A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system

Shen, Yunbing; Jiang, Long; Iyer, Vaishnavi Srinivasan; Raposo, Bruno; Dubnovitsky, Anatoly; Boddul, Sanjaykumar V.; Kasza, Zsolt; Wermeling, Fredrik

doi:10.1016/j.csbj.2021.09.020

Cited by 3 publications

(4 citation statements)

References 54 publications

(66 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3A ). To do this, we resorted to an in vivo CRISPR-based approach that we previously published ( 23 ) and adopted it to the KRN+ splenocyte transfer model. By electroporating sgRNAs targeting genes of interest into Cas9+ BM cells, followed by transplanting these modified BM cells into irradiated recipients, we generate what we refer to as immuno-CRISPR (iCR) mice.…”

Section: Resultsmentioning

confidence: 99%

“…As we had established the model’s premises, we wanted to test if we could apply the immuno-CRISPR (iCR) approach we previously developed ( 23 ). This approach is based on modifying BM cells with CRISPR-Cas9, targeting a gene of interest to generate a controlled genetically heterogeneous BM cell population.…”

Section: Discussionmentioning

confidence: 99%

“…BM cells were prepared as described before ( 17 ). For BM transplantation, roughly 10 6 BM cells were i.v.…”

Section: Methodsmentioning

confidence: 99%

“…2.10 sgRNA, primer design, and Indel analysis sgRNAs were designed as described previously (17). Briefly, the Green Listed software http://greenlisted.cmm.ki.se/ (21) and Synthego CRISPR design tool were utilized to design sgRNAs (Table S2).…”

Section: Flow Cytometry Analysis and Sortingmentioning

confidence: 99%

See 3 more Smart Citations

Dnmt3amutations limit normal and autoreactive Tfh differentiation

Shen,

Li,

Boddul

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation, strongly associated with the activity of autoreactive CD4+ T cells.DNMT3Amutations are the most common somatic mutations found in the hematopoietic system of patients with rheumatoid arthritis. However, the role of DNMT3A in CD4+ T cells and CD4+ T follicular helper (Tfh) cells is poorly understood. Since somatic mutations are not identified in standard genome-wide association studies, somatic mutations’ impact on the etiology of diseases could be underestimated. Here, we thoroughly characterized and used the KRN+ splenocyte transfer model of autoimmune joint inflammation and inactivatedDnmt3ausing CRISPR-Cas9 and standard Cre/loxP approaches. Experiments with competitive bone marrow (BM) chimeras identified a positive role forDnmt3ain Tfh differentiation, which was validated by comparing mice withDnmt3amutations in CD4+ cells to animals with WTDnmt3a. In conclusion, We identify thatDnmt3amutations limit normal and autoreactive Tfh differentiation.Key findings–Dnmt3amutations limit Tfh differentiation, which could contribute to reduced immune responses in individuals with somaticDNMT3Amutations.–Deep characterization of the KRN+ splenocyte transfer model defines a dynamic process leading to reproducible autoimmune joint inflammation.–The immuno-CRISPR (iCR) methodology can be used to test the role of candidate genes in disease models.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…BM cells were prepared as described before ( 17 ). For BM transplantation, roughly 10 6 BM cells were i.v.…”

Section: Methodsmentioning

confidence: 99%

Section: Flow Cytometry Analysis and Sortingmentioning

confidence: 99%

See 2 more Smart Citations

Dnmt3amutations limit normal and autoreactive Tfh differentiation

Shen,

Li,

Boddul

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Modulating T-cell activation with antisense oligonucleotides targeting lymphocyte cytosolic protein 2

Iyer

Boddul

Johnsson

et al. 2022

Journal of Autoimmunity

View full text Add to dashboard Cite

CRISPR/Cas9-mediated gene disruption determines the roles of MITF and CITED2 in human mast cell differentiation

Mo,

Wermeling,

Nilsson

et al. 2024

Blood Advances

View full text Add to dashboard Cite

The differentiation of hematopoietic stem and progenitor cells into the various cell lineages is regulated by cell-intrinsic factors and the progenitors' microenvironment. Experiments in mice have allowed the identification of transcriptional modulators that control mast cell differentiation. However, a comprehensive approach that allows efficient disruption of individual transcriptional modulators in primary human hematopoietic progenitors coupled with a mast cell formation readout has not been described. Here, we report a simple electroporation- and ribonucleoprotein-based knockout system that allows the identification of genes that are required and dispensable for human mast cell differentiation. We show that the transcription factor MITF is upregulated in human mast cell progenitors and reveal that the loss of MITF results in the suppressed formation of mast cells. By contrast, CITED2, another transcriptional modulator that is upregulated along the mast cell differentiation trajectory, was dispensable for human mast cell differentiation. Taken together, we report a CRISPR/Cas9-based framework that serves to identify genes involved in regulating the formation of human mast cells, and the results uncover the role of two transcriptional modulators in controlling human mast cell differentiation.

show abstract

A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system

Abstract: Graphical abstract

Cited by 3 publications

References 54 publications

Dnmt3amutations limit normal and autoreactive Tfh differentiation

Dnmt3amutations limit normal and autoreactive Tfh differentiation

Modulating T-cell activation with antisense oligonucleotides targeting lymphocyte cytosolic protein 2

CRISPR/Cas9-mediated gene disruption determines the roles of MITF and CITED2 in human mast cell differentiation

Contact Info

Product

Resources

About