CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes

By Luke A. Gilbert, Matthew H. Larson, Leonardo Morsut, Zairan Liu, G. Brar, S. E. Torres, Noam Stern-Ginossar, Onn Brandman, E. Whitehead, Jennifer A. Doudna and 3 more. Cell, 2013

3,520 citations recorded by the source corpus.

SUMMARY The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells with the site of delivey determined solely by a co-expressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence and reveal the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/3c80e1c07ea3d94e38c1b5a35f91364f3142c5a8

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Citations in the corpus

Interpreting thelanguage of histone and DNA modifications (2014, background): “Our ability to now site-specifically modify the chromatin landscape in complex eukaryotic systems using re-purposed genome editing tools like zinc-finger (ZF) nuclease, TALE nuclease, and clustered regulatory interspaced short palindromic repeat (CRISPR) technologies [228–231] is an important step t”
Transcriptional enhancers: from properties to genome-wide predictions (2014, background): “Through these domains, transcriptional activators (part Aa ), repressive transcription factors (TFs) 117,137,139 (part Ab ) or chromatin-modifying domains 138,139 (part Ac ) can be recruited to genomic regulatory regions to modulate the transcription of target genes.”
Activity-by-Contact model of enhancer-promoter regulation from thousands of CRISPR perturbations (2019, methodology): “We assumed the gRNAs targeting the TSS of the assayed gene have a “true” effect size of 85% (based on previous observations that show CRISPRi effects of 80–90% across a panel of genes 8 ), but that some portion of the FlowFISH signal is due to non-specific binding of the probe.”
The new frontier of genome engineering with CRISPR-Cas9 (2014, positive): “Specifically, fusion of dCas9 to effector domains including VP64 or KRAB allowed stable and efficient transcriptional activation or repression, respectively, in human and yeast cells (129).”
The KRAB Domain of ZNF10 Guides the Identification of Specific Amino Acids That Transform the Ancestral KRAB-A-Related Domain Present in Human PRDM9 into a Canonical Modern KRAB-A Domain (2022, methodology): “In conjunction with CRISPR/Cas the ZNF10-KRAB is used as a tool for modulating and redirecting transcriptional gene regulation in basic research (CRISPRi; [27,28]), and applied in oncology (e.”
Highly Specific Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements (2015, positive): “…to DNA, KRAB recruits a heterochromatin-forming complex that causes histone methylation and deacetylation 25–28 . dCas9-KRAB fusions have effectively silenced non-coding RNAs and single genes when targeted to promoter regions, 5’ untranslated regions, and proximal enhancer elements 6, 9, 16, 18 .”