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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References
Lander, E.S. et al. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).
Peisajovich, S.G., Garbarino, J.E., Wei, P. & Lim, W.A. Rapid diversification of cell signaling phenotypes by modular domain recombination. Science 328, 368–372 (2010).
Chothia, C., Gough, J., Vogel, C. & Teichmann, S.A. Evolution of the protein repertoire. Science 300, 1701–1703 (2003).
Jinek, M. et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Doudna, J.A. & Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).
Hsu, P.D., Lander, E.S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
Guilinger, J.P., Thompson, D.B. & Liu, D.R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).
Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).
Anders, C., Niewoehner, O., Duerst, A. & Jinek, M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014).
Tanenbaum, M.E., Gilbert, L.A., Qi, L.S., Weissman, J.S. & Vale, R.D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).
Davis, K.M., Pattanayak, V., Thompson, D.B., Zuris, J.A. & Liu, D.R. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat. Chem. Biol. 11, 316–318 (2015).
Reynolds, K.A., McLaughlin, R.N. & Ranganathan, R. Hot spots for allosteric regulation on protein surfaces. Cell 147, 1564–1575 (2011).
Edwards, W.R., Busse, K., Allemann, R.K. & Jones, D.D. Linking the functions of unrelated proteins using a novel directed evolution domain insertion method. Nucleic Acids Res. 36, e78 (2008).
Schultz, J. et al. Specific interactions between the syntrophin PDZ domain and voltage-gated sodium channels. Nat. Struct. Biol. 5, 19–24 (1998).
Dueber, J.E. et al. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759 (2009).
Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
Oakes, B.L., Nadler, D.C. & Savage, D.F. Protein engineering of Cas9 for enhanced function. Methods Enzymol. 546, 491–511 (2014).
Briner, A.E.E.E. et al. Guide RNA functional modules direct Cas9 activity and orthogonality. Mol. Cell 56, 333–339 (2014).
Stein, V. & Alexandrov, K. Synthetic protein switches: design principles and applications. Trends Biotechnol. 33, 101–110 (2015).
Shiau, A.K. et al. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95, 927–937 (1998).
Tanenbaum, D.M., Wang, Y., Williams, S.P. & Sigler, P.B. Crystallographic comparison of the estrogen and progesterone receptor's ligand binding domains. Proc. Natl. Acad. Sci. USA 95, 5998–6003 (1998).
Wärnmark, A. et al. Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor-α. J. Biol. Chem. 277, 21862–21868 (2002).
Tucker, C.L. & Fields, S. A yeast sensor of ligand binding. Nat. Biotechnol. 19, 1042–1046 (2001).
Tsai, S.Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).
McIsaac, R.S. et al. Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast. Nucleic Acids Res. 41, e57 (2013).
Feil, R. et al. Ligand-activated site-specific recombination in mice. Proc. Natl. Acad. Sci. USA 93, 10887–10890 (1996).
Dow, L.E. et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat. Biotechnol. 33, 390–394 (2015).
González, F. et al. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15, 215–226 (2014).
Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551 (1992).
Gossen, M. et al. Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769 (1995).
Kearns, N.A. et al. Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. Development 141, 219–223 (2014).
Jiang, F., Zhou, K., Ma, L., Gressel, S. & Doudna, J.A. A Cas9-guide RNA complex preorganized for target DNA recognition. Science 348, 1477–1481 (2015).
Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
Zalatan, J.G. et al. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339–350 (2015).
Shechner, D.M., Hacisuleyman, E., Younger, S.T. & Rinn, J.L. Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat. Methods 12, 664–670 (2015).
Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).
Zetsche, B., Volz, S.E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).
Truong, D.-J.J. et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res. 43, 6450–6458 (2015).
Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).
Tiscornia, G., Singer, O. & Verma, I.M. Production and purification of lentiviral vectors. Nat. Protoc. 1, 241–245 (2006).
Lin, S., Staahl, B.T., Alla, R.K. & Doudna, J.A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).
Fellmann, C. et al. An optimized microRNA backbone for effective single-copy RNAi. Cell Rep. 5, 1704–1713 (2013).
Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471 (1998).
Sanjana, N.E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).
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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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/).
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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.
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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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DOI: https://doi.org/10.1038/nbt.3528
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