Sagai et al., 2005 Sagai T.

Hosoya M.

Mizushina Y.

Tamura M.

Shiroishi T. Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb. We used CRISPR/Cas9 technology to replicate the enhancer deletion to exclude possible effects of the neomycin cassette and genetic background on the phenotype observed by Sagai et al. ().

(A) Schematic overview of the strategy. A 4.5 kb mouse genomic region containing the ZRS (red) is shown together with the vertebrate sequence conservation track (dark blue). The sgRNA recognition site is indicated in purple. A mouse with a CRISPR/Cas9-induced 1324 bp deletion (chr5:29,314,497-29,315,820; mm10) similar to the deletion from Sagai et al. was selected for further analysis. Genotyping primers are indicated as blue arrows (F and R). A 3780 bp ‘donut‘ transgenic reporter was used to detect residual enhancer activity outside of the deleted region.

(B) Representative E11.5 transgenic mouse embryo injected with a reporter under control of the ‘donut‘ sequence. No reproducible limb activity was detected in 6/6 independent transgenic embryos.

(C) PCR genotyping of the ZRS enhancer knockout mice.

(D) The PCR product from ZRSΔ/Δ mice was sequenced to identify the deletion breakpoint.

(E) Gross phenotypes of two week old ZRSWT/Δ (top) and ZRSΔ/Δ (bottom) mice. The body sizes are comparable, but ZRSΔ/Δ mice have truncated limbs. Scale bars, 10 mm.