The new frontier of genome engineering with CRISPR-Cas9

Doudna, Jennifer A.; Charpentier, Emmanuelle

doi:10.1126/science.1258096

Cited by 5,456 publications

(4,130 citation statements)

References 153 publications

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“…This blocks accessibility of PI(3)P for PI(3)P-dependent motor proteins and PI(3)P metabolizing enzymes (Petiot, Faure et al 2003). To allow dissociation of the FYVE-probe from endosomes, a single FYVE domain under a weak promoter (Hayakawa, Hayes et al 2004;Stuffers, Malerod et al 2010) or knockin of the GFP-tagged FYVE domain using CRISPR/ Cas9 technology within a genomic safe harbour locus (Doudna and Charpentier 2014) should be used. Thus, it would match binding constants of endogenous…”

Section: Pi Conversion In Endosomal Exocytosis Parallels Switching Ofmentioning

confidence: 99%

A phosphoinositide conversion mechanism for exit from endosomes

Ketel¹,

Krauß²,

Nicot³

et al. 2016

Nature

165

177

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I owe special thanks to Marnix Wieffer and Michael Krauß, who spent a lot of time helping me to get started in the lab, to overcome numerous experimental problem, I was not sure how to deal with, and frequently joined in discussions about my project with excellent advice. I am really grateful for your experimental support, Michael, and the time you invested in the project during our paper revision.Further, I would like to thank Jocelyn Laporte and his group, especially Anne-Sophie Nicot, at the IGBMC in Strasbourg for their support and a very fruitful collaboration, and Carsten Schultz and his group at the EMBL in Heidelberg for their support and constant supply with PIP/AMs. I owe special thanks to Dmytro Puchkov for the ultrastructural analysis and Eberhard Krause for mass spectrometry done within this project. I also need to acknowledge Markus Wenk and Federico Torta at the Singapore Lipidomics Incubator for joining the project at a very late stage, when we urgently needed help from lipidomics specialists. It was a pity that experiments did not work out as planned.I would further like to express my gratitude to two very skilled technicians in the AG Haucke lab: Silke Zillmann and Delia Löwe. Without your help it would have been impossible to achieve so much within the limited amount of time I had during the paper revision. by VPS34-IN1 treatment................................................... 52 2.3.11 Fluorescence microscopy................................................................................. 53 2.3.12 Flow cytometry............................................................................................ III SummaryPhosphoinositides (PIs) are a minor class of short-lived phospholipids that serve as crucial signposts of membrane identity. Thereby, PIs full fill important functions in cell signaling and membrane transport. PI 4-phosphates such as phosphatitylinositol-4-phosphate (PI(4)P) and phosphatitylinositol-4,5-bisphosphate (PI(4,5)P 2 ) are enriched at the plasma membrane (PM), on secretory organelles and lysosomes, while PI 3-phosphates, i.e.phosphatitylinositol-3-phosphate (PI(3)P), are a hallmark of the endosomal system.Directional transport between these compartments, thus, requires regulated PI conversion.However, PI conversion in exit from PI(3)P-enriched endosomes en route to the PI(4)P-and PI(4,5)P 2 -positive PM in endosomal recycling remained unknown.Here, we report that cargo exit from endosomes requires removal of PI(3)P by the PI(3)P 3-phosphatase myotubularin 1 (MTM1), and concomitant PI(4)P synthesis by PI 4-kinase type II α (PI4K2α). Loss of MTM1 causes endosomal accumulation of PI(3)P and PI(3)P effector proteins, i.e. sorting nexins, Kif16b-mediated outward traffic of PI(3)P containing endosomes and sub-plasmalemmal accumulation of exocytosis-deficient endosomes. As PI4K2α associates with MTM1 and thereby facilitates membrane recruitment of MTM1, these phenotypic changes are mimicked by loss of PI4K2α. The conversion of PI(3)P-to-PI(4)P is paralleled by a switch i...

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Section: Pi Conversion In Endosomal Exocytosis Parallels Switching Ofmentioning

confidence: 99%

A phosphoinositide conversion mechanism for exit from endosomes

Ketel¹,

Krauß²,

Nicot³

et al. 2016

Nature

165

177

View full text Add to dashboard Cite

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“… The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing 1–4 . High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown 5–9 .…”

mentioning

confidence: 99%

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Chen¹,

Dagdas²,

Kleinstiver³

et al. 2017

Nature

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971

877

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The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1–4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5–9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

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“…In early 2013, several papers, including some describing how the technology could be used to edit the genomes of human stem cells and to alter a whole organism (the zebrafish), were an early indication of the coming tsunami 2,3 . By the end of 2014, scientists had -among other things -used CRISPR-Cas9 to enhance pest resistance in wheat, reproduce the carcinogenic effects of specific chromosome translocations in mouse lungs and correct a mutation in adult mice that in humans causes the disease hereditary tyrosinaemia [4][5][6] .…”

Section: Growing Excitementmentioning

confidence: 99%

Genome-editing revolution: My whirlwind year with CRISPR

Doudna

2015

Nature

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The new frontier of genome engineering with CRISPR-Cas9

Cited by 5,456 publications

References 153 publications

A phosphoinositide conversion mechanism for exit from endosomes

A phosphoinositide conversion mechanism for exit from endosomes

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Genome-editing revolution: My whirlwind year with CRISPR

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