CRISPR-Cas9 is a versatile genome-editing technology widely used to investigate genetic function. To broaden its in vivo applications, we have developed a Cre-dependent Cas9 knock-in mouse model. This model supports a variety of uses, including but not limited to:

1.Delivery of guide RNAs via adeno-associated virus or lentivirus vectors;

2.Early-stage screening of gRNAs in vivo;

3.Combination with multiplexed sgRNA delivery to induce multiple genetic alterations within the same animal.

To develop a strain that constitutively expresses Cas9, we established a B‑Cas9‑tdTomato mouse line. Validation experiments confirmed the detection of tdTomato and Cas9 proteins at varying levels in multiple tissues, including the brain, heart, spleen, liver, lungs, kidneys, pancreas, bladder, stomach, and intestines.

B-CAG-Cas9 tdTomato flox mice (fl/fl)

A CAG-LSL-Cas9-tdTomato-WPRE-pA cassette was inserted between exon 1 and exon 2 of the Gt(ROSA)26Sor locus in B-CAG-Cas9 tdTomato flox mice.

B-CAG-Cas9 tdTomato mice (Mut/Mut)

A CAG-LSL-Cas9-tdTomato-WPRE-pA cassette was inserted between exon 1 and exon 2 of the Gt(ROSA)26Sor locus in B-CAG-Cas9 tdTomato flox mice. To generate the constitutive Cas9-expressing strain, B-CAG-Cas9-tdTomato flox mice were crossed with mice systemically expressing Cre recombinase, thereby excising the STOP cassette and activating Cas9 and tdTomato expression.

B-CAG-Cas9 tdTomato flox mice (fl/fl)

The protein expression analysis of tdTomato in B-CAG-Cas9 tdTomato flox mice and B-CAG-Cas9 tdTomato mice by IHC. Brain, heart, spleen, liver, lung, kidney, pancreas, bladder, stomach, small intestine and large intestine were collected from B-CAG-Cas9 tdTomato flox mice (fl/fl) and homozygous B-CAG-Cas9 tdTomato mice (Mut/Mut) and analyzed by IHC with mCherry/TdTomato Rabbit Polyclonal Antibody (HUABIO, HA500049). The results indicate that only the B-Rosa26-Cas9 tdTomato mice can express tdTomato protein.

The protein expression analysis of Cas9 in homozygous B-CAG-Cas9 tdTomato flox mice and B-CAG-Cas9 tdTomato mice by western blot. Brain, heart, spleen, lung, kidney and pancreas were collected from B-CAG-Cas9 tdTomato flox mice (fl/fl) and homozygous B-CAG-Cas9 tdTomato mice (Mut/Mut) and analyzed by western blot with anti-Flag antibody (Sigma-Aldrich，F1804). Cas9 was detectable in brain, heart, spleen, lung, kidney, pancreas of homozygous B-CAG-Cas9 tdTomato mice, but not in B-CAG-Cas9 tdTomato flox mice. fl/fl: B-CAG-Cas9 tdTomato flox mice; Mut/Mut: B-CAG-Cas9 tdTomato mice.

The protein expression analysis of Cas9 in homozygous B-CAG-Cas9 tdTomato flox mice and B-CAG-Cas9 tdTomato mice by western blot. Colon, stomach, bladder and liver were collected from B-CAG-Cas9 tdTomato flox mice (fl/fl) and homozygous B-CAG-Cas9 tdTomato mice (Mut/Mut) and analyzed by western blot with anti-Flag antibody (Sigma-Aldrich，F1804). Cas9 was detectable in colon, stomach, bladder, liver of homozygous B-CAG-Cas9 tdTomato mice, but not in B-CAG-Cas9 tdTomato flox mice. fl/fl: B-CAG-Cas9 tdTomato flox mice; Mut/Mut: B-CAG-Cas9 tdTomato mice.