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 analyzing the role of the targeted gene in the studied cell population. Here, we present a rapid experimental approach to functionally analyze a CRISPR-targeted cell population that does not involve generating clonal cell lines. As a simple readout, we leverage the CRISPR-induced genetic heterogeneity and use sequencing to identify how different genotypes are enriched or depleted related 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 knowledge to also perform these experiments. As proof of principle, we present examples studying the role of different genes for various aspects related to hematopoietic cells (T cell development in vivo and activation in vitro, macrophage phagocytosis, and 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.
Highlights
‐ CRISPR generates genetic heterogeneity at the targeted site.
‐ Genetic heterogeneity complicates identifying the role of a targeted gene.
‐ Heterogeneity can be quantified by Sanger sequencing with sufficient sensitivity.
‐ Enrichment of specific genotypes can be used to identify roles for targeted genes.
‐ Competitive experiments show the potential of genotype enrichment as a discovery tool.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- (AA)
- Amino acid
- (BM)
- bone marrow
- (CRISPR)
- Clustered Regularly Interspaced Short Palindromic Repeats
- (ctrl)
- control
- (FLA)
- fragment length analysis
- (GMP)
- granulocyte-macrophage precursor
- (HSC)
- hematopoietic stem cell
- (iCR)
- immuno-CRISPR
- (IDAA)
- Indel Detection by Amplicon Analysis
- (ICE)
- Inference of CRISPR Edits
- (InDel)
- insertion or deletion
- (KO)
- knockout
- (Lin)
- lineage
- (NGS)
- next-generation sequencing
- (PBMC)
- peripheral blood mononuclear cells
- (RCC assay)
- rapid CRISPR competitive assay
- (sgRNA)
- single guide RNA
- (shRNA)
- short hairpin RNA
- (siRNA)
- small interfering RNA
- (TIDE)
- Tracking of Indels by Decomposition
- (TRAC)
- T cell receptor alpha chain constant
- (WT)
- wild type