Fig. 1

Designs for creating conditional alleles through CRISPR-mediated targeting with ssODN donor DNA. a Schematic for illustrating conditional targeting designs. Cas9 (gray) complexed with sgRNA (dark blue) binds to complementary DNA (blue) on the target strand after recognition of the PAM site (red). b Symmetrical design utilized two 60 bp homology arms, excluding the sgRNA target sequence and PAM site. The symmetrical ssODN donors were designed to be complementary to the target strand. c Asymmetrical design utilized a 36 bp PAM-distal and a 91 bp PAM-proximal homology arms [35]. The asymmetrical ssODN donors were designed to be complementary to the non-target strand. d Diagram illustrating the position of the loxP site insertion within the sgRNA target sequence in the ssODN donor. The loxP sequence was always inserted one base away from the Cas9 cut site, disrupting the sgRNA sequence in the ssODN donor and thereby preventing re-cutting by Cas9 after targeting