X-CHIME: Context Dependent Germline Knockout in Immune Cells

By Guest Blogger

Annotation of genes in immune cells typically involves the creation of germline knockout mice, which is time-consuming, as it only changes one gene at a time. CRISPR-based systems enable gene knockout in immune cells in a high-throughput manner, but these systems have not been widely employed in vivo. The CHIME and new X-CHIME systems, developed and deposited at Addgene by Arlene Sharpe’s lab, allow for wide deployment of in vivo gene knockout in immune systems.  


We previously developed CHIME (CHimeric IMmune Editing system) to enable the constitutive, ubiquitous knockout of single genes in immune cells in vivo (LaFleur et al., 2019a and LaFleur et al., 2019b). CHIME works through transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic stem cells with a lentiviral vector containing a gRNA. These transduced stem cells can be implanted in irradiated recipients to create mice with gene knockouts in their immune systems.

However, many genes have different functions in different cell types, at different times, and in combination with different genes. Having CRISPR systems for assessing genes in defined contexts would be quite useful for interrogating these scenarios.


We recently developed and deposited four new systems to fill this gap. To enable context-specific knockouts, we developed X-CHIME, a suite of four systems which utilizes custom mouse strains and lentiviral expression vectors (Table 1).

The four systems




Transduction Marker

Mouse Strain


Knocks out a pair of genes


Violet-excited GFP (vex)



Inducible knockout of a single gene


Violet-excited GFP (vex)

Rosa26-FlpO-ERT2; H11-Cas9


Knocks out a single gene in defined cell types (lineage-based)


Violet-excited GFP (vex)

Lineage-Cre; Rosa26-Cas9


Knocks out pairs of genes sequentially


Violet-excited GFP (vex)

Rosa26-FlpO-ERT2; H11-Cas9

Table 1: X-CHIME systems

Proof of Concept

These systems were benchmarked on positive control genes in CD4+ and CD8+ T cells. To demonstrate the power of these systems to investigate in vivo mechanisms, we targeted two phosphatases, PTPN1 and PTPN2, which are of interest as cancer immunotherapy targets but are embryonic lethal, hindering studies post embryogenesis. Using C-CHIME and S-CHIME, we discovered that double knockout of PTPN1 and PTPN2 in immune cells in a developing or developed immune system was lethal (LaFleur et al., 2024).


Schematic showing the X-CHIME approach. Cas9-expression LSK from X-CHIME donors leads to X-CHIME vector lentiviral transduction, then transfer LSK to irradiated WT recipients, then 8 weeks immune reconstitution in a mouse leads to X-CHIME BMC.

Figure 1: Schematic showing the X-CHIME approach. Figure from LaFleur, et al. 2024.


The X-CHIME deposit also includes pXPR_071, a plasmid that leads to higher titer lentivirus preparations than the first generation CHIME (pXPR_053).

Find X-CHIME plasmids here!


0136_Martin_Lafleur_001 (1)-minMarty LaFleur is a Postdoctoral Fellow in Arlene Sharpe’s laboratory at Harvard Medical School and is interested in improving CD8+ T cell responses to cancer.



References and resources


LaFleur, M. W., Lemmen, A. M., Streeter, I. S. L., Nguyen, T. H., Milling, L. E., Derosia, N. M., Hoffman, Z. M., Gillis, J. E., Tjokrosurjo, Q., Markson, S. C., Huang, A. Y., Anekal, P. V., Montero Llopis, P., Haining, W. N., Doench, J. G., & Sharpe, A. H. (2024). X-CHIME enables combinatorial, inducible, lineage-specific and sequential knockout of genes in the immune system. Nature Immunology, 25(1), Article 1. https://doi.org/10.1038/s41590-023-01689-6

LaFleur, M.W., Nguyen, T.H., Coxe, M.A., Miller, B.C., Yates, K.B., Gillis, J.E., Sen, D.R., Gaudiano, E.F., Al Abosy, R., Freeman, G.J., Haining, W.N., & Sharpe, A.H. (2019). PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity. Nature Immunology 20, 1335–1347. https://doi.org/10.1038/s41590-019-0480-4

LaFleur, M. W., Nguyen, T. H., Coxe, M. A., Yates, K. B., Trombley, J. D., Weiss, S. A., Brown, F. D., Gillis, J. E., Coxe, D. J., Doench, J. G., Haining, W. N., & Sharpe, A. H. (2019). A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system. Nature Communications, 10(1), 1668. https://doi.org/10.1038/s41467-019-09656-2

More resources on the Addgene blog

CRISPR101: Validating Your Genome Edit

How to Design Your gRNA for CRISPR Genome Editing

CRISPR Cheat Sheat

Resources from Addgene.org


CRISPR: Protocol for Genome Deletions in Mammalian Cell Lines



Topics: CRISPR, Other Plasmid Tools

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