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Simplifying the combined use of CRISPR-Cas9 and Cre-loxP technologies for the efficient generation of targeted conditional gene knockouts in mammalian cells

Tzahi Noiman, Chaim Kahana
doi: https://doi.org/10.1101/236679
Tzahi Noiman
1Department of Molecular Genetics, the Weizmann Institute of Science, Rehovot 76199, Israel
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Chaim Kahana
1Department of Molecular Genetics, the Weizmann Institute of Science, Rehovot 76199, Israel
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  • For correspondence: Chaim.kahana@weizmann.ac.il
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ABSTRACT

Gene knockout technologies have contributed fundamentally to our understanding of the cellular functions of various genes. Two prevalent systems used for the efficient elimination of the expression of specific genes are the Cre-LoxP system and the CRISPR-Cas9 system. Here we present a simple method that combines the use of CRISPR-Cas9 and Cre-loxP for the conditional deletion of essential genes in mammalian cells. First, an inducible Cre recombinase is stably expressed in the cells. Next CRISPR-Cas9 is used to knockout an essential gene, whose function is complemented by stable expression of a FLAG-tagged version of the same protein encoded from a floxed transcription unit containing silent mutations, making it refractory to the CRISPR-Cas9 guide. This FLAG-tagged protein can be deleted by activating the expressed Cre protein enabling evaluation of the cellular consequences of its deletion. We have further used this system to evaluate potential mutants of the tested gene.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted December 19, 2017.
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Simplifying the combined use of CRISPR-Cas9 and Cre-loxP technologies for the efficient generation of targeted conditional gene knockouts in mammalian cells
Tzahi Noiman, Chaim Kahana
bioRxiv 236679; doi: https://doi.org/10.1101/236679
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Simplifying the combined use of CRISPR-Cas9 and Cre-loxP technologies for the efficient generation of targeted conditional gene knockouts in mammalian cells
Tzahi Noiman, Chaim Kahana
bioRxiv 236679; doi: https://doi.org/10.1101/236679

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