In vivo genome editing restores haemostasis in a mouse model of haemophilia

Li, Hojun; Haurigot, Virginia; Doyon, Yannick; Li, Tianjian; Wong, Sunnie; Bhagwat, Anand S.; Malani, Nirav; Anguela, Xavier M.; Sharma, Rajiv P.; Ivanciu, Lacramiora; Murphy, Samuel L.; Finn, Jonathan D.; Khazi, Fayaz R.; Zhou, Shangzhen; Paschon, David E.; Rebar, Edward J.; Bushman, Frederic D.; Gregory, Philip D.; Holmes, Michael C.; High, Katherine A.

doi:10.1038/nature10177

Cited by 501 publications

(411 citation statements)

References 28 publications

(41 reference statements)

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“…ZFN-based gene therapy for HIV infection is currently under phase 2 clinical trials, yielding some successful cases (8). Other studies suggested that ZF-based gene control could also be therapeutically effective for hemophilia (9) and Parkinson's disease (10). For the success of these technologies, however, two issues, toxicity and low efficiency, should be resolved (4,5,11).…”

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confidence: 99%

Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble

Zandarashvili

Esadze

Vuzman

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

145

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Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins. Our current study of the zinc-finger protein Egr-1 (also known as Zif268) and its nuclease derivatives reveals kinetic and thermodynamic roles of the dynamic conformational equilibrium between two modes during the DNAscanning process: one mode suitable for search and the other for recognition. By mutagenesis, we were able to shift this equilibrium, as confirmed by NMR spectroscopy. Using fluorescence and biochemical assays as well as computational simulations, we analyzed how the shifts of the conformational equilibrium influence binding affinity, target search kinetics, and efficiency in displacing other proteins from the target sites. A shift toward the recognition mode caused an increase in affinity for DNA and a decrease in search efficiency. In contrast, a shift toward the search mode caused a decrease in affinity and an increase in search efficiency. This accelerated site-specific DNA cleavage by the zinc-finger nuclease, without enhancing off-target cleavage. Our study shows that appropriate modulation of the dynamic conformational ensemble can greatly improve zinc-finger technology, which has used Egr-1 (Zif268) as a major scaffold for engineering.protein-DNA interactions | DNA scanning | target search | kinetics | dynamics A rtificial transcription factors and DNA-modifying enzymes have gained popularity as powerful means for artificial gene regulation and genome editing (1-7). Successful applications were reported on artificial zinc-finger (ZF) proteins engineered to exhibit a desired sequence specificity in binding to DNA (1-5). Artificial ZF transcription factors, comprising engineered ZFs and transactivation or repression domains, are used to regulate particular genes (1-3). ZF nucleases (ZFNs), comprising engineered ZFs and a FokI nuclease domain (ND), can site-specifically cleave DNA at particular sequences and allow for genome editing in vivo (4, 5). ZFN-based gene therapy for HIV infection is currently under phase 2 clinical trials, yielding some successful cases (8). Other studies suggested that ZF-based gene control could also be therapeutically effective for hemophilia (9) and Parkinson's disease (10). For the success of these technologies, however, two issues, toxicity and low efficiency, should be resolved (4, 5, 11). Regarding the latter issue, some studies (11)(12)(13)(14) suggest that, despite high affinities for target DNA, the artificial proteins do not necessa...

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mentioning

confidence: 99%

Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble

Zandarashvili

Esadze

Vuzman

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

145

View full text Add to dashboard Cite

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“…Genome editing has become an important technique to study human disease in in vivo models 7 and may one day become an established therapeutic approach to correct disease-causing mutations 8 . Here we have utilized transcription activator-like effector nuclease (TALEN)-mediated genome editing to introduce the naturally occurring À 175T4C HPFH mutation into the g-globin promoter in erythroid cell lines.…”

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confidence: 99%

Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin

et al. 2015

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“…66 Another option is to integrate the wild-type copy of the gene into the endogenous locus, allowing for endogenous gene regulation, which is expected to be more physiological. 67 While the majority of liver diseases are caused by loss of function genetic mutations, there are other liver diseases that are initiated by aberrant or excessive activity of a gene. For example, familial hypercholesterolemia can be caused by nonsense mutations in PCSK9 gene.…”

Section: Key Pointmentioning

confidence: 99%

Therapeutic editing of hepatocyte genome in vivo

Lujambio

2017

Journal of Hepatology

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In vivo genome editing restores haemostasis in a mouse model of haemophilia

Cited by 501 publications

References 28 publications

Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble

Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble

Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin

Therapeutic editing of hepatocyte genome in vivo

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