Abstract
Annotation of immunologic gene function in vivo typically requires the generation of knockout mice, which is time consuming and low throughput. We previously developed CHimeric IMmune Editing (CHIME), a CRISPR–Cas9 bone marrow delivery system for constitutive, ubiquitous deletion of single genes. Here we describe X-CHIME, four new CHIME-based systems for modular and rapid interrogation of gene function combinatorially (C-CHIME), inducibly (I-CHIME), lineage-specifically (L-CHIME) or sequentially (S-CHIME). We use C-CHIME and S-CHIME to assess the consequences of combined deletion of Ptpn1 and Ptpn2, an embryonic lethal gene pair, in adult mice. We find that constitutive deletion of both PTPN1 and PTPN2 leads to bone marrow hypoplasia and lethality, while inducible deletion after immune development leads to enteritis and lethality. These findings demonstrate that X-CHIME can be used for rapid mechanistic evaluation of genes in distinct in vivo contexts and that PTPN1 and PTPN2 have some functional redundancy important for viability in adult mice.
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Data availability
The data and materials that support the findings of this study are available from the corresponding author upon reasonable request. All 10× scRNA sequencing data from this study has been deposited in the Broad Institute’s Single Cell Portal and are accessible through the accession code SCP1945. Mus musculus genome GRCm39 is accessible at https://ftp.ensembl.org/pub/release-106/fasta/mus_musculus/dna. Gene annotations from Ensembl release 106 are accessible at https://ftp.ensembl.org/pub/release-106/gtf/mus_musculus/. Source data are provided with this paper.
Code availability
The custom code used for scRNA-seq analyses has been deposited on GitHub.
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Acknowledgements
This work was supported by U19AI133524 from the National Institute of Allergy and Infectious Diseases to A.H.S. and J.G.D., U54 CA225088 from the National Cancer Institute to A.H.S., and P01 AI108545 from the National Institute of Allergy and Infectious Diseases to A.H.S. A.H.S. also received funding for this work from Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. We thank the Dana-Farber/Harvard Cancer Center in Boston, MA, for use of the Rodent Histopathology Core, which performed necropsies. Dana-Farber/Harvard Cancer Center is supported in part by NCI Cancer Center Support Grant #NIH 5 P30 CA06516. We thank the Center for Computational and Integrative Biology (CCIB) at Massachusetts General Hospital for the use of the CCIB DNA Core Facility (Cambridge, MA), which performed CRISPR sequencing for indel assessment. We thank the Microscopy Resources on the North Quad (MicRoN) core, RRID:SCR_019195 at Harvard Medical School for microscopy and analysis services.
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Contributions
M.W.L., W.N.H. and A.H.S. conceived the project. M.W.L., A.M.L., I.S.L.S., T.H.N., N.M.D. and J.E.G. designed experiments. M.W.L., A.M.L., I.S.L.S., T.H.N., L.E.M., N.M.D., Z.M.H., J.E.G. and Q.T. acquired and analyzed data. M.W.L., T.H.N. and A.H.S. wrote the manuscript with contributions from A.M.L., I.S.L.S., N.M.D. and L.E.M. All authors edited the manuscript. P.V.A. and P.M.L. assisted with imaging and analysis. S.C.M. and A.Y.H. performed 10× scRNA-seq analyses. J.G.D. assisted with methodology. A.H.S. secured funding and supervised the project.
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Competing interests
A.H.S. has patents/pending royalties on the PD-1 pathway from Roche and Novartis. A.H.S. is on advisory boards for Elpiscience, Selecta, Bicara, Monopteros, Fibrogen, Alixia, IOME, Corner Therapeutics, GlaxoSmithKline, Amgen and Janssen. A.H.S. has received research funding from Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA related to this project and Vertex, Moderna, Quark/Iome, AbbVie and Erasca unrelated to this project. J.G.D. consults for Microsoft Research, Abata Therapeutics, Servier, Maze Therapeutics, BioNTech, Sangamo and Pfizer. J.G.D. consults for and has equity in Tango Therapeutics. J.G.D. serves as a paid scientific advisor to the Laboratory for Genomics Research, funded in part by GlaxoSmithKline. J.G.D. receives funding support from the Functional Genomics Consortium: AbbVie, Bristol Myers Squibb, Janssen, Vir Biotechnology, and Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. J.G.D.’s interests were reviewed and are managed by the Broad Institute in accordance with its conflict-of-interest policies. W.N.H. is an employee of and holds equity in Arsenal Biosciences. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Additional data related to Fig. 1.
