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

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.

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

“…For instance, Cas9 variants with higher specificity could decrease indel frequency [66,67], and the knock-in efficiency and repair accuracy may be predicted based on the target sequence [28,29]. Also, alternative delivery methods such as ribonucleoproteins (RNPs) [68] might increase the efficiency of DNA delivery.…”

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

“…For instance, Cas9 variants that are more accurate could increase specificity and decrease indel frequency (50,51), and the indel frequency may be predicted based on the target sequence (25,26). Also, alternative delivery methods such as ribonucleoproteins (RNPs) (52), might increase the efficiency of targeted integration.…”

The transcription factor E2A drives neural differentiation in pluripotent cells

“…As E2A is regulated by Id1 on the protein level, rather than the transcriptional level, we generated an endogenously tagged ES cell line ( Figure 1B) using CRISPR/Cas9 targeting to follow the expression of E2A protein during differentiation. Based on a strategy previously developed to tag neural stem cells with high efficiency (Dewari et al, 2018), guide RNA (sgRNA) was designed to cut proximal to the stop codon in the 3'UTR of E2A (Supplementary Figure 1A), and was co-transfected into wild-type ES cells with recombinant Cas9 protein (rCas9) and a single-stranded donor DNA template (ssODN) encoding the V5 tag flanked by homology arms (Supplementary Figure 1B ).…”

“…For ssODN design, the V5 tag sequence was flanked by ~75bp homology arms and a PAM-blocking mutation (NGG>NGC) was introduced into the 3'UTR sequence. Ribonucleoprotein (RNP) complexes were assembled as described in (Dewari et al, 2018). Briefly, crRNA and tracrRNA oligos were mixed in equimolar concentration, heated at 95°C for 5 min and allowed to cool to room temperature to anneal.…”