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Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch

Abstract

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

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Figure 1: Mapping the insertion potential of Cas9 with the PDZ domain.
Figure 2: Creation of a switch-like Cas9 though insertion of the ER-LBD in E. coli.
Figure 3: Validation of arC9 in eukaryotic cells.

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Acknowledgements

We thank S. Qi (Stanford) and J. Dueber (UC Berkeley) for providing the E. coli strain and the PDZ and SH3 domains, respectively. We would like to thank M. O'Connell, S. Sternberg, A. Wright and S. Higgins for productive discussions and readings of the manuscript. This work was supported by a NIH New Innovator Award (1DP2EB018658-01) and a Basil O'Connor Starter Scholar Research Award from the March of Dimes (D.F.S.) and by the National Science Foundation (IQJEDMS001 to J.A.D.); A.F. is funded by a National Science Foundation Graduate Research Fellow, and B.T.S. is funded by a Roche Postdoctoral Fellowship (RPF 311).

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Authors and Affiliations

Authors

Contributions

B.L.O., D.C.N., C.F., J.A.D. and D.F.S. designed the research. B.L.O., D.C.N., C.F., A.F. and B.T.S. performed the experiments. A.F. performed the computational analysis. B.L.O., A.F., C.F. and D.F.S. analyzed the data. B.L.O., C.F., J.A.D. and D.F.S. wrote the paper. Reagents described in this work are available on Addgene (https://www.addgene.org/David_Savage/).

Corresponding author

Correspondence to David F Savage.

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Competing interests

The authors have submitted a patent disclosure on this work. J.A.D. is employed by HHMI and works at the University at California Berkeley, which have patents pending for CRISPR technologies on which she is an inventor. J.A.D. is the executive director of the Innovative Genomics Initiative at UC Berkeley and UCSF. J.A.D. is a cofounder of Editas Medicine, Intellia Therapeutics and Caribou Biosciences and a scientific advisor to Caribou, Intellia, eFFECTOR Therapeutics and Driver.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18 (PDF 7933 kb)

Supplementary Table 1

Significant PDZ-insertion data (XLSX 36 kb)

Supplementary Table 2

All sequences (5′-3′) (XLSX 42 kb)

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Oakes, B., Nadler, D., Flamholz, A. et al. Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch. Nat Biotechnol 34, 646–651 (2016). https://doi.org/10.1038/nbt.3528

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