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

Authors:
Dewari PS, Southgate B, Mccarten K, Monogarov G, O'Duibhir E, Quinn N, Tyrer A, Leitner MC, Plumb C, Kalantzaki M, Blin C, Finch R, Bressan RB, Morrison G, Jacobi AM, Behlke MA, von Kriegsheim A, Tomlinson S, Krijgsveld J, Pollard SM.
In:
Source: eLife
Publication Date: (2018)
Issue: 11;7: 1-29
Research Area:
Neurobiology
Stem Cells
Gene Expression
Regenerative medicine
Cells used in publication:
Neural stem cell (NSC), mouse
Species: mouse
Tissue Origin: brain
Embryonic stem cell (ES), mouse
Species: mouse
Tissue Origin: embryo
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
Platform:
96-well Shuttle™ System
4D-Nucleofector™ X-Unit
Experiment

We used 4D Amaxa nucleofection system for the delivery of CRISPR ingredients. For NS cells and GNS cells, approximately 1.5  105 cells were resuspended in 20 mL of Lonza SG cell line buffer and were mixed with the complete RNP mix and electroporated using the DN-100 program (two conse-cutive pulses for mouse NS cells) or using EN-138 program (one pulse for human GBM-derived cells).
For embryonic stem cells, approximately 6  104 cells in 20 mL of Lonza P3 primary cell buffer were used for each transfection with different programs: one pulse of program CA-120 for mouse ESCs; program CB-150 for human ESCs. After the electroporation, cells were transferred into a 6-well plate
and allowed to recover for 3–5 days and later seeded into 96-well plates (1–2  104 cells per well) for ICC.

Abstract

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.