CRISPR-Directed In Vitro Gene Editing of Plasmid DNA Catalyzed by Cpf1 (Cas12a) Nuclease and a Mammalian Cell-Free Extract

Authors: Sansbury B, Wagner A, Nitzan E, Tarcic G, Kmiec E.
Date: 1 April, 2018
Journal: The CRISPR Journal 1(2): 191-202.

Abstract:
Extraordinary efforts are underway to offer greater versatility and broader applications for CRISPR-directed gene editing. Here, we report the establishment of a system for studying this process in a mammalian cell-free extract prepared from HEK-293 human embryonic kidney cells. A ribonucleoprotein (RNP) particle and a mammalian cell-free extract coupled with a genetic readout are used to generate and identify specific deletions or insertions within a plasmid target. A Cpf1 (Cas12a) RNP induces a double-stranded break, and the cell-free extract provides the appropriate enzymatic activities to direct specific deletion through resection and homology directed repair in the presence of single- and double-stranded donor DNA. This cell-free system establishes a foundation to study the heterogeneous products of gene editing, as well as the relationship between nonhomologous end joining and homology directed repair and related regulatory circuitries simultaneously in a controlled environment.

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