the foreign genes in large fragments,we first constructed and optimized the DSB induction and recombination system mediated by I-SceⅠ restriction endonuclease.By introducing a GIM cassette with I-SceⅠ as the core,various yeast strains with single DSB or double DSBs of different spacing were constructed.This project initially speculated that when two DSBs are produced synchronously and are closely spaced,the sequence in the middle of the double DSBs will fall off,and therefore is conducive to the integration of large fragments of foreign genes.However,results showed that it will not cause a loss of the DNA between two DSBs even if the distance is very short.In addition,the current study found that the recombination efficiency near the upstream DSB was significantly elevated with the presence of the downstream DSB,and the closer the distance,the more efficient the gene conversion.The successful integration of foreign genes and the expression of the target proteins proved that the model has practical application potential.In addition,we found that the shorter the distance between double DSBs,the more difficult it is to repair and the more lethal to cells.Based on this phenomenon,this project screened the key genes of HR and NHEJ repair pathways to determine their impacts on DNA repair toward double DSBs with different spacing.Results showed that when the doubleDSB spacing was less than 1000 bp,the cell growth was inhibited and more cell death,while when the spacing was greater than 1000 bp,the cell growth was not much affected,indicating that double DSBs with closer distances could affect each other’s repair.However,after knocking out RAD51,a key gene in the HR pathway,the growth of the strain was inhibited even when double-DSB spacing is as large as 2000 bps.However,the growth of the strain with a spacing of 4000 bp was no different from the wild type,indicating that the distance between the two DSBs affected its repair mechanism,which the repair of DSBs is more dependent on the HR pathway than the single-DSB strains when the two DSBs are more close.We further knocked out the three key genes for resection,MRE11,RAD50 and XRS2.Resection is the key steps of DSB repair and recombination.Although these three proteins bind together to form the MRX complex,the deletion of the MRE11 gene showed stronger impact than the others when dealing with two DSBs that were far away from each other.These results revealed some novel functions of these genes in repairing multiple DSBs.In summary,this thesis attempted to construct a yeast gene editing system that can mediate the integration and recombination of large fragments of exogenous genes by inducing the expression of I-SceⅠ endonuclease to simultaneously generate multiple DSBs on the same yeast chromosome.In addition,the impact of the relative spatial position of multiple DSB s on repair and recombination efficiency was studied,providing some important insights for further optimization of this recombination system as well as for understanding the repair mechanism of multiple DSBs. |
