Maybe you can turn me on: CRISPRa-based strategies for therapeutic applications

Abstract

Since the revolutionary discovery of the CRISPR-Cas technology for programmable genome editing, its range of applications has been extended by multiple biotechnological tools that go far beyond its original function as "genetic scissors". One of these further developments of the CRISPR-Cas system allows genes to be activated in a targeted and efficient manner. These gene-activating CRISPR-Cas modules (CRISPRa) are based on a programmable recruitment of transcription factors to specific loci and offer several key advantages that make them particularly attractive for therapeutic applications. These advantages include inter alia low off-target effects, independence of the target gene size as well as the potential to develop gene- and mutation-independent therapeutic strategies. Herein, I will give an overview on the currently available CRISPRa modules and discuss recent developments, future potentials and limitations of this approach with a focus on therapeutic applications and in vivo delivery.

Keywords: CRISPR-mediated transcriptional activation; CRISPRa; Cas9-MPH; Cas9-SAM; Cas9-VP64; Cas9-VPR; Gene activation; Gene therapy; Transcriptional activation.

Fig. 1

Overview of CRISPRa modules that have been successfully applied to treat genetic or acquired diseases. Right panel, strategies and expression cassettes used for in vivo delivery of the individual modules. The red asterisk highlights the expression cassette which exceeds the packaging capacity of rAAV vectors (4.7 kb). TRE tetracycline response element. UbProm ubiquitous promoter (e.g., CAG or EF1a). pA polyadenylation signal, U6 human Pol III promoter, CMV cytomegalovirus immediate-early promoter, RHO human Rhodopsin promoter, N- or C-SpdCas9 N-or C-terminal part of split SpdCas9, N-or C-int N-or C-terminal part of split intein, dgRNA “dead” sgRNA with short spacer sequence (14–15 bp) harboring a MS2 stem-loop