Journal Article

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex

Konermann, Silvana, Trevino, Alexandro E., Habib, Naomi, Nishimasu, Hiroshi, Nureki, Osamu, Joung, Julia, Abudayyeh, Omar O., Barcena, Clea, Brigham, Mark D., Gootenberg, Jonathan S., Hsu, Patrick D., Zhang, Feng

2015

Overview

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology. The CRISPR-Cas9 system, a powerful tool for genome editing, has been engineered to activate endogenous gene transcription specifically and potently on a genome-wide scale and applied to a large-scale gain-of-function screen for studying melanoma drug resistance. CRISPR-Cas9 used for gene-expression regulation The CRISPR-Cas9 system has emerged as a powerful tool for genome editing and transcriptional regulation of specific genes. Feng Zhang and colleagues have successfully modified the system to specifically and potently activate endogenous gene transcription on a genome-wide scale, such that it can be used for large-scale functional genomics screens. Application to a genome-wide screen of melanoma cells for genes which when overexpressed can confer resistance to a BRAF inhibitor demonstrates the feasibility of such screens, and also led to the discovery of potential new resistance mechanisms.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 45/23 / 631/337/572/2102 / Cell Line, Tumor / Clustered Regularly Interspaced Short Palindromic Repeats - genetics / CRISPR-Associated Proteins - genetics / CRISPR-Associated Proteins - metabolism / CRISPR-Cas Systems - genetics / Crystal structure / DNA, Complementary - biosynthesis / DNA, Complementary - genetics / Drug resistance / Drug Resistance, Neoplasm - drug effects / Drug Resistance, Neoplasm - genetics / Enzymes / Epigenetics / Gene expression / Gene Expression Regulation, Neoplastic - genetics / Gene Library / Genetic aspects / Genetic engineering / Genetic Engineering - methods / Genetic Loci - genetics / Genetic regulation / Genetic research / Genetic Testing / Genetic transcription / Genome, Human - genetics / Genomes / Genomics / Health aspects / Humanities and Social Sciences / Humans / Indoles - pharmacology / Melanoma / Melanoma - drug therapy / Melanoma - genetics / multidisciplinary / Nucleotide sequence / Proteins / Proto-Oncogene Proteins B-raf - antagonists & inhibitors / Reproducibility of Results / RNA, Untranslated - biosynthesis / RNA, Untranslated - genetics / RNA, Untranslated - metabolism / Science / Sulfonamides - pharmacology / Transcription factors / Transcriptional Activation - genetics / Up-Regulation - genetics