Supplementary Figure 1: Different methods to generate conditional KOs.

(a) Schematic drawing of the conventional strategy to generate conditional knockouts (KOs) utilizing CRISPR. Cas9 and gRNAs are used to flank the exon of one allele of the gene of interest with loxP sites. To achieve biallelic targeting the second allele needs to be floxed via either a second round of recombination or breeding of mice. Orange triangles, loxP sites; DS, Drug selection gene; HAL, Homologous arm left; HAR, homologous arm right; PGK, phosphoglycerate kinase

Promoter.

(b) Conditionals by inversion (COIN) strategy to generate conditional KOs. A ‘flippable’ reporter gene combined with a drug selection cassette (DS) is inserted into an exon or intron of the gene of interest via homologous recombination. The drug selection cassette is flanked by FRT sites and is removed prior to inversion of the reporter cassette. The reversion of orientation (of the reporter cassette) is mediated by Cre recombinase and converts the lox71 and lox66 sites to a lox72 and loxP site respectively. Following inversion the 3’ splice site (3’SS) together with the polyA signal now in sense direction, abrogate the transcription of the gene.

(c) Generation of conditional KOs in mouse zygotes. Co-injection of Cas9 mRNA, different gRNAs and DNA vectors/oligos in mouse zygotes allows generation of a conditional allele by flanking the exon of interest with loxP sites. The gRNA sequence is red, capitalised and underlined, while the PAM is in green and the template oligos containing the loxP sites (light blue text with orange background) to be inserted are in light blue.

(d) Inducible Cas9 systems. In the first system (i), cells express the Tet-On 3G transactivator in an inactive form and Cas9 is not expressed. Addition and binding of doxycycline to the Tet-On 3G transactivator induces a conformational change allowing it to bind to the Tet responsive element 3G and initiate expression of the Cas9 protein. The constant transcription of the gRNA in combination with the induced expression of Cas9 allows gene editing. It is noteworthy that there is a similar system based on the same principle (although using M2rtTA rather than 3G) except that the gRNA is not constitutively expressed but needs to be provided through transfection. In the second system (ii) the Cas9 protein is split in two parts. Following translation the C-terminal part having two nuclear localization signals (NLS) is transported into the nucleus whereas the N-terminal part, having a nuclear export signal (NES) is kept in the cytoplasm. In addition the N- and C-terminal part of Cas9 are fused to FKBP (FK506 binding protein 12) and FRB (FKBP rapamycin binding) domains respectively. Thus, in the presence of rapamycin the FKBP and FRB domains will bring the Cas9 domains together, allowing their reassembly and import into the nucleus. Upon gRNA transfection the reassembled Cas9 can induce double stranded breaks.