Enhancing gene editing specificity by attenuating DNA cleavage kinetics

Miller, Jeffrey C.; Patil, Deepak P.; Xia, Danny F; Paine, Charles B; Fauser, Friedrich; Richards, Hunter W.; Shivak, David A.; Bendaña, Yuri R.; Hinkley, Sarah J.; Scarlott, Nicholas A.; Lam, Stephen; Reik, Andreas; Zhou, Yuanyue; Paschon, David E.; Li, Patrick; Wangzor, Tenzin; Lee, Gary; Zhang, Lei; Rebar, Edward J.

doi:10.1038/s41587-019-0186-z

Cited by 45 publications

(40 citation statements)

References 62 publications

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“…Creating similar epiRILs in wheat and barley would provide substantial resources not only for identifying ideal epigenetic variation in crops, but also for fully using the potential of epigenetics in crop improvement [123][124][125][126]. Besides, genome-editing enzymes such as transcription activator-like effector nucleases (TALENs) and CRISPR-Cas9 system have been used to engineer epigenomes in a sequence-specific manner in mammalian systems [127][128][129][130]. In Arabidopsis, Johnson et al directed DNA methylation to target DNA sequences and caused expected phenotype changes through fusing ZFNs with the SRA domain-containing protein SUVH9-a protein integral to RNA-directed DNA methylation (RdDM) [131].…”

Section: Discussionmentioning

confidence: 99%

Insight into the Role of Epigenetic Processes in Abiotic and Biotic Stress Response in Wheat and Barley

Kong

Liu

Wang

et al. 2020

IJMS

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Environmental stresses such as salinity, drought, heat, freezing, heavy metal and even pathogen infections seriously threaten the growth and yield of important cereal crops including wheat and barley. There is growing evidence indicating that plants employ sophisticated epigenetic mechanisms to fine-tune their responses to environmental stresses. Here, we provide an overview of recent developments in understanding the epigenetic processes and elements—such as DNA methylation, histone modification, chromatin remodeling, and non-coding RNAs—involved in plant responses to abiotic and biotic stresses in wheat and barley. Potentials of exploiting epigenetic variation for the improvement of wheat and barley are discussed.

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Section: Discussionmentioning

confidence: 99%

Insight into the Role of Epigenetic Processes in Abiotic and Biotic Stress Response in Wheat and Barley

Kong

Liu

Wang

et al. 2020

IJMS

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“…Kinetic models suggest that the cleavage of off-target sequences can be reduced by accelerating the dissociation of the enzyme from the DNA or by reducing its catalytic activity [94]. Following these predictions, an enhancement of cleavage specificity has been realized by designing enzymes with attenuated cleavage rate or by fusing Cas9 to inhibitor proteins [95,96]. In the above model, the specificity is affected by both the binding and catalytic reactions because they are coupled, since both of them involve the same molecular species, the enzyme.…”

Section: Conclusion-optimization Of Transcriptional Activation and Rmentioning

confidence: 99%

Tuning up Transcription Factors for Therapy

Becskei

2020

Molecules

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The recent developments in the delivery and design of transcription factors put their therapeutic applications within reach, exemplified by cell replacement, cancer differentiation and T-cell based cancer therapies. The success of such applications depends on the efficacy and precision in the action of transcription factors. The biophysical and genetic characterization of the paradigmatic prokaryotic repressors, LacI and TetR and the designer transcription factors, transcription activator-like effector (TALE) and CRISPR-dCas9 revealed common principles behind their efficacy, which can aid the optimization of transcriptional activators and repressors. Further studies will be required to analyze the linkage between dissociation constants and enzymatic activity, the role of phase separation and squelching in activation and repression and the long-range interaction of transcription factors with epigenetic regulators in the context of the chromosomes. Understanding these mechanisms will help to tailor natural and synthetic transcription factors to the needs of specific applications.

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“…Kinetic models suggest that cleavage of off-target sequences can be reduced by increasing the dissociation from the DNA binding or by reducing the catalytic activity [86]. Such an enhancement of cleavage specificity has been realized in the meantime, by attenuating cleavage rate of the endonuclease or by fusing Cas9 to inhibitor proteins [87,88]. In the above model, the specificity is affected by the dissociation rate and the enzymatic turnover because the binding and catalytic reactions are coupled.…”

Section: Conclusion: Optimization Of Transcriptional Activation and mentioning

confidence: 99%

Tuning up Transcription Factors for Therapy

Becskei

2020

Preprint

View full text Add to dashboard Cite

The recent developments in the delivery and design of transcription factors put their therapeutic applications within reach, exemplified by cell replacement, cancer differentiation and T-cell based cancer therapies. The success of such applications depends on the efficacy and precision in the action of transcription factors. The biophysical and genetic characterization of the paradigmatic prokaryotic repressors, LacI and TetR and the designer transcription factors, TALE and CRISPR-Cas9 revealed common rules, which can help the optimization of activators and repressors. Further studies will be required to analyze the linkage between dissociation constants and enzymatic activity, the role of phase separation and squelching in activation and repression, and the long-range interaction of transcription factors with epigenetic regulators in the context of the chromosomes. Understanding these mechanisms will help to tailor systematically optimized designer transcription factors to the needs of specific applications.

show abstract

Enhancing gene editing specificity by attenuating DNA cleavage kinetics

Cited by 45 publications

References 62 publications

Insight into the Role of Epigenetic Processes in Abiotic and Biotic Stress Response in Wheat and Barley

Insight into the Role of Epigenetic Processes in Abiotic and Biotic Stress Response in Wheat and Barley

Tuning up Transcription Factors for Therapy

Tuning up Transcription Factors for Therapy

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