Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions

Abstract

A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

“…Aptazyme riboswitches have also been used by Tang et al to control gRNA function, enabling theophylline-induced genome editing and guanine-dependent targeting of transcriptional activators and achieving 5-6-fold regulation in each application [188]. Lin et al recently used short trigger RNAs, including an endogenous miRNA, to modulate gRNA function in HEK293T cells, although as with aptazyme switches oligonucleotides