title: Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity.
abstract: Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94158, USA.
authors.forename: Duy P
authors.initials: DP
authors.lastname: Nguyen
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, California 94143, USA.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Miyaoka
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143, USA.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Gilbert
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, California 94143, USA.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Mayerl
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94158, USA.
authors.forename: Brian H
authors.initials: BH
authors.lastname: Lee
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143, USA.|Howard Hughes Medical Institute, University of California, San Francisco, California 94158, USA.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Weissman
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, California 94143, USA.|Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143, USA.|Department of Medicine, University of California, San Francisco, San Francisco, California 94143, USA.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Conklin
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94158, USA.|Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Wells
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000378:metabolism*; D044127:Epigenesis, Genetic; D004965:Estrogen Antagonists / Q000494:pharmacology; D000072669:Gene Editing / Q000379:methods*; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000494:pharmacology; D010957:Plasmids; D011485:Protein Binding; D000072417:Protein Domains; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000187:drug effects / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000378:metabolism*; D013211:Staphylococcus aureus; D013297:Streptococcus pyogenes; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D004965:Estrogen Antagonists; D005938:Glucocorticoids; D018160:Receptors, Cytoplasmic and Nuclear; D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid; C411470:glucocorticoid receptor alpha; D013629:Tamoxifen; C016601:afimoxifene; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases