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Small molecule-triggered Cas9 protein with improved genome-editing specificity.

Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR - Nat. Chem. Biol. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

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.

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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 ratio4–6, 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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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 ratio4–6, 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.

Mentions: Overall on-target genome modification frequency of intein-Cas9(S219) and intein-Cas9 (C574) in the presence of 1 µM 4-HT was similar to that of wild-type Cas9 (Fig. 2a, Supplementary Tables 2 and 3, and Supplementary Data Set 1). On-target modification frequency in the presence of 4-HT was 3.4- to 7.3-fold higher for intein-Cas9(S219), and 3.6- to 9.6-fold higher for intein-Cas9(C574), than in the absence of 4-HT, whereas modification efficiency for wild-type Cas9 was 1.2- to 1.8-fold lower in the presence of 4-HT (Fig. 2a). Both intein-Cas9 variants exhibited a low level of background activity in the absence of 4-HT, consistent with previous reports20–22. Western blot analysis of intein-Cas9(S219) from transfected HEK293 cells confirmed the presence of spliced product at the earliest assayed time point (4 h) following 4-HT treatment; no spliced product was detected in the absence of 4-HT (Supplementary Fig. 2). Together, these results indicate that intein-Cas9(S219) and intein-Cas9(C574) are slightly less active than wild-type Cas9 in the presence of 4-HT, likely due to incomplete splicing (Supplementary Fig. 2), but much less active in the absence of 4-HT.


Small molecule-triggered Cas9 protein with improved genome-editing specificity.

Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR - Nat. Chem. Biol. (2015)

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 ratio4–6, 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4402137&req=5

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 ratio4–6, 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.
Mentions: Overall on-target genome modification frequency of intein-Cas9(S219) and intein-Cas9 (C574) in the presence of 1 µM 4-HT was similar to that of wild-type Cas9 (Fig. 2a, Supplementary Tables 2 and 3, and Supplementary Data Set 1). On-target modification frequency in the presence of 4-HT was 3.4- to 7.3-fold higher for intein-Cas9(S219), and 3.6- to 9.6-fold higher for intein-Cas9(C574), than in the absence of 4-HT, whereas modification efficiency for wild-type Cas9 was 1.2- to 1.8-fold lower in the presence of 4-HT (Fig. 2a). Both intein-Cas9 variants exhibited a low level of background activity in the absence of 4-HT, consistent with previous reports20–22. Western blot analysis of intein-Cas9(S219) from transfected HEK293 cells confirmed the presence of spliced product at the earliest assayed time point (4 h) following 4-HT treatment; no spliced product was detected in the absence of 4-HT (Supplementary Fig. 2). Together, these results indicate that intein-Cas9(S219) and intein-Cas9(C574) are slightly less active than wild-type Cas9 in the presence of 4-HT, likely due to incomplete splicing (Supplementary Fig. 2), but much less active in the absence of 4-HT.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

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.

Show MeSH
Related in: MedlinePlus