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Fig. 5 | BMC Biology

Fig. 5

From: Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles

Fig. 5

Designs for creating and genotyping conditional alleles through CRISPR-mediated targeting with lssDNA donors. a Schematic for illustrating lssDNA 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). A double-stranded DNA template is purchased with 100–200 bp homology arms. The 5′ homology arm begins with a GGG and ends with the 5′ sgRNA cut site. The 3′ homology arm terminates at an appropriate cDNA primer. b Genotyping schemes for detecting floxed and null alleles. Orange triangles represent loxP sites, with representative homology sequence color coded on blue DNA strand. Genotyping for a null allele uses primers P1 and P3 from each loxP genotyping reaction, which can also detect a full-length wild-type product (not shown) due to the smaller distance between loxP sites than typical designs from ssODN targeting attempts

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