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Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse

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Resumo:Genetically modified model organisms are valuable tools for probing gene function, dissecting complex signaling networks, studying human disease, and more. CRISPR/Cas9 technology has significantly democratized and reduced the time and cost of generating genetically modified models to the point that small gene edits are now routinely and efficiently generated in as little as two months. However, generation of larger and more sophisticated gene-modifications continues to be inefficient. Alternative ways to provide the replacement DNA sequence, method of Cas9 delivery, and tethering the template sequence to Cas9 or the guide RNA (gRNA) have all been tested in an effort to maximize homology-directed repair for precise modification of the genome. We present two CRISPR/Cas9 methods that have been used to successfully generate large and complex gene-edits in mouse. In the first method, the Cas9 enzyme is used in conjunction with two sgRNAs and a long single-stranded DNA (lssDNA) template prepared by an alternative protocol. The second method utilizes a tethering approach to couple a biotinylated, double-stranded DNA (dsDNA) template to a Cas9-streptavidin fusion protein. •Alternative method for generating long, single-stranded DNA templates for CRISPR/Cas9 editing.•Demonstration that using two sgRNAs with Cas9-streptavidin/biotinylated-dsDNA is feasible for large DNA modifications.
Autores principais:Pineault, Kyriel M.
Outros Autores:Novoa, Ana; Lozovska, Anastasiia; Wellik, Deneen M.; Mallo, Moises
Ano:2019
País:Portugal
Tipo de documento:artigo
Tipo de acesso:acesso aberto
Instituição associada:Repositório do GIMM – Gulbenkian Institute for Molecular Medicine
Idioma:inglês
Origem:ARCA

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