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dc.contributor.authorShen, Yunbingen_US
dc.contributor.authorJiang, Longen_US
dc.contributor.authorIyer, Vaishnavi Srinivasanen_US
dc.contributor.authorRaposo, Brunoen_US
dc.contributor.authorDubnovitsky, Anatolyen_US
dc.contributor.authorBoddul, Sanjaykumar V.en_US
dc.contributor.authorKasza, Zsolten_US
dc.contributor.authorWermeling, Fredriken_US
dc.identifier.citationShen, 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.
dc.description.abstractCRISPR/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.en_US
dc.description.sponsorshipNanyang Technological Universityen_US
dc.relation.ispartofComputational and Structural Biotechnology Journalen_US
dc.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 (
dc.subjectScience::Biological sciencesen_US
dc.titleA rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic systemen_US
dc.typeJournal Articleen
dc.contributor.schoolSchool of Physical and Mathematical Sciencesen_US
dc.description.versionPublished versionen_US
dc.subject.keywordsClustered Regularly Interspaced Short Palindromic Repeats (CRISPR)en_US
dc.subject.keywordsSequence Analysisen_US
dc.description.acknowledgementWe are grateful to Drs. Helena Malmgren, Lisa Westerberg, Taras Kreslavskiy, Laura Plant and Sudeepta Panda for valuable discussions and input. The ER-Hoxb8 construct was a gift from Mark P. Kamps, University of California, San Diego. The pSIRV-NF-jBeGFP construct was a gift from Peter Steinberger, Medical University of Vienna. This research was partly funded by grants from the Swedish Research Council, the Swedish Cancer Society, Karolinska Institutet, Magnus Bergvalls stiftelse, Stiftelsen Professor Nanna Svartz fond, Felix Mindus contribution to Leukemia Research (to FW), the China Scholarship Council (to LJ and YS), and the Nanyang Y. Shen, L. Jiang, Vaishnavi Srinivasan Iyer et al. Computational and Structural Biotechnology Journal 19 (2021) 5360–5370 5369 Technological University–Karolinska Institutet Joint PhD Programme (to VSI).en_US
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