title: 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.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Oakes
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Dana C
authors.identifier: 0000-0002-9792-145X
authors.initials: DC
authors.lastname: Nadler
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Avi
authors.initials: A
authors.lastname: Flamholz
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Christof
authors.initials: C
authors.lastname: Fellmann
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Brett T
authors.initials: BT
authors.lastname: Staahl
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.|Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA.|Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California, USA.|Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.|Department of Chemistry, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.|Department of Chemistry, University of California, Berkeley, Berkeley, California, USA.|Energy Biosciences Institute, University of California, Berkeley, Berkeley, California, USA.
authors.forename: David F
authors.identifier: 0000-0003-0042-2257
authors.initials: DF
authors.lastname: Savage
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites; D000076987:CRISPR-Associated Protein 9; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics*; D005812:Genes, Switch / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000235:genetics* / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D011485:Protein Binding; D000072417:Protein Domains; D015202:Protein Engineering / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases