As CRISPR-Cas systems advance toward clinical application, it is essential to identify all the outcomes of gene-editing activity in human cells. Reports highlighting the remarkable success of homology-directed repair (HDR) in the treatment of inherited diseases may inadvertently underreport the collateral activity of this remarkable technology. We are utilizing an in vitro gene-editing system in which a CRISPR-Cas complex provides the double-stranded cleavage and a mammalian cell-free extract provides the enzymatic activity to promote non-homologous end joining, micro-homology mediated end joining, and homology-directed repair. Here, we detail the broad spectrum of gene-editing reaction outcomes utilizing Cas9 and Cas12a in combination with single-stranded donor templates of the sense and nonsense polarity. This system offers the opportunity to see the range of outcomes of gene-editing reactions in an unbiased fashion, detailing the distribution of DNA repair outcomes as a function of a set of genetic tools.
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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