Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization

Şentürk, Şerif; Shirole, Nitin H.; Nowak, Dawid G.; Corbo, Vincenzo; Pal, Debjani; Vaughan, Alexander; Tuveson, David A.; Trotman, Lloyd C.; Kinney, Justin B.; Sordella, Raffaella

doi:10.1038/ncomms14370

Cited by 141 publications

(118 citation statements)

References 38 publications

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“…To provide further validation to our findings, we also employed an inducible CRISPR-Cas9 system to inactivate p53 in in-vitro and in-vivo model systems (Senturk et al, 2016) (Figure 3H and I and Figure 3—figure supplement 7). In these cases, the inactivation of p53 also resulted in a substantial decrease in cell viability only in those cells expressing TP53 exon-6 truncating mutations.…”

Section: Resultsmentioning

confidence: 85%

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TP53 exon-6 truncating mutations produce separation of function isoforms with pro-tumorigenic functions

Shirole

Pal

Kastenhuber

et al. 2016

eLife

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TP53 truncating mutations are common in human tumors and are thought to give rise to p53-null alleles. Here, we show that TP53 exon-6 truncating mutations occur at higher than expected frequencies and produce proteins that lack canonical p53 tumor suppressor activities but promote cancer cell proliferation, survival, and metastasis. Functionally and molecularly, these p53 mutants resemble the naturally occurring alternative p53 splice variant, p53-psi. Accordingly, these mutants can localize to the mitochondria where they promote tumor phenotypes by binding and activating the mitochondria inner pore permeability regulator, Cyclophilin D (CypD). Together, our studies reveal that TP53 exon-6 truncating mutations, contrary to current beliefs, act beyond p53 loss to promote tumorigenesis, and could inform the development of strategies to target cancers driven by these prevalent mutations.DOI: http://dx.doi.org/10.7554/eLife.17929.001

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Section: Resultsmentioning

confidence: 85%

“…For DD-Cas9 System: DD-Cas9 (Destabilized Cas9) system was designed in lab by (Senturk et al, 2016). sgRNAs were designed using an algorithm on …”

Section: Methodsmentioning

confidence: 99%

TP53 exon-6 truncating mutations produce separation of function isoforms with pro-tumorigenic functions

Shirole

Pal

Kastenhuber

et al. 2016

eLife

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“…This makes base editing responsive to a ligand, although the system exhibited some activity without ligand and was somewhat less efficient than standard sgRNAs. Additional means of inducing Cas9 activity, such as light (Nihongaki et al, 2015; Polstein and Gersbach, 2015), cell surface interactions (Dingal et al, 2017), and other small molecules (Gao et al, 2016; Senturk et al, 2017; Zetsche et al, 2015b), should be transferrable to base editors and enable fine control of editing dynamics.…”

Section: Section 2: Applications Of Base Editingmentioning

confidence: 99%

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

et al. 2017

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The past several years have seen an explosion in development of applications for the CRISPR-Cas9 system, from efficient genome editing, to high-throughput screening, to recruitment of a range of DNA and chromatin-modifying enzymes. While homology-directed repair (HDR) coupled with Cas9 nuclease cleavage has been used with great success to repair and re-write genomes, recently developed base editing systems present a useful orthogonal strategy to engineer nucleotide substitutions. Base editing relies on recruitment of cytidine deaminases to introduce changes (rather than double stranded breaks and donor templates), and offers potential improvements in efficiency while limiting damage and simplifying the delivery of editing machinery. At the same time, these systems enable novel mutagenesis strategies to introduce sequence diversity for engineering and discovery. Here, we review the different base editing platforms, including their deaminase recruitment strategies and editing outcomes, and compare them to other CRISPR genome editing technologies. Additionally, we discuss how these systems have been applied in therapeutic, engineering, and research settings. Lastly, we explore future directions of this emerging technology.

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“…Consequentially, this delay would reduce the efficiency of CRISPR/Cas9-based genome editing by reducing the frequency of modified cells, and the beneficial effect of removal of Cas9 from its target sequence via destabilization of Cas9 itself would be due to reducing this cell cycle delay. Such a destabilized Cas9 has been previously generated by fusing FKBP12-L106P destabilization domain (23) fused to the N-terminus of wildtype Cas9 to generate DD-Cas9 (13,24). DD-Cas9 can be inducibly and reversibly stabilized by the addition of the small molecule Shield-1 (13,23,24).…”

Section: Introductionmentioning

confidence: 99%

“…Such a destabilized Cas9 has been previously generated by fusing FKBP12-L106P destabilization domain (23) fused to the N-terminus of wildtype Cas9 to generate DD-Cas9 (13,24). DD-Cas9 can be inducibly and reversibly stabilized by the addition of the small molecule Shield-1 (13,23,24).…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9 Treatment Causes Extended TP53-Dependent Cell Cycle Arrest In Human Cells

Geisinger

Stearns

2019

Preprint

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While the mechanism of CRISPR/Cas9 cleavage is understood, the large variation in mutant recovery for a given target sequence between cell lines is much less clear. We hypothesized that this variation may be due to differences in how the DNA damage response affects cell cycle progression. We used incorporation of EdU as a marker of cell cycle progression to analyze the response of several human cell lines to CRISPR/Cas9 treatment with a single guide directed to a unique locus. Cell lines with functionally wild-type TP53 exhibited higher levels of cell cycle arrest compared to lines without. Chemical inhibition of TP53 protein combined with TP53 and RB1 transcript silencing alleviated induced arrest in TP53 +/+ cells. This arrest is driven in part by Cas9 binding to DNA. Additionally, wild-type Cas9 induced fewer 53BP1 foci in TP53 +/+ cells compared to TP53 -/cells, suggesting that differences in break sensing are responsible for cell cycle arrest variation. We conclude that CRISPR/Cas9 treatment induces a cell cycle arrest dependent on functional TP53 as well as Cas9 DNA binding and cleavage. Our findings suggest that transient inhibition of TP53 may increase genome editing efficiency in primary and TP53 +/+ cell lines.

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Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization

Cited by 141 publications

References 38 publications

TP53 exon-6 truncating mutations produce separation of function isoforms with pro-tumorigenic functions

TP53 exon-6 truncating mutations produce separation of function isoforms with pro-tumorigenic functions

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

CRISPR/Cas9 Treatment Causes Extended TP53-Dependent Cell Cycle Arrest In Human Cells

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