(a) Gating strategy for isolation of LSK from bone marrow for all BMC studies. (b) Gating strategy for isolation of Vex+ CD4+ or Vex+ CD8+ T cells from mouse peripheral blood or spleens. (c) Representative flow cytometry plots of CD2 and CD28 expression on Vex+ CD4+ splenocytes from BMCs containing two non-targeting control gRNAs (left) or Cd2 and Cd28 gRNAs (right). (d, e) Quantification of CD2 (d) or CD28 (e) expression on Vex+ CD4+ splenocytes from (c) as well as relevant single gRNA controls. (f) Quantification of the percentage of cells lacking expression of both CD2 and CD28 on Vex+ CD4+ splenocytes from (c) as well as relevant single gRNA controls. Note ++ indicates two control gRNAs in (d-f). (g) Gating strategy for splenic red-pulp macrophages from mouse spleens. Data are representative of two experiments with at least n = 4 mice per group. Bar graphs represent the mean and error bars represent s.d. Calculated knockout efficiency is denoted above bars for indicated comparisons.
Extended Data Fig. 2 Additional data related to Fig. 2.
(a) Schematic of I-CHIME loxP vector before and after Cre-mediated excision. (b-c) Quantification of CD2 expression on (b) Vex+ CD4+ and (c) Vex+ CD8+ splenocytes from Cre-ERT2 I-CHIME BMCs containing a constitutive control or Cd2-targeted gRNA, or inducible control or Cd2-targeted gRNA treated with vehicle, 1 mg Tamoxifen (1x in panel), or 2 mg Tamoxifen (2x in panel). (d) Quantification of indels in Cd2 following next-generation sequencing of Vex+ CD8+ splenocytes as in (c). (e) Quantification of % excision of PCR products following an I-CHIME vector-specific PCR of Vex+ CD8+ splenocytes isolated from BMCs containing a non-targeting control or Cd2 gRNA and treated with vehicle control, 1 mg Tamoxifen (1x in panel), or 2 mg Tamoxifen (2x in panel). (f) Representative flow cytometry plots of CD2 expression on Vex+ CD8+ splenocytes from BMCs containing a non-targeting control or Cd2 gRNA. BMCs were created using H11-Cas9 donors or R26-Cas9 donors. (g) Quantification of CD2 expression on Vex+ CD4+ and Vex+ CD8+ splenocytes from (f). (h) Quantification of CD2 expression on Vex+ CD8+ peripheral blood T cells from FlpO-ERT2 I-CHIME BMCs containing an inducible control or Cd2-targeted gRNA treated with vehicle (3 days or 5 days), 1 mg Tamoxifen (3 days or 5 days) (1x Tamoxifen in panel), or 2 mg Tamoxifen (3 days) (2x Tamoxifen in panel). Serial measurements were taken from the same mice. (i) Quantification of indels in Cd2 following next-generation sequencing of Vex+ CD8+ T cells and Vex+ LSKs (as in Fig. 2g). The bar graph represents pooled data from two independent experiments (p = 0.0146). (j) TapeStation gel of PCR products following an I-CHIME vector-specific PCR of Vex+ CD8+ splenocytes isolated from BMCs containing a non-targeting control or Cd2 gRNA and treated with Tamoxifen or vehicle control. Genomic DNA that is 0% excised and 100% excised were included as controls. Data are representative of one experiment with at least n = 2 mice per group except (i), which is representative of two experiments with at least n = 3 mice per group. Bar graphs represent the mean and error bars represent s.d. Calculated knockout efficiency is denoted above bars for indicated comparisons. Statistical significance was assessed by a two-sided Student’s paired t-test (i).
Extended Data Fig. 3 Specific deletion of genes in CD8+ T cells using L-CHIME.
