The AID/APOBEC (activation induced deaminase/ apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) protein family is a group of evolutionarily conserved cytidine deaminases, enzymes that catalyze the deamination of cytidine to uridine on single-stranded polynucleotides, which play a role in various physiological functions. Members of this family, genes APOBEC3A and APOBEC3B are stacked in tandem with other APOBEC3 members on chromosome 22 in the human genome. APOBEC3 family proteins are part of the first line of defense against many exogenous and endogenous retroviruses, retroelements and DNA viruses. They also play a role in cancer, where aberrant DNA mechanisms can lead to tumor mutations.
Protein A3A strongly inhibits retrotransposons and viruses. Its antiviral activity is expressed against AAV, HSV-1, HPV, HBV, HCMV, HIV-1 and HTLV-1. A3A also plays a function in cancer, where it causes mutagenic changes on transiently exposed ssDNA through somatic mutation.
Protein A3B inhibits retroelements and exhibits antiviral activity against HBV, HIV, HTLV-1, EBV, KSHV and HSV-1. It is also involved in the process of mutagenesis of the cell genome. A3B expression correlates with cell cycle and DNA repair genes.
As part of the master's thesis, we prepared A3A and A3B kock-out HeLa single-cell cell line. For this purpose we used CRISPR/Cas9 system for target genome editing. Plasmid constructs, which code guide RNA and protein Cas9 (pX458-A3Ag1, A3Ag2 and A3Bg2) were used for transfection of human HeLa cells. Single-cell lines were established through appropriate dilution of cells, so that new lines were formed from one cell. The success of the experiment was confirmed by Sanger sequencing of the genomic locus with the knocked-out gene. Preparation of single-cell A3B knocked-out HeLa cell line was unsuccessful. We successfully generated A3A knock-out single-cell line of HeLa cells. The developed cell line will serve as basis for research of viruses and their behavior within human cells.
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