A Simple and Efficient CRISPR Technique for Protein Tagging

Zeng, Fanning; Beck, Valerie; Schuierer, Sven; Garnier, Isabelle; Manneville, Carole; Agarinis, Claudia; Morelli, Lapo; Quinn, Lisa; Knehr, Judith; Roma, Guglielmo; Bassilana, Frédéric; Nash, Mark S.

doi:10.3390/cells9122618

Cited by 6 publications

(4 citation statements)

References 26 publications

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“…These fusion proteins have been used in combination with untagged vimentin or transfected into vimentin expressing cells in order to study in vivo filament dynamics in live cells [ 40 ], fluorescence recovery after photobleaching [ 43 ] and fluorescence energy transfer to study intermolecular interactions [ 48 ]. Apart from these fluorescent tags, there are non-fluorescent tags such as N-Myc (EQKLISEEDL) [ 49 , 50 ], FLAG (DYKDDDDK) and 3 × FLAG [ 51 , 52 ], HA Tag (YPYDVPDYA) [ 53 ], Spot Tag (PDRVRAVSHWSS) [ 54 , 55 ], V5 Tag (GKPIPNPLLGLDST) [ 56 ] and His Tag (HHHHHH) [ 57 , 58 ]. These are small non-fluorescent peptides, which are fused in frame with the protein of interest so the fusion proteins can be tracked using tag-specific antibodies and therefore they are not suitable for live cell imaging.…”

Section: Discussionmentioning

confidence: 99%

Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly

Usman

Aldehlawi²,

Nguyen

et al. 2022

IJMS

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Vimentin, a type III intermediate filament protein, is found in most cells along with microfilaments and microtubules. It has been shown that the head domain folds back to associate with the rod domain and this association is essential for filament assembly. The N-terminally tagged vimentin has been widely used to label the cytoskeleton in live cell imaging. Although there is previous evidence that EGFP tagged vimentin fails to form filaments but is able to integrate into a pre-existing network, no study has systematically investigated or established a molecular basis for this observation. To determine whether a tag would affect de novo filament assembly, we used vimentin fused at the N-terminus with two different sized tags, AcGFP (239 residues, 27 kDa) and 3 × FLAG (22 residues; 2.4 kDa) to assemble into filaments in two vimentin-deficient epithelial cells, MCF-7 and A431. We showed that regardless of tag size, N-terminally tagged vimentin aggregated into globules with a significant proportion co-aligning with β-catenin at cell–cell junctions. However, the tagged vimentin aggregates could form filaments upon adding untagged vimentin at a ratio of 1:1 or when introduced into cells containing pre-existing filaments. The resultant filament network containing a mixture of tagged and untagged vimentin was less stable compared to that formed by only untagged vimentin. The data suggest that placing a tag at the N-terminus may create steric hinderance in case of a large tag (AcGFP) or electrostatic repulsion in case of highly charged tag (3 × FLAG) perhaps inducing a conformational change, which deleteriously affects the association between head and rod domains. Taken together our results shows that a free N-terminus is essential for filament assembly as N-terminally tagged vimentin is not only incapable of forming filaments, but it also destabilises when integrated into a pre-existing network.

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Section: Discussionmentioning

confidence: 99%

Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly

Usman

Aldehlawi²,

Nguyen

et al. 2022

IJMS

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“…This alleviates the need for homology templates and enables the tagging of endogenous loci using a single generic donor plasmid. Since then, another CRISPR/Cas9-based gene tagging, which is called "Homology independent gene Tagging (HiTag)" [69], has been provided.…”

Section: Gene Tagging Using I-gonadmentioning

confidence: 99%

Recent Advances in the Production of Genome-Edited Animals Using i-GONAD, a Novel in vivo Genome Editing System, and Its Possible Use for the Study of Female Reproductive Systems

Sato,

Morohoshi,

Ohtsuka

et al. 2023

OBM Genet

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Gene-engineered animals created using gene-targeting technology have long been recognized as beneficial, valid, and valuable tools for exploring the function of a gene of interest, at least in early 2013. This approach, however, suffers from laborious and time-consuming tasks, such as the production of successfully targeted embryonic stem (ES) cells, their characterization, production of chimeric blastocysts carrying these gene-modified ES cells, and transplantation of those manipulated blastocysts to the recipient (pseudopregnant) females to deliver chimeric mice. Since the appearance of genome editing technology, which is now exemplified by the CRISPR/Cas9 system, in late 2013, significant advances have been made in the generation of genome-edited animals through pronuclear microinjection (MI) of genome-editing components into fertilized eggs (zygotes) or electroporation (EP) of zygotes in the presence of these reagents. However, these procedures require the transfer of genome-edited embryos into the reproductive tracts of recipient females for further development. Genome editing via oviductal nucleic acids delivery (GONAD) and its modified version, called “improved GONAD (i-GONAD),” were developed as an alternative to the MI- or EP-based genome-edited animal production and now recognized to be very convenient and straightforward as genome editing can only be performed in vivo (within the oviductal lumen where fertilized embryos exist). This system also enables the simultaneous transfection of epithelial cells lining the oviductal lumen. In this review, we summarize the recent advances in GONAD/i-GONAD and their derivatives and discuss the potential of these technologies to study various biological systems related to female reproduction.

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“…To date, the CRISPaint system has been tested in a handful of human cells 22,23,25 . In this study, we sought to expand the application of CRISPaint into mESCs which are a powerful and versatile model.…”

Section: Adapting Crispaint For C-terminal Knock-ins In Mescsmentioning

confidence: 99%

“…To circumvent these constraints, NHEJ has been recently introduced for FP knock-in in mammalian cells [18][19][20][21][22][23][24][25][26] . One method, named CRISPR-assisted insertion tagging (CRISPaint) 22 is especially streamlined because it uses universal donor plasmids, such that the only cloning required is construction of the gene-specific sgRNA.…”

Section: Introductionmentioning

confidence: 99%

Efficient and rapid fluorescent protein knock-in with universal donors in mammalian stem cells

Shi

Kopparapu

Ohler

et al. 2022

Preprint

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Fluorescent protein (FP) tagging is a foundational approach in cell biology because it allows observation of protein distribution, dynamics, and interaction with other proteins in living cells. However, the typical approach using overexpression of tagged proteins can perturb cell behavior and introduce localization artifacts. To preserve native expression, fluorescent proteins can be inserted directly into endogenous genes. This approach has been standard in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous fluorescent protein tagging has not been widely used in mammalian cells due to inefficient homology-directed repair (HDR). Here, we describe a streamlined method for efficient and fast integration of FP tags into native loci via non-homologous end joining (NHEJ) in mouse embryonic stem cells. Our protocol minimizes cloning with universal donors, allows for N or C-terminal tagging of endogenous proteins, and requires less than 2 weeks from transfection to imaging, thereby improving the applicability of FP knock-in in mammalian cells.

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A Simple and Efficient CRISPR Technique for Protein Tagging

Cited by 6 publications

References 26 publications

Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly

Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly

Recent Advances in the Production of Genome-Edited Animals Using <i>i</i>-GONAD, a Novel <i>in vivo</i> Genome Editing System, and Its Possible Use for the Study of Female Reproductive Systems

Efficient and rapid fluorescent protein knock-in with universal donors in mammalian stem cells

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