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, 34 (6), 646-51

Profiling of Engineering Hotspots Identifies an Allosteric CRISPR-Cas9 Switch

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Profiling of Engineering Hotspots Identifies an Allosteric CRISPR-Cas9 Switch

Benjamin L Oakes et al. Nat Biotechnol.

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.

Figures

Figure 1
Figure 1. Mapping the insertion potential of Cas9 with the Alpha-Syntrophin PDZ protein interaction domain
(a) Generation of the transposon-based domain insertion library. (b) Fold change values for insertions at specific amino acid sites derived from sequencing data over two rounds of screening. A positive value indicates the preference of the domain insertion at a site to remain in the library after screening for function. A negative value indicate a loss of the clone with an insertion at the site. More significant P values (DESeq, multiple hypothesis testing corrected) are represented as darker color bars. Positive values which attain 102 represent sites that were not sequenced before screening. Negative bars that extend into the shaded region represent clones which have been cleared from the library (i.e. not observed after screening). (c) Log2 fold change values from (b) mapped onto the structure of Cas9 (PDB ID:4UN3). (d) GFP repression activity of individual PDZ insertion sites. Values represent biological replicates with standard deviation (n=3), constructs are in order of decreasing fold change from sequencing. Positive and negative controls dCas9 and vector only are colored orange and grey, respectively. N.D. stands for no difference detected from dCas9, t-test (p > 0.01), (* = p < 0.01) (e) Cleavage activity of clones via an E. coli based transformation assay. Cas9 activity results in genomic cleavage and lower CFU/mL, values represent biological triplicates with standard deviation (n=3). Positive and negative controls Cas9 and dCas9 are colored orange and grey, respectively. N.D. stands for no difference detected from WT Cas9, t-test (p > 0.01), (* = p < 0.01)
Figure 2
Figure 2. Creation of a switch-like Cas9 though insertion of the Estrogen Receptor ligand binding domain
(a) Schematic of the screen / counter-screen procedure to select for ligand responsive Estrogen Receptor ligand binding domain (ER-Cas9) insertions. (b) Dose-response curve to 4-HT. darC9:231 has an IC50 of 440 ± 70 nM (S.D) and a Hill coefficient of 1.04 as expected for non-cooperative binding of 4-HT to ER-LBD. (c) Single cell analysis of darC9:231 binding in response to increasing concentrations of 4-HT. Flow cytometry data tracks ensemble data demonstrating a > 9 fold switch between darC9 based GFP repression plus and minus 4-HT (darC9 GFP signal without 4-HT mean: 24,310 (au) and with 100 µM 4-HT: 2,631 (au) (d) Dose response of darC9:231 binding to various ligands (B–E and DES are beta-estradiol & diethylstilbestrol). Response is normalized to vector control fluorescence under the same conditions. (e) Switching of arC9:231 cleavage activity. Transformation assays demonstrate that ligand dependent arC9 switching also extends to cleavage activity, t-test (** p-value <0.01). All experiments performed in E. coli.
Figure 3
Figure 3. Validating arC9 in eukaryotic cells
(a) Schematic of the arC9:231 expression constructs and GFP disruption assay. (b) Quantification of EGFP disruption at 72 hours for Cas9 and arC9 with a N- and C-terminal nuclear localization signal (NLS) (n=3). These data demonstrate a ~6 fold increase in EGFP disruption. Background activity of arC9 is 10.9 ± 0.5% (S.D.) while EGFP disruption in the presence of 300 nM 4-HT increases to 66 ± 1% (S.D.) Error bars represent one standard deviation of biological replicates. (c) Quantification EGFP disruption at 72 hours for Cas9 and arC9 without an NLS (n=3). Background activity of arC9 is not significantly different from a non-targeting negative control, t-test. EGFP disruption in the presence of 4-HT increases to 30 ± 2% (S.D.) this represents at least a 24-fold increase in arC9 activity in the presence of 300 nM 4-HT, t-test (*** p values < 0.001). (d) Dose response of arC9 w/o NLS normalized to maximum activity. IC50 is 1.0 ± 0.2 nM (S.D). (e) T7EI assay of Cas9 and arC9 mediated indel formation at the EMXI locus at 72 hours. Cas9 with the targeting guide causes indels regardless of treatment condition, arC9 only cleaves a genomic locus in the presence of 4-HT. Quantification and error bars represent the standard deviation of biological replicates (n=3). N.D. signifies not detected, below the detection limit of the assay.

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