An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

被引：39

作者：

Dewari, Pooran Singh ^{[1
,2
]}

Southgate, Benjamin ^{[1
,2
]}

Mccarten, Katrina ^{[1
,2
]}

Monogarov, German ^{[3
]}

O'Duibhir, Eoghan ^{[1
,2
]}

Quinn, Niall ^{[4
]}

Tyrer, Ashley ^{[1
,2
]}

Leitner, Marie-Christin ^{[1
,2
]}

Plumb, Colin ^{[1
,2
]}

Kalantzaki, Maria ^{[1
,2
]}

Blin, Carla ^{[1
,2
]}

Finch, Rebecca ^{[1
,2
]}

Bressan, Raul Bardini ^{[1
,2
]}

Morrison, Gillian ^{[1
,2
]}

Jacobi, Ashley M. ^{[5
]}

Behlke, Mark A. ^{[5
]}

von Kriegsheim, Alex ^{[4
]}

Tomlinson, Simon ^{[1
,2
]}

Krijgsveld, Jeroen ^{[3
]}

Pollard, Steven M. ^{[1
,2
]}

机构：

[1] Univ Edinburgh, Edinburgh Canc Res United Kingdom Ctr, Edinburgh, Midlothian, Scotland

[2] Univ Edinburgh, MRC Ctr Regenerat Med, Edinburgh, Midlothian, Scotland

[3] Heidelberg Univ, German Canc Res Ctr, Heidelberg, Germany

[4] Univ Edinburgh, Inst Genet & Mol Med, Edinburgh Canc Res United Kingdom Ctr, Edinburgh, Midlothian, Scotland

[5] Integrated DNA Technol Inc, Coralville, IA USA

来源：

ELIFE | 2018年 / 7卷

基金：

英国生物技术与生命科学研究理事会; 英国工程与自然科学研究理事会; 英国医学研究理事会;

关键词：

MASS-SPECTROMETRY; HIGH-THROUGHPUT; GUIDE RNAS; CHIP-SEQ; GENOME; CRISPR-CAS9; PROTEINS; DELIVERY; DNA; COMPLEXES;

D O I：

10.7554/eLife.35069

中图分类号：

Q [生物科学];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of 5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

引用

页数：29

共 53 条

[1] Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice [J].