(a) Schematic of L-CHIME vector before and after Cre-mediated excision. (b) Schematic for creation of L-CHIME BMCs. (c) Representative flow cytometry plots of CD2 expression on splenic CD8+ T cells from BMCs created using Cas9ON bone marrow (no Cre present) and the L-CHIME vector containing control or Cd2-targeting gRNAs. (d) Quantification of CD2 expression on Vex+ CD4+ and Vex+ CD8+ splenocytes from BMCs in (c). (e) (Left) Representative gating of Ly6c+ monocytes from the blood. (Right) Quantification of indels in Cd2 following next-generation sequencing of Vex+ Ly6c+ monocytes from BMCs in (c). (f) Representative flow cytometry plots of CD2 expression on Vex+ CD4+ and Vex+ CD8+ T cells isolated from BMCs created using E8I-Cre Cas9ON bone marrow (Cre present only in CD8+ T cells) and the L-CHIME vector containing control or Cd2-targeting gRNAs. (g) Quantification of CD2 expression on Vex+ CD4+ and Vex+ CD8+ splenocytes from BMCs in (f). (h) TapeStation gel of PCR products following an L-CHIME vector-specific PCR of Vex+ CD4+ and Vex+ CD8+ splenocytes containing a non-targeting control or Cd2 gRNA. Genomic DNA that is 0% excised and 100% excised were included as controls. (i) Quantification of % excision as in (h). (j) Quantification of CD2 expression on Vex+ CD4+ and Vex+ CD8+ tumor-infiltrating lymphocytes from BMCs as in (f) challenged with MC38 tumors. (k) CD8+ Vex+ splenocytes were isolated from L-CHIME BMCs containing control or Cd2 gRNAs and were either activated with CD3 and CD28 cross-linking antibodies and IL-2 or maintained in a naive state with IL-7. Quantification of CD2 expression on day 3 post culture. The red dotted line represents the average CD2 expression on the Cd2-targeted CD8+ Vex+ splenocytes prior to culture (p = 0.0131). Data are representative of two experiments with at least n = 3 mice per group. Bar graphs represent the mean and error bars represent s.d. Calculated knockout efficiency is denoted above bars for indicated comparisons and in (k) each Cd2 gRNA is compared to the control gRNA for the corresponding activation condition. Statistical significance was assessed by a two-sided Student’s unpaired t-test (k).
Extended Data Fig. 4 Additional data related to Fig. 3.
(a) Quantification of CD2 (left) or CD28 (right) expression on Vex+ CD8+ peripheral blood T cells from (Fig. 3d). Serial measurements were taken from the same mice. (b) Quantification of CD2 (left) or CD28 (right) expression on Vex+ CD4+ peripheral blood T cells from (Fig. 3d). Serial measurements were taken from the same mice. (c) TapeStation gel of PCR products following an S-CHIME vector-specific PCR of Vex+ CD8+ splenocytes containing (in the inducible gRNA cassette) a non-targeting control or Cd2 gRNA and treated with Tamoxifen or vehicle control. Genomic DNA that is 0% excised and 100% excised were included as controls. (d) Quantification of % excision in (c). Knockout data are representative of two experiments with at least n = 5 mice per group. Excision data are representative of one experiment with at least n = 2 mice per group. Bar graphs represent the mean and error bars represent s.d. Calculated knockout efficiency is denoted above bars for indicated comparisons.
Extended Data Fig. 5 Additional data related to Fig. 4.
(a) Quantification of peripheral blood leukocytes assessed by flow cytometry from BMCs (as in Fig. 4a) 4 weeks after bone marrow implantation (p < 0.0001). (b) Quantification of Vex expression on CD45+ leukocytes from peripheral blood 4 weeks after bone marrow implantation. (c) Quantification of indels in Ptpn1 (left) and Ptpn2 (right) following next-generation sequencing of peripheral blood cells from BMCs (as in Fig. 4a) 4 weeks after bone marrow implantation. (d, e) CBA analysis of serum cytokines from BMCs (as in Fig. 4a) 2 weeks (d) or 4 weeks (e) after bone marrow implantation (*Ctrl-1/Ctrl-2 vs. Ptpn1/Ptpn2 p = 0.0480, *Ctrl-1/Ptpn2 vs. Ptpn1/Ptpn2 p = 0.0342, ****p < 0.0001). (f) Weight loss curves normalized to initial starting weight after bone marrow implantation for BMCs (as in Fig. 4a). Repeat measurements were taken from the same mice (**p = 0.0027, ****p < 0.0001). (g) Hematoxylin and eosin (10x magnification) scanned images of representative BMC bone marrow sections 4 weeks after bone marrow implantation. Scale bar indicates 1000 µm. (h) Hematoxylin and eosin (10x magnification) scanned images of BMC spleens 4 weeks post bone marrow implantation. Scale bar indicates 1000 µm. (i) Quantification of white pulp area in spleens in (h) (**Ctrl-1/Ctrl-2 vs. Ptpn1/Ptpn2 p = 0.0021, *Ptpn1/Ctrl-2 vs. Ptpn1/Ptpn2 p = 0.0259, **Ctrl-1/Ptpn2 vs. Ptpn1/Ptpn2 p = 0.0029). The bar graph represents pooled data from two independent experiments. Data are representative of two experiments with at least n = 3 mice per group. Bar graphs represent the mean and error bars represent s.d. Statistical significance was assessed by one-sided two-way ANOVA (a, d, e, f) or one-sided one-way ANOVA (i).
Extended Data Fig. 6 Additional data related to Fig. 4.
(a) Heat map of genes used to annotate UMAP clusters of 10x scRNA-seq from bone marrow samples of BMCs (as in Fig. 4f). Note values added to the heat map represent the intensity of saturated values that exceed the intensity scale. (b) UMAP clustering of 10x scRNA-seq from spleen samples of BMCs (as in Fig. 4a). (c) Heat map of genes used to annotate UMAP clusters of 10x scRNA-seq from spleen samples of BMCs (as in Extended Data Fig. 6b). (d, e) Quantification of cell cluster proportions from the spleen within each cluster (d) or within each BMC group (e). Data are representative of one experiment with n = 3 mice per group.
Extended Data Fig. 7 Additional data related to Fig. 5.
(a) Weight loss curves normalized to initial starting weight after bone marrow implantation for BMCs (as in Fig. 5a). Serial measurements were taken from the same mice. (b) Quantification of peripheral blood leukocytes assessed by flow cytometry from BMCs (as in Fig. 5a) 4 weeks after bone marrow implantation (CD11b: ***Ctrl-1/Ctrl-2 vs. Ptpn1/FRT-Ptpn2 p = 0.0002, ***Ctrl-2/FRT-Ptpn2 vs. Ptpn1/FRT-Ptpn2 p = 0.0008; CD19: ***p = 0.0002, ****p < 0.0001). (c) Quantification of Vex expression on CD45+ leukocytes from peripheral blood 4 weeks after bone marrow implantation. (d, e) CBA analysis of serum cytokines from BMCs one week prior to (d) or one week after (e) Tamoxifen administration (p < 0.0001). (f) Ptpn1 and Ptpn2 indel percentage in the blood from BMCs created by nucleofecting bone marrow stem cells with Ptpn1/Ptpn2 gRNAs, mixing with non-nucleofected bone marrow stem cells, and implanting into irradiated recipients. (g) Survival curve of BMCs as in (f) (p = 0.0005). (h) Quantification of pathologic findings for indicated organs from BMCs in (g). Data are representative of two experiments with at least n = 5 mice per group, except (f) which is representative of two experiments with at least n = 3 mice per group. The graph in (h) represents pooled data from two independent experiments. Bar graphs represent the mean and error bars represent s.d. Statistical significance was assessed by one-sided two-way ANOVA (a, b, d, e), two-sided Mantel-Cox test (g), or two-sided Fisher’s exact test (h).
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LaFleur, M.W., Lemmen, A.M., Streeter, I.S.L. et al. X-CHIME enables combinatorial, inducible, lineage-specific and sequential knockout of genes in the immune system. Nat Immunol 25, 178–188 (2024). https://doi.org/10.1038/s41590-023-01689-6
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DOI: https://doi.org/10.1038/s41590-023-01689-6
