Gene activation by dCas9-CBP and the SAM system differ in target preference

Abstract

Gene overexpression through the targeting of transcription activation domains to regulatory DNA via catalytically defective Cas9 (dCas9) represents a powerful approach to investigate gene function as well as the mechanisms of gene control. To date, the most efficient dCas9-based activator is the Synergistic Activation Mediator (SAM) system whereby transcription activation domains are directly fused to dCas9 as well as tethered through MS2 loops engineered into the gRNA. Here, we show that dCas9 fused to the catalytic domain of the histone acetyltransferase CBP is a more potent activator than the SAM system at some loci, but less efficient at other locations in Drosophila cells. Our results suggest that different rate-limiting steps in the transcription cycle are affected by dCas9-CBP and the SAM system, and that comparing these activators may be useful for mechanistic studies of transcription as well as for increasing the number of hits in genome-wide overexpression screens.

Epigenetic editing: Dissecting chromatin function in context

“…Moreover, Cpf1 utilizes a thymidine-rich PAM, such as TTTN, thus providing a good alternative to SpdCas9 when targeting C/G-poor genomic regions as their full-length counterparts, while guarding against the confounding effects of recruiting other regulatory proteins. [25,82] In recent years, the development of the dCas9 -SunTag or -SAM systems has enabled simultaneous recruitment of several copies of the same effector or of different transcriptional regulators simultaneously to a target locus [83][84][85] (Figure 2). Despite the caveat of recruiting unphysiologically large amount of proteins, the transient targeting of multiple effectors has proven a valuable principle to maximize the quantitative level of on-target epigenome editing [79,[86][87][88] and to minimize dCas9 off-target activity.…”

“…In recent years, the development of the dCas9 ‐SunTag or ‐SAM systems has enabled simultaneous recruitment of several copies of the same effector or of different transcriptional regulators simultaneously to a target locus [ 83–85 ] (Figure 2). Despite the caveat of recruiting unphysiologically large amount of proteins, the transient targeting of multiple effectors has proven a valuable principle to maximize the quantitative level of on‐target epigenome editing [ 79,86–88 ] and to minimize dCas9 off‐target activity.…”

Chromatin Manipulation and Editing: Challenges, New Technologies and Their Use in Plants

“…In non-mammalian species, the direct fusion of dCas9 with the histone acetyltransferase domain CBP was reported to be a more potent activator than a SAM system targeting three different transcription activation domains in Drosophila cells. Interestingly, the opposite was observed at promoters or enhancers pre-marked with H3K27 acetylation [ 150 ].…”

Diversification of the CRISPR Toolbox: Applications of CRISPR-Cas Systems Beyond Genome Editing

“…54 Additional research continues to improve CRISPR-based gene regulation techniques. 55,56 The generation of chimeric dCas9 variants with proteins capable of altering epigenetic marks further expands the capabilities of CRISPR for gene regulation. This strategy closely resembles epigenetic modulation with ZFPs and TALEs.…”