GENOME ENGINEERING TOOLS TO GENERATE ISOGENIC CELLULAR MODEL OF AMYOTROPHIC LATERAL SCLEROSIS

Genome editing systems TALENs and CRISPR/Cas9 are powerful tools to solve critical problems of biomedicine such as creation of human hereditary diseases cellular models and searching for therapy. In this issue we produced and improved tools to generate isogenic cell lines panel with mutations in SOD1 modeling familial forms of amyotrophic lateral sclerosis (ALS). We obtained 7 paires of vectors that expressed TALENs and 8 ones expressed components of CRISpR/Cas9 system to produce 8 double-stranded breaks in 8 sites of SOD1. We designed donor molecules single-stranded oligonucleotide localized near the double-stranded breaks to generate 11 singlenucleotide substitution. We observed that all genome editing tools disrupted SOD1 gene, with CRISPR/Cas9 is being more effective than TALEN in all investigated sites. We demonstrated that single-stranded oligonucleotide carrying phosphorotioate bondes improved homology-directed repair in the region of double-stranded breaks as a template for Ala89Val substitution.

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