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 tumour-derived stem cells. Biallelictagged 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 sixty 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.

Acquisition of chromosome 1q duplication in parental and genome‐edited human‐induced pluripotent stem cell‐derived neural stem cells results in their higher proliferation rate in vitro and in vivo

“…This is in agreement with previous studies in ZFN-modified human foetal NSCs, 31 TALEN-modified hiPSC-NSCs 30 and CRISPR/Cas9-targeted human 7,32 and mouse brain-derived NSCs. 32,39 However, the chromosomal integrity and functional consequences that chromosomal aberrations might induce in gene targeted NSCs in vitro and in vivo have not been addressed in these previous studies. Here, we show that prolonged culture of hiPSC-NSCs leads to the acquisition of dup(1)q independently of whether they were genetically modified by ZFNs or not, and that this aberration increases NSC proliferation in vitro as well as in vivo in the first weeks after transplantation most likely by activation of the AKT3 signalling pathway.…”

Concise Review: Modeling Neurodegenerative Diseases with Human Pluripotent Stem Cell-Derived Microglia

“…This can be prevented by using silencing resistant promoters to drive transgene expression, with a promoter combined with sequences that prevent silencing such as the UCOE sequence or by introducing a selection cassette and apply constant selection pressure (such as puromycin, neomycin, zeocin) as previously done for RFP iPSC‐derived microglia before adding them to the neuronal cocultures . Another option is to gene‐edit the endogenous gene locus to coexpress a fluorescent reporter or directly introduce a tag such as the short FLAG tag to visualize the endogenous protein . Furthermore, modeling tools are inducible promoters allowing to switch genes on or off in the differentiated cells .…”