• 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.

Flow cytometry analysis of leukocyte subpopulations in immune organs and peripheral blood of CRBN humanized mice. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6N and CRBN humanized mice (8 weeks 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.

Flow cytometry analysis of T-cell subpopulations in immune organs and peripheral blood of CRBN humanized mice. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6N and CRBN humanized mice (8 weeks 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 analysis of CRBN humanized mice. Three-week-old CRBN humanized mice were grouped by sex (10 males and 10 females). Body weight was measured weekly on the same day for 13 consecutive weeks. The minimum and maximum body weights presented in the table were calculated as mean ± SD. The body weight data followed an approximately normal distribution, with approximately 68% of values falling within one standard deviation (± SD) of the mean.

Complete blood count (CBC) analysis of CRBN humanized mice. Values are expressed as mean ± SD.

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

Blood biochemical analysis of CRBN humanized mice. Values are expressed as mean ± SD.

Organs of female CRBN humanized mice (10 weeks old, n = 10).

Organs of male CRBN humanized mice (10-week-old, n = 10).

Histopathological analysis of major organs in CRBN humanized mice. Major organs were collected from 10-week-old CRBN humanized mice (male, n = 10; female, n = 10) and analyzed by hematoxylin and eosin (H&E) staining.

In vivo efficacy of NEK7-molecular glue (by the client) in an LPS- and nigericin-induced mouse peritonitis model using CRBN humanized mice. CRBN humanized mice were orally dosed either vehicle or the test compound twice daily (BID), starting 24 hours prior to stimulation. On the day of stimulation, mice received a final dose, followed by lipopolysaccharide (LPS, 1 mg/kg, i.p.) 1 hour later and nigericin (3 mg/kg, i.p.) 3 hours after LPS administration. Two hours later, spleens and peritoneal lavage fluid were collected for analysis.

Analysis of of spleen and peritoneal lavage fluid by western blot and ELISA in CRBN humanized mice. (A) Spleen tissue lysates were collected and analyzed by Western blot using an anti-NEK7 antibody. Relative NEK7 protein expression levels were quantified by densitometric analysis and are presented. (B) Peritoneal lavage fluid was collected, and mouse IL-1β levels were measured by ELISA. Values are expressed as mean ± SEM. Statistical significance was determined using an unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

In vivo toxicity evaluation of CC-885 in CRBN humanized mice. (A) CRBN humanized mice and wild-type C57BL/6N mice were administered CC-885 intraperitoneally once daily. Mice were euthanized 11 hours after the final dose on the following day. Lymph nodes, liver, and large intestine tissues were collected for analysis.

In vivo toxicity evaluation of CC-885 in CRBN humanized mice. Lymph nodes, liver, and large intestine tissues were collected for analysis. (A) Survival rate. (B) Body weight and body weight changes during treatment. (C) Histopathological analysis. Scale bar: 100 μm.

In vivo toxicity evaluation of CC-885 in CRBN humanized mice. CRBN humanized mice and wild-type C57BL/6N mice were administered CC-885 intraperitoneally once daily. Mice were euthanized 6 hours after the final dose on the following day. Skeletal muscle, heart, and blood samples were collected for analysis.

Skeletal muscle, heart and blood of CRBN humanized mice were collected for analysis. Skeletal muscle and heart tissue lysates were collected from all group, then analyzed by western blot with anti-CRBN antibody , anti-GSPT1 antibody and anti-CK1α antibody. 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.)

Q1: What are CRBN humanized mice used for?

They are designed for evaluating human CRBN-binding drugs, including small molecules, molecular glues, and PROTAC-based degraders, under physiologically relevant human CRBN expression.

Q2: Do CRBN humanized mice exhibit normal baseline physiology?

Yes. CBC, biochemistry, organ weight, and histopathology show no abnormalities, supporting their suitability for long-term studies.

Q3: Why do CRBN humanized mice respond differently to CC-885?

CC-885 selectively binds human CRBN, resulting in degradation of substrates such as GSPT1. This on-target effect occurs only in CRBN humanized mice, not WT animals.

Q4: Can these mice be used for immunomodulatory drug testing?

Yes. Lenalidomide-induced IL-2 upregulation is observed only in CRBN humanized mice, reflecting the human-specific CRBN dependence of its immunomodulatory activity.

Q5: Are CRBN humanized mice suitable for oncology studies?

Absolutely. Tumor cell line inoculation combined with CRBN-dependent therapeutics allows in vivo evaluation of efficacy, safety, and mechanism of action.