CRISPR/Cas9-based knockout pipeline for reverse genetics in mammalian cell 
culture.

Spiegel, Aleksandra; Bachmann, Mandy; Jiménez, Gabriel Jurado; Sarov, Mihail

doi:10.1016/j.ymeth.2019.04.016

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CRISPR/Cas9-based knockout pipeline for reverse genetics in mammalian cell culture.

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Bachmann, Mandy
Max Planck Institute for Molecular Cell Biology and Genetics, Max Planck Society;

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Sarov, Mihail
Max Planck Institute for Molecular Cell Biology and Genetics, Max Planck Society;

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Citation

Spiegel, A., Bachmann, M., Jiménez, G. J., & Sarov, M. (2019). CRISPR/Cas9-based knockout pipeline for reverse genetics in mammalian cell culture. Methods (San Diego, Calif.), 164-165, 49-58. doi:10.1016/j.ymeth.2019.04.016.

Cite as: https://hdl.handle.net/21.11116/0000-0006-7D74-C

Abstract

We present a straightforward protocol for reverse genetics in cultured mammalian cells, using CRISPR/Cas9-mediated homology-dependent repair (HDR) based insertion of a protein trap cassette, resulting in a termination of the endogenous gene expression. Complete loss of function can be achieved with monoallelic trap cassette insertion, as the second allele is frequently disrupted by an error-prone non-homologous end joining (NHEJ) mechanism. The method should be applicable to any expressed gene in most cell lines, including those with low HDR efficiency, as the knockout alleles can be directly selected for.