A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system
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 genotyp...
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doaj-7ef9e66cdc654f80b5bdd9d389d86c632021-10-02T04:00:49ZengElsevierComputational and Structural Biotechnology Journal2001-03702021-01-011953605370A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic systemYunbing Shen0Long Jiang1Vaishnavi Srinivasan Iyer2Bruno Raposo3Anatoly Dubnovitsky4Sanjaykumar V. Boddul5Zsolt Kasza6Fredrik Wermeling7Department of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, SwedenDepartment of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, SwedenDepartment of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden; School of Physical and Mathematical Sciences, Nanyang Technological University, SingaporeDepartment of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, SwedenDepartment of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden; Science for Life Laboratory, Department of Medicine Solna, Karolinska Institutet, Stockholm, SwedenDepartment of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, SwedenDepartment of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, SwedenDepartment of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden; Corresponding author at: Center for Molecular Medicine, L8:03, Karolinska University Hospital, 171 76 Stockholm, Sweden.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.http://www.sciencedirect.com/science/article/pii/S2001037021004050CRISPRSequence analysisDrug target discoveryCell assayIn vivo modelHematopoiesis |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yunbing Shen Long Jiang Vaishnavi Srinivasan Iyer Bruno Raposo Anatoly Dubnovitsky Sanjaykumar V. Boddul Zsolt Kasza Fredrik Wermeling |
spellingShingle |
Yunbing Shen Long Jiang Vaishnavi Srinivasan Iyer Bruno Raposo Anatoly Dubnovitsky Sanjaykumar V. Boddul Zsolt Kasza Fredrik Wermeling 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 CRISPR Sequence analysis Drug target discovery Cell assay In vivo model Hematopoiesis |
author_facet |
Yunbing Shen Long Jiang Vaishnavi Srinivasan Iyer Bruno Raposo Anatoly Dubnovitsky Sanjaykumar V. Boddul Zsolt Kasza Fredrik Wermeling |
author_sort |
Yunbing Shen |
title |
A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system |
title_short |
A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system |
title_full |
A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system |
title_fullStr |
A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system |
title_full_unstemmed |
A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system |
title_sort |
rapid crispr competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system |
publisher |
Elsevier |
series |
Computational and Structural Biotechnology Journal |
issn |
2001-0370 |
publishDate |
2021-01-01 |
description |
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. |
topic |
CRISPR Sequence analysis Drug target discovery Cell assay In vivo model Hematopoiesis |
url |
http://www.sciencedirect.com/science/article/pii/S2001037021004050 |
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