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|Title:||A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system||Authors:||Shen, Yunbing
Iyer, Vaishnavi Srinivasan
Boddul, Sanjaykumar V.
|Issue Date:||2021||Source:||Shen, Y., Jiang, L., Iyer, V. S., Raposo, B., Dubnovitsky, A., Boddul, S. V., Kasza, Z. & Wermeling, F. (2021). A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system. Computational and Structural Biotechnology Journal, 19, 5360-5370. https://dx.doi.org/10.1016/j.csbj.2021.09.020||Journal:||Computational and Structural Biotechnology Journal||Abstract:||CRISPR/Cas9 can be used as an experimental tool to inactivate genes in cells. However, a CRISPR-targeted cell population will not show a uniform genotype of the targeted gene. Instead, a mix of genotypes is generated - from wild type to different forms of insertions and deletions. Such mixed genotypes complicate analysis of the role of the targeted gene in the studied cell population. Here, we present a rapid and universal experimental approach to functionally analyze a CRISPR-targeted cell population that does not involve generating clonal lines. As a simple readout, we leverage the CRISPR-induced genetic heterogeneity and use sequencing to identify how different genotypes are enriched or depleted in relation to the studied cellular behavior or phenotype. The approach uses standard PCR, Sanger sequencing, and a simple sequence deconvoluting software, enabling laboratories without specific in-depth experience to perform these experiments. As proof of principle, we present examples studying various aspects related to hematopoietic cells (T cell development in vivo and activation in vitro, differentiation of macrophages and dendritic cells, as well as a leukemia-like phenotype induced by overexpressing a proto-oncogene). In conclusion, we present a rapid experimental approach to identify potential drug targets related to mature immune cells, as well as normal and malignant hematopoiesis.||URI:||https://hdl.handle.net/10356/153758||ISSN:||2001-0370||DOI:||10.1016/j.csbj.2021.09.020||Rights:||© 2021 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).||Fulltext Permission:||open||Fulltext Availability:||With Fulltext|
|Appears in Collections:||SPMS Journal Articles|
Updated on Jul 6, 2022
Updated on Jul 6, 2022
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