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, 11 (5), 316-8

Small Molecule-Triggered Cas9 Protein With Improved Genome-Editing Specificity

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Small Molecule-Triggered Cas9 Protein With Improved Genome-Editing Specificity

Kevin M Davis et al. Nat Chem Biol.

Abstract

Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.

Figures

Figure 1
Figure 1
Insertion of an evolved ligand-dependent intein enables small-molecule control of Cas9. (a) Intein insertion renders Cas9 inactive. Upon 4-HT binding, the intein undergoes conformational changes that trigger protein splicing and restore Cas9 activity. (b) The evolved intein was inserted to replace each of the colored residues. Intein-inserted Cas9 variants at S219 and C574 (green) were used in subsequent experiments. (c) Genomic EGFP disruption activity of wild-type Cas9 and intein-Cas9 variants in the absence or presence of 4-HT. Intein-Cas9 variants are identified by the residue replaced by the intein. Error bars reflect the standard deviation of three biological replicates.
Figure 2
Figure 2
Genomic DNA modification by intein-Cas9(S219), intein-Cas9(C574), and wild-type Cas9. (a) Indel frequency from high-throughput DNA sequencing of amplified genomic on-target sites in the absence or presence of 4-HT. Note that a significant number of indels were observed at the CLTA on-target site even in the absence of a targeting sgRNA (Supplementary Table 7). (b–d) DNA modification specificity, defined as on-target:off-target indel frequency ratio, normalized to wild-type Cas9. Cells were transfected with 500 ng of the Cas9 expression plasmid. P-values are < 10−15 for the Fisher exact test (one-sided up) on comparisons of indel modification frequency in the presence versus the absence of 4-HT for intein-Cas9(S219) and intein-Cas9(C574). P-values were adjusted for multiple comparisons using the Benjamini-Hochberg method, and are listed in Supplementary Table 3. Error bars reflect the range of two independent experiments conducted on different days.

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