Abstract
CRISPR-Cas9-based genome editing enables the rapid genetic manipulation of any genomic locus without the need for gene targeting by homologous recombination. Here we describe a conditional transgenic approach that allows temporal control of CRISPR-Cas9 activity for inducible genome editing in adult mice. We show that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9D10A (Cas9n) variant can regulate the frequency and size of target gene modifications, respectively. Further, we show that this inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and, thus, provides a flexible and fast platform to study loss-of-function phenotypes in vivo.
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Acknowledgements
We thank J.Simon for technical assistance with animal colonies, and other members of the Lowe laboratory for advice and discussions. We thank the MSKCC transgenic core facility for production of transgenic animals by blastocyst injection and the MSKCC Genomics core facility for library preparation and Illumina sequencing. This work was supported by a program project grant number CA-013106 from the National Cancer Institute (NCI). L.E.D. was supported by a K22 Career Development Award from the NCI/ National Institutes of Health (NIH) under award number 1K22CA181280-01. K.P.O'R. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the NIH under award number T32GM07739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program. G.L. was supported by an American Cancer Society postdoctoral fellowship PF-13-037-01 and NIH F32 grant 1F32CA177072-01. S.W.L. is an investigator of the Howard Hughes Medical Institute and the Geoffrey Beene Chair for Cancer Biology.
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L.E.D. conceived of the project, performed and analyzed experiments and wrote the paper. J.F. performed and analyzed experiments and wrote the paper. K.P.O'R., A.M., G.L. and D.F.T. performed and analyzed experiments. E.R.K. and N.D.S. developed informatics pipelines and analyzed data. S.W.L. supervised experiments, analyzed data and wrote the paper.
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Dow, L., Fisher, J., O'Rourke, K. et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat Biotechnol 33, 390–394 (2015). https://doi.org/10.1038/nbt.3155
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DOI: https://doi.org/10.1038/nbt.3155
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