Spatiotemporal control of CRISPR/Cas9 gene editing

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) gene editing technology, as a revolutionary breakthrough in genetic engineering, offers a promising platform to improve the treatment of various genetic and infectious diseases because of its simple design and powerful ability to edit different loci simultaneously. However, failure to conduct precise gene editing in specific tissues or cells within a certain time may result in undesirable consequences, such as serious off-target effects, representing a critical challenge for the clinical translation of the technology. Recently, some emerging strategies using genetic regulation, chemical and physical strategies to regulate the activity of CRISPR/Cas9 have shown promising results in the improvement of spatiotemporal controllability. Herein, in this review, we first summarize the latest progress of these advanced strategies involving cell-specific promoters, small-molecule activation and inhibition, bioresponsive delivery carriers, and optical/thermal/ultrasonic/magnetic activation. Next, we highlight the advantages and disadvantages of various strategies and discuss their obstacles and limitations in clinical translation. Finally, we propose viewpoints on directions that can be explored to further improve the spatiotemporal operability of CRISPR/Cas9.

Deciphering the Retinal Epigenome during Development, Disease and Reprogramming: Advancements, Challenges and Perspectives

“…Second, by leveraging all known cis - and trans -regulatory sites, CRISPR-Cas9 can be repurposed for simultaneous, reversible gene activation or silencing by conjugating the catalytically inactive caspase9 dCas9 to an effector protein [ 276 , 277 , 278 ]. A short guide RNA (sgRNA) with a protospacer adjacent motif (PAM) complementary to the targeted genomic sites can then direct the dCas9-KRAB-Effector transcriptional suppresser [ 279 ] or the dCas9-SAM synergistic activation mediator [ 280 ], to specific genetic loci of interest.…”

Tailoring Cardiac Synthetic Transcriptional Modulation Towards Precision Medicine

“…Taming Cas9 expression or activity using pharmacological approaches might therefore be a prospective route for temporally restricted or pulsed endogenous gene activation, possibly reducing expected immune responses upon constitutive Cas9 expression (159) and deserves a future validation in in vivo models. Bioengineering of Cas9 proteins as well as elaborate cell-type specific and temporally resolved Cas9 expression systems (151) are therefore essential ventures for safe and limited CRISPR/Cas9 based applications (71). Protein-based anti-CRISPRs, which are accessory proteins with fewer than 200 amino acids called "anti-CRISPRs, " can function as antagonists of CRISPR systems and achieve context-specific inhibition of Cas9.…”

Advanced Nanotheranostics of CRISPR/Cas for Viral Hepatitis and Hepatocellular Carcinoma

“…A given stimulus to control spatial and temporal delivery has received lots of attention. [ 140 ] Stimuli‐responsive nanoparticles can facilitate endosome escape and liberate the CRISPR cargo with remote control. Moreover, designing ligands for ASGPR‐mediated hepatocyte targeting has been investigated most in the relative applications.…”