Single stranded oligos

LentiGuide-Puro was digested with BsmBI and purified by gel extraction (Macherey-Nagel). Single stranded oligos (Sigma) containing the guide sequence and 25nt overlap with digested LentiGuide-Puro on each side (Supplementary Table 1) were cloned with the Gibson cloning kit (NEB). Chemo-competent Stbl3 cells (Thermofisher) were transformed with the Gibson products by heat shock. Upon minipreps (Macherey-Nagel), the plasmids were verified by Sanger sequencing (ABI).

Guide RNA (gRNA) sequences were synthesised as single-stranded oligos (Sigma-Aldrich), annealed and cloned in the vectors PX458 (pSpCas9(BB)-2A-GFP, Addgene plasmid # 48138) or PX461(pSpCas9n(BB)-2A-GFP, Addgene plasmid # 48140), both a gift from Feng Zhang^{41 (link)}. Recombinant plasmids were verified by Sanger sequencing and electroporated into E14 cells using the Nucleofector II platform (Lonza) (programme A-013). At 48 h post electroporation, GFP-expressing cells were FACS-sorted and cloned by serial dilution in 96-well plates. Single-cell colonies were allowed to grow for a week and subsequently screened by PCR. Colonies detected as recombinant were further analysed by Sanger sequencing, by PCR amplifying the targeted locus, cloning the PCR amplicons in the pCR2.1 TOPO plasmid (Thermo Fisher Scientific) and transforming 10-beta competent Escherichia coli bacteria (New England Biolabs). Plasmid inserts from 20 bacterial colonies were sequenced for each ES cell colony to verify homozygosity.