CRISPR Activation/Interference Screen to Identify Genetic Networks in HDAC-Inhibitor-Resistant Cells

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2022 Oct 18

PMID 36255641

Authors

Paul Jung

Laura Schmalbrock

Matthias Wirth

Affiliations

Soon will be listed here.

Abstract

Epigenetic alterations have been identified in various tumor types. In part, these alterations are mediated via increased histone deacetylase activity. Although preclinical results of monotherapies with histone deacetylase inhibitors (HDACi) are promising, success in clinical trials is limited. Reasons for these limitations may be de novo or acquired resistance to HDAC inhibitors that could be overcome with rational combination therapies. This requires knowledge of resistance mechanism along with the involved genetic networks. One way to identify such genetic networks is the implementation of a CRISPR-based technology allowing transcriptional repression (CRISPRi) and activation (CRISPRa) at a genome-wide scale. We describe a simple approach to amplify and validate sgRNA libraries, generate a myeloid progenitor cell line expressing catalytically dead Cas9 (dCas9) fusion proteins with transcriptional effectors to repress or activate genetic regions of interest and demonstrate a complementary genome-wide HDACi resistance screening approach. Furthermore, we present bioinformatics tools for quality control and analysis of the sequencing data.

Citing Articles

CRISPR-Cas-mediated transcriptional modulation: The therapeutic promises of CRISPRa and CRISPRi.

Bendixen L, Jensen T, Bak R Mol Ther. 2023; 31(7):1920-1937.

PMID: 36964659 PMC: 10362391. DOI: 10.1016/j.ymthe.2023.03.024.

References

Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik V, Paschka P, Roberts N . Genomic Classification and Prognosis in Acute Myeloid Leukemia. N Engl J Med. 2016; 374(23):2209-2221. PMC: 4979995. DOI: 10.1056/NEJMoa1516192. View

Hsu P, Lander E, Zhang F . Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014; 157(6):1262-1278. PMC: 4343198. DOI: 10.1016/j.cell.2014.05.010. View

Doudna J, Charpentier E . Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014; 346(6213):1258096. DOI: 10.1126/science.1258096. View

Gilbert L, Larson M, Morsut L, Liu Z, Brar G, Torres S . CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013; 154(2):442-51. PMC: 3770145. DOI: 10.1016/j.cell.2013.06.044. View

Konermann S, Brigham M, Trevino A, Joung J, Abudayyeh O, Barcena C . Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2014; 517(7536):583-8. PMC: 4420636. DOI: 10.1038/nature14136. View

Chavez A, Scheiman J, Vora S, Pruitt B, Tuttle M, Iyer E . Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 2015; 12(4):326-8. PMC: 4393883. DOI: 10.1038/nmeth.3312. View

Zalatan J, Lee M, Almeida R, Gilbert L, Whitehead E, La Russa M . Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell. 2014; 160(1-2):339-50. PMC: 4297522. DOI: 10.1016/j.cell.2014.11.052. View

Tanenbaum M, Gilbert L, Qi L, Weissman J, Vale R . A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014; 159(3):635-46. PMC: 4252608. DOI: 10.1016/j.cell.2014.09.039. View

Hilton I, DIppolito A, Vockley C, Thakore P, Crawford G, Reddy T . Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol. 2015; 33(5):510-7. PMC: 4430400. DOI: 10.1038/nbt.3199. View

10.

Cheng A, Wang H, Yang H, Shi L, Katz Y, Theunissen T . Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 2013; 23(10):1163-71. PMC: 3790238. DOI: 10.1038/cr.2013.122. View

11.

Friedman J, Fredericks W, Jensen D, Speicher D, Huang X, Neilson E . KAP-1, a novel corepressor for the highly conserved KRAB repression domain. Genes Dev. 1996; 10(16):2067-78. DOI: 10.1101/gad.10.16.2067. View

12.

Kim S, Chen Y, OLeary E, Witzgall R, Vidal M, Bonventre J . A novel member of the RING finger family, KRIP-1, associates with the KRAB-A transcriptional repressor domain of zinc finger proteins. Proc Natl Acad Sci U S A. 1996; 93(26):15299-304. PMC: 26399. DOI: 10.1073/pnas.93.26.15299. View

13.

Moosmann P, Georgiev O, Le Douarin B, Bourquin J, Schaffner W . Transcriptional repression by RING finger protein TIF1 beta that interacts with the KRAB repressor domain of KOX1. Nucleic Acids Res. 1996; 24(24):4859-67. PMC: 146346. DOI: 10.1093/nar/24.24.4859. View

14.

Polstein L, Perez-Pinera P, Kocak D, Vockley C, Bledsoe P, Song L . Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators. Genome Res. 2015; 25(8):1158-69. PMC: 4510000. DOI: 10.1101/gr.179044.114. View

15.

Chavez A, Tuttle M, Pruitt B, Ewen-Campen B, Chari R, Ter-Ovanesyan D . Comparison of Cas9 activators in multiple species. Nat Methods. 2016; 13(7):563-567. PMC: 4927356. DOI: 10.1038/nmeth.3871. View

16.

Gilbert L, Horlbeck M, Adamson B, Villalta J, Chen Y, Whitehead E . Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014; 159(3):647-61. PMC: 4253859. DOI: 10.1016/j.cell.2014.09.029. View

17.

Morita S, Kojima T, Kitamura T . Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 2000; 7(12):1063-6. DOI: 10.1038/sj.gt.3301206. View

18.

Lee J, Hapel A, Ihle J . Constitutive production of a unique lymphokine (IL 3) by the WEHI-3 cell line. J Immunol. 1982; 128(6):2393-8. View

19.

Pfaffl M . A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001; 29(9):e45. PMC: 55695. DOI: 10.1093/nar/29.9.e45. View

20.

Joung J, Konermann S, Gootenberg J, Abudayyeh O, Platt R, Brigham M . Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc. 2017; 12(4):828-863. PMC: 5526071. DOI: 10.1038/nprot.2017.016. View