• CRBN interacts with DNA damage-binding protein 1 (DDB1), Cullin 4 (Cul4A or Cul4B), and Regulator of Cullins 1 (RoC1) to form a functional E3 ubiquitin ligase complex. Within this complex, CRBN functions as the substrate receptor and targets proteins for proteolysis through the ubiquitin–proteasome pathway.
• The coding sequence (CDS) of the human CRBN gene encoding the full-length protein was inserted into mouse Crbn at exons 2–3. The CRBN humanized mice express the human CRBN protein, while mouse Crbn is no longer expressed.
• Mouse Crbn mRNA was detectable in the cortex of wild-type mice (+/+). Human CRBN mRNA was detectable in CRBN humanized mice (H/H) but not in wild-type mice.
• Naive CD4⁺ T cells derived from CRBN humanized mice exhibited increased IL-2 secretion after lenalidomide treatment that targets human CRBN, while no such change was observed in cells from wild-type mice.
• CC-885, a CRBN modulator, exhibited marked toxicity exclusively in CRBN humanized mice, with no detectable toxicity in wild-type mice.
• Body weight, complete blood count, and blood biochemistry of male and female CRBN humanized mice were analyzed. The main organs were dissected, weighed, and analyzed by H&E staining. No abnormalities were observed in the organs examined (brain, heart, lung, liver, spleen, stomach, small intestine, colon, kidney, uterus, ovary, and testis).
• Tumor cell lines inoculated into CRBN humanized mice can be used to study the in vivo efficacy and safety of CRBN small-molecule drugs, CRBN-based molecular glue drugs, or CRBN-based PROTAC drugs.

• Fully human CRBN CDS knock-in, enabling precise evaluation of human CRBN-binding small molecules, molecular glues, and PROTACs.
• Physiological human CRBN expression across multiple tissues, with mouse Crbn expression eliminated.
• Genetically confirmed human mRNA and protein expression, ensuring accurate human-specific target engagement studies.
• Strict on-target drug response, including selective sensitivity to CC-885 only in CRBN humanized mice.
• Immune cell functional relevance, demonstrated by lenalidomide-induced IL-2 upregulation specifically in humanized animals.
• Normal hematology, biochemistry, organ morphology, and gross anatomy, supporting safe long-term study designs.
• Ideal translational model for human CRBN-targeting drug discovery and mechanistic studies.

• Genetic validation: Human CRBN CDS successfully inserted into mouse Crbn exons 2–3; mouse Crbn mRNA is absent in humanized mice.
• mRNA validation: Human CRBN mRNA expressed across multiple tissues; mouse Crbn is only present in WT animals.
• Protein validation: Western blot confirms human CRBN expression; anti-CRBN antibody detects both mouse and human proteins.
• Functional validation: Lenalidomide upregulates IL-2 in CD4⁺ T cells only from CRBN humanized mice.
• Pharmacological validation: CC-885 toxicity and GSPT1 degradation observed exclusively in CRBN humanized mice.
• Pathology validation: Organs show expected drug-induced histopathology only in humanized mice; WT mice remain unaffected.
• General health validation: Normal CBC, biochemistry, organ weight, and H&E profiles in untreated humanized mice.

• In vivo efficacy testing of CRBN-binding small molecules, molecular glues, and CRBN-based PROTACs.
• On-target toxicity modeling, such as CC-885–induced degradation and tissue pathology specific to human CRBN.
• Mechanistic studies of ubiquitination and protein degradation, enabling investigation of CRBN-substrate regulation.
• Immunomodulatory drug evaluation, including compounds affecting T-cell activation or cytokine induction.
• Translational biomarker discovery, such as GSPT1 degradation and IL-2 secretion signatures.
• Tumor model studies, including CRBN-dependent therapeutic efficacy and safety profiling.
• Comparative human vs mouse pathway studies, leveraging human-only drug responses.

CRBN

• The coding sequence (CDS) of human CRBN gene that encodes the full-length protein was inserted into the mouse Crbn exons 2-3. The CRBN humanized mice will express the human CRBN protein, while mouse Crbn will no longer be expressed.

Strain-specific analysis of CRBN mRNA expression in wild-type C57BL/6 mice and CRBN humanized mice by RT-PCR. Total RNA was isolated from the heart, liver, spleen, lung, kidney, stomach, small intestine, colon, and cortex of wild-type C57BL/6 mice (+/+) and homozygous CRBN humanized mice (H/H). cDNA was synthesized by reverse transcription, followed by PCR amplification using mouse- or human-specific CRBN primers. Sequencing of the short PCR products confirmed that the amplified sequences matched the corresponding database reference sequences.

qPCR analysis of CRBN/Crbn mRNA expression in wild-type C57BL/6N mice and CRBN humanized mice. Total RNA was isolated from various tissues of wild-type C57BL/6N mice (+/+) (female, 7 weeks old, n = 3) and homozygous CRBN humanized mice (H/H) (female, 7 weeks old, n = 3). cDNA was synthesized by reverse transcription, followed by quantitative PCR (qPCR) using primers targeting an identical sequence shared by human CRBN and mouse Crbn in wild-type C57BL/6N and homozygous CRBN humanized mice. Relative mRNA expression levels were normalized to those in the heart of wild-type C57BL/6N mice. Values are expressed as mean ± SEM. Statistical significance was determined by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Strain-specific analysis of CRBN protein expression in homozygous CRBN humanized mice by Western blot. Cortex, liver, spleen, lung, kidney, stomach, small intestine, colon, heart, and ear tissues were collected from wild-type C57BL/6 mice (+/+) and homozygous CRBN humanized mice (H/H). CRBN protein expression was analyzed by Western blot using an anti-CRBN antibody.

Naive CD4⁺ T cells derived from CRBN humanized mice exhibited increased IL-2 secretion after treatment with lenalidomide. Naive CD4⁺ T cells were collected from wild-type C57BL/6 mice (+/+) and homozygous CRBN humanized mice (H/H), then stimulated with DMSO or lenalidomide in vitro for 24 hours. Supernatants were collected, and IL-2 production was measured by ELISA.

• The percentages of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hCRBN mice were similar to those in C57BL/6N mice.
• Humanization of CRBN does not affect normal immune cell development or distribution.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6N and B-hCRBN mice (female, 8-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous B-hCRBN mice were comparable to those in C57BL/6N mice.
• Humanization of CRBN does not affect normal T cell development, differentiation or distribution.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6N and B-hCRBN mice (female, 8-week-old, n = 3). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Growth curve of B-hCRBN mice. Three-week-old mice were grouped by sex (10 males and 10 females). Body weight was measured weekly for 13 weeks on the same day each week. The minimum and maximum body weights shown in the table were calculated from the mean ± SD. The growth curve follows a normal distribution, with approximately 68% of values falling within ± SD.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of B-hCRBN mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of B-hCRBN mice are shown. Values are expressed as mean ± SD.

• No abnormalities were observed.

Organs of female B-hCRBN mice (10-week-old, n = 10).

• No abnormalities were observed.

Organs of male B-hCRBN mice (10-week-old, n = 10).

• No obvious abnormalities were observed in any organs examined (brain, heart, lung, liver, spleen, stomach, small intestine, large intestine, kidney, ovary, uterus ,testis).

Histopathological analysis of organs in B-hCRBN mice. Major organs from B-hCRBN mice were collected at 10 weeks of age and analyzed by H&E staining (male, n = 10; female, n = 10).

In vivo Efficacy of NEK7-molecular glue (by the client) in mouse peritonitis model. B-hCRBN mice were dosed orally, BID, with vehicle or compound, starting 24h prior to stimulation. On the stimulation day, animals received a final dose, followed 1h later by LPS (1mg/kg, i.p.), and nigericin (3mg/kg, i.p.) 3h after LPS. Two hours later, spleens and peritoneal lavage fluid were collected for analysis.

• The NEK7-molecular glue can efficiently induce degradation of the NEK7 and IL-1β proteins in B-hCRBN mice.

Analysis of spleen and peritoneal lavage fluid by WB and ELISA. (A) Spleen tissue lysates were collected and analyzed by western blot with anti-NEK7 antibody. Relative NEK7 protein levels were quantified by densitometric analysis and presented.(B) Peritoneal lavage fluid were collected and mouse IL-1β was analyzed by ELISA. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test.  *P < 0.05, **P < 0.01, ***P < 0.001.

In vivo toxicity of CC-885. (A) B-hCRBN mice and C57BL/6N mice were administered the CC-885 intraperitoneally once daily, mice were euthanized 11 hours after the final dose on the following day, Lymph node, liver and large intestine were collected for analysis.

• The results show that CC-885 exhibits marked toxicity exclusively in B-hCRBN mice, with no detectable toxicity observed in wild-type mice.

In vivo toxicity of CC-885. Lymph node, liver and large intestine were collected for analysis. (A) Percent survival. (B) Body weight and body weight change during the treatment. (C) Histopathological analysis. Scale bar: 100 μm.

In vivo toxicity of CC-885. B-hCRBN mice and C57BL/6N mice were administered the CC-885 intraperitoneally once daily, mice were euthanized 6 hours after the final dose on the following day, skeletal muscle, heart and blood were collected for analysis.

• CC-885 induced degradation of the GSPT1 protein in B-hCRBN mice (G4), but not in wild-type mice.
• CC-885 exhibits hematological toxicity in B-hCRBN mice, but not in wild-type mice.

Skeletal muscle, heart and blood were collected for analysis. Skeletal muscle and heart tissue lysates were collected from all group, then analyzed by western blot with anti-CRBN antibody (CST, #71810), anti-GSPT1 antibody (Abcam, ab234433) and anti-CK1α antibody (Abcam, ab302638). 40 μg total proteins were loaded for western blotting analysis. (A) Western blot analysis of GSPT1 protein expression. (B) Biochemical test and complete blood count (Additional hematological parameters are not shown.)

Q: What is the strategy of B-hCRBN mice (113236)?

A: B-hCRBN mice were constructed by inserting human CRBN starting from the 24th amino acid of mouse Crbn. Since the first 23 amino acids are completely identical between human and mouse, the human sequence was inserted from the 24th amino acid onward. In the mouse, amino acids 1–23 in the second exon were retained, while the remainder of exon 2 and all of exon 3 were knocked out.

Q: What is the difference between B-hCRBN mice (113236) and I391V point mutation mice?

A: Regarding B-hCRBN mice, we have generated a full human CRBN protein replacement, which also includes the I391V point mutation. Therefore, we have not developed a separate point mutation mouse model. The reasons are as follows:

• Functional limitation of point mutation: The I391V mutation only restores drug binding, but does not address other amino acid differences between human and mouse CRBN, which may affect the stability and degradation efficiency of the CRBN complex.
• Advantage of full humanization: The B-hCRBN mouse expresses complete human CRBN protein, ensuring natural and efficient interaction with human DDB1 and accurate drug binding to the human CRBN pocket, better mimicking human physiology.
• Different utility for studies: The point mutation mouse may be suitable for pharmacodynamic studies when the drug specifically targets the 391 site. However, for preclinical toxicology and safety evaluation, the fully humanized model is superior because it better recapitulates human physiological conditions.