Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays. J Vis Exp 2019 Jan 07(143)
Date
01/22/2019Pubmed ID
30663717DOI
10.3791/58710Scopus ID
2-s2.0-85060180448 (requires institutional sign-in at Scopus site) 1 CitationAbstract
Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.
Author List
Deshpande A, Chen BR, Zhao L, Saddoris K, Kerr M, Zhu N, Mali P, Deshpande AJAuthor
Nan Zhu PhD Assistant Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsCRISPR-Cas Systems
Gene Editing
Humans
Mice
Mice, Knockout