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.

IntroductionIn October 2017, Scotland legalised the home use of misoprostol for the purpose of early medical abortion (EMA). Women up to 9+6 weeks’ gestation can now self-administer the drug at home, 24–48 hours after receiving mifepristone in the clinic.ObjectiveTo evaluate the impact of this change on the uptake and success rate of EMA, and on the provision of effective contraception on discharge.MethodsA prospective observational study was conducted to compare the outcomes of two cohorts of women in the 6 months before and 6 months after the introduction of home administration of misoprostol. The main outcome measures were uptake of EMA, success of EMA and provision of long-acting reversible contraception (LARC) to women undergoing EMA.ResultsThere was a statistically significant increase in the uptake of EMA from 698/1075 (64.9%) women in the first study period to 823/1146 (71.8%) in the second study period. There was no statistically significant difference in the success rate of EMA: 99.3% and 98.9% in clinic and home misoprostol cohorts, respectively. There was also no statistically significant difference in the proportion of women provided with LARC: 37.7% and 33.7% in clinic and home misoprostol cohorts, respectively.ConclusionsSelf-administration of misoprostol at home increased uptake of EMA, with no effect on the high success rate that was previously seen with clinic administration of misoprostol. In addition, the reduced number of visits associated with home use of misoprostol has not affected the provision of effective contraception to women.

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.