Construction Of Mito-CRISPR/Cas9 System And Validation Of Mitochondrial Target Delivery Function

Mitochondria are important organs of cellular aerobic energy metabolism.Mitochondrial studies are important for animal high altitude adaptability and tolerance to hypoxia stress;more and more diseases are found to be caused by mutations in the mitochondrial genome.Therefore,mitochondrial genome editing has gradually become a research hotspot for scientists.With the rapid development and improvement of CRISPR/Cas9 technology,in view of the successful application of nuclease techniques such as zinc finger nuclease(Zinc-Finger Nucleases,ZFNs)and TALE nuclease(Transcription Activator-Like Effector Nucleases,TALENs)in mammalian mitochondrial genome editing,people began to try to modify the CRISPR/Cas9 system for mitochondrial genome editing.CRISPR/Cas9 system mainly uses Cas9 nuclease and single-stranded guided RNA(sg RNA)complexes to achieve efficient genome editing by more effectively inducing double-strand breaks at specific sites in genomic DNA.The repair mechanism of its double-strand breaks is unclear for mitochondrial genomic DNA(mt DNA),and cells increase their copy number to normal levels while clearing the mt DNA of the breaks.moreover,the mechanism of RNA entry into mitochondria is unclear,and sg RNA mitochondrial targeted delivery is difficult when using CRISPR/Cas9 system for mt DNA editing.this study aimed at mammalian mitochondrial editing,modified the sg RNA and Cas9 in the CRISPR/Cas9 system for mitochondrial targeted delivery,and initially established a Mito-CRISPR/Cas9 mitochondrial genome editing platform.the main results are as follows:(1)Four sg RNA expression vectors for mitochondrial targeted delivery were constructed,and RP stem ring sequence,3' UTR sequence and termination sequence were added,respectively.after transfection of cells,mitochondrial RNA were extracted for RT-PCR semi-quantitative detection.the sg RNA of the four groups could enter mitochondria effectively,among which the g RUP group had the highest efficiency.(2)three GFP and Cas9 expression vectors for mitochondrial targeted delivery were constructed,and different mitochondrial localization signals were added,respectively.after transfection of three different GFP expression vectors into cells,three GFP carrying different mitochondrial localization signals were detected by Western Blot experiments,which indirectly indicated that the modified Cas9 could be used for mitochondrial genome editingThis study established the Mito-CRISPR/Cas9 mitochondrial genome editing platform through the transformation of the CRISPR/Cas9 system,and laid the foundation for basic biology,medicine and animal breeding research based on mt DNA editing.