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...

Full description

Bibliographic Details
Main Authors: Yunbing Shen, Long Jiang, Vaishnavi Srinivasan Iyer, Bruno Raposo, Anatoly Dubnovitsky, Sanjaykumar V. Boddul, Zsolt Kasza, Fredrik Wermeling
Format: Article
Language:English
Published: Elsevier 2021-01-01
Series:Computational and Structural Biotechnology Journal
Subjects:
Online Access:http://www.sciencedirect.com/science/article/pii/S2001037021004050
id doaj-7ef9e66cdc654f80b5bdd9d389d86c63
record_format Article
spelling 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
work_keys_str_mv AT yunbingshen arapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT longjiang arapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT vaishnavisrinivasaniyer arapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT brunoraposo arapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT anatolydubnovitsky arapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT sanjaykumarvboddul arapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT zsoltkasza arapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT fredrikwermeling arapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT yunbingshen rapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT longjiang rapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT vaishnavisrinivasaniyer rapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT brunoraposo rapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT anatolydubnovitsky rapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT sanjaykumarvboddul rapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT zsoltkasza rapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
AT fredrikwermeling rapidcrisprcompetitiveassayforinvitroandinvivodiscoveryofpotentialdrugtargetsaffectingthehematopoieticsystem
_version_ 1716859463268302848