The development of CRISPR/Cas9-based genome engineering represents a major breakthrough for the gene therapy field (1). The hope is that many severe monogenic diseases could be cured by correcting the disease-causing mutations. Several CRISPR/Cas9-based clinical trials have started, for example, in patients with sickle cell anemia and beta-Thalassemia (2, 3).

We have identified that CRISPR/Cas9 can enrich for cells with mutations in a broad p53-linked interactome and that gene expression can predict this enrichment (4). As mutations in p53, and many of the linked genes we found to be enriched by CRISPR/Cas9, are connected to cancer development, the enrichment could be problematic for the clinical CRISPR used.

In this project, the aim is to identify robust gene expression biomarkers that can predict how cells respond to CRISPR/Cas9. Such biomarkers are important to develop better and more safe CRISPR/Cas9 tools.

The student will learn to work with CRISPR/Cas9, including the design of sgRNAs, mutation analysis, and different delivery methods (electroporation, transfection). The student will also learn how to work with cell cultures of different cell types and RT-qPCR to measure gene expression. Due to the technical nature of the project, the project is not suitable for too short rotations in the lab.

Information about how the group work with CRISPR can be found in these references (5-8).


References

1. M. H. Porteus, A New Class of Medicines through DNA Editing. N Engl J Med 380, 947-959 (2019).
2. E. B. Esrick et al., Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease. N Engl J Med 384, 205-215 (2021).
3. H. Frangoul et al., CRISPR-Cas9 Gene Editing for Sickle Cell Disease and beta-Thalassemia. N Engl J Med 384, 252-260 (2021).
4. L. Jiang et al., CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based Therapies. Cancer Res 82, 36-45 (2022).
5. Y. Shen et al., A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system. Comput Struct Biotechnol J 19, 5360-5370 (2021).
6. S. K. Panda et al., IL-4 controls activated neutrophil FcgammaR2b expression and migration into inflamed joints. Proc Natl Acad Sci U S A 117, 3103-3113 (2020).
7. V. S. Iyer et al., Designing custom CRISPR libraries for hypothesis-driven drug target discovery. Comput Struct Biotechnol J 18, 2237-2246 (2020).
8. S. K. Panda et al., Green listed-a CRISPR screen tool. Bioinformatics 33, 1099-1100 (2017).