Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors

Natsume, Toyoaki; Kiyomitsu, Tomomi; Saga, Yumiko; Kanemaki, Masato T.

doi:10.1016/j.celrep.2016.03.001

Cited by 583 publications

(793 citation statements)

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“…1A,B). AIDtagged proteins are degraded upon addition of indole-3-acetic acid (referred to here as auxin [IAA]) in a manner dependent on plant Tir1 protein (Nishimura et al 2009;Natsume et al 2016). HCT116 cells were chosen for this experiment due to their diploid nature.…”

Section: Resultsmentioning

confidence: 99%

Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity

et al. 2018

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Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5 ′ → 3 ′ exonuclease Xrn2 or the polyadenylation signal (PAS) endonuclease CPSF73 (cleavage and polyadenylation specificity factor 73). The ability to rapidly control Xrn2 reveals a clear and general role for it in cotranscriptional degradation of 3 ′ flanking region RNA and transcriptional termination. This defect is characterized genome-wide at high resolution using mammalian native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage, and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or small nuclear RNA (snRNA) gene termination even though both RNA classes undergo 3 ′ end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive readthrough transcription than Xrn2 elimination, it likely plays a more underpinning role in termination.

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Section: Resultsmentioning

confidence: 99%

Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity

et al. 2018

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“…First applied to yeast, the method involves knock-in of the AID at either end of the POI, so that the fusion protein can be rapidly and efficiently depleted upon addition of auxin to the culture medium and conditional expression of the plant SCF TIR1 ubiquitin ligase [54]. The auxin degron technology has 9 proven its potential to study the biological function of proteins in higher eukaryotes.…”

Section: Small-molecule Dependent Degronsmentioning

confidence: 99%

“…The auxin degron technology has 9 proven its potential to study the biological function of proteins in higher eukaryotes. For example, it has been recently applied to induce rapid and conditional depletion of essential genes, for which knockouts or small-interfering RNAs are not suitable, in human and embryonic stem cells by introducing the AID-POI fusion using the CRISPR/Cas9-based method [54].…”

Section: Small-molecule Dependent Degronsmentioning

confidence: 99%

Recognition of substrate degrons by E3 ubiquitin ligases and modulation by small-molecule mimicry strategies

Lucas

Ciulli

2017

Current Opinion in Structural Biology

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“…However, the efficiency of precise introduction of large fragments by homologous recombination (HR) is still not ideal 4,5 . Previous reports have suggested methods to increase large fragment knock-in efficiency in zygotes, by Cas9-RNP injection 6 , with templates activating micro-homology mediated end joining or long-homology mediated end joining pathways 4,7 , with long singlestranded DNA templates or by chemically manipulating DNA repair pathways 8,9 .…”

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“…Some methods produce imprecise junctions at editing sites 7 , while others are limited by practical considerations and the size of the DNA fragment that can be inserted (up to 2 kb) 8 .…”

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Efficient generation of targeted large insertions in mouse embryos using 2C-HR-CRISPR

Pósfai

Rossant

2017

Preprint

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Rapid and efficient generation of large fragment targeted knock-in mouse models is still a major hurdle in mouse genetics. Here we developed 2C-HR-CRISPR, a highly efficient gene editing method based on introducing CRISPR reagents into mouse embryos at the 2-cell stage, taking advantage of the likely increase in HR efficiency during the long G2 phase and open chromatin structure of the 2-cell embryo. With 2C-HR-CRISPR and a modified biotinstreptavidin approach to localize repair templates to target sites, we rapidly targeted 20 endogenous genes that are expressed in mouse blastocysts with fluorescent reporters and generated reporter mouse lines. We showcase the . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/204339 doi: bioRxiv preprint first posted online Oct. 16, 2017; 2 first live triple-color blastocyst with all three lineages differentially reported.Additionally, we demonstrated efficient double targeting, enabling rapid assessment of the auxin-inducible degradation system for probing protein function in mouse embryos. These methods open up exciting avenues for exploring cell fate decisions in the blastocyst and later stages of development.We also suggest that 2C-HR-CRISPR can be a better alternative to random transgenesis by ensuring transgene insertions at defined 'safe harbor' sites.The development of endonuclease-mediated genome editing technologies, in particular CRISPR/Cas9, has revolutionized mouse genetics by opening up the possibility to bypass the long and labor intensive embryonic stem cell-based methods and achieve precise genome editing directly in zygote stage embryos 1 .CRISPR/Cas9-mediated genome editing has shown consistently high efficiency in generating indels, point mutations and small insertions in zygotes 2,3 , largely making use of the non-homologous end-joining (NHEJ) and homology directed repair (HDR) pathways. However, the efficiency of precise introduction of large fragments by homologous recombination (HR) is still not ideal 4,5 . Previous reports have suggested methods to increase large fragment knock-in efficiency in zygotes, by Cas9-RNP injection 6 , with templates activating micro-homology mediated end joining or long-homology mediated end joining pathways 4,7 , with long singlestranded DNA templates or by chemically manipulating DNA repair pathways 8,9 .However, only a limited number of successfully targeted loci have been reported to . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/204339 doi: bioRxiv preprint first posted online Oct. 16, 2017; 3 date 4 . Some methods produce imprecise junctions at editing sites 7 , while others are limited by practi...

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Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors

Cited by 583 publications

References 35 publications

Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity

Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity

Recognition of substrate degrons by E3 ubiquitin ligases and modulation by small-molecule mimicry strategies

Efficient generation of targeted large insertions in mouse embryos using 2C-HR-CRISPR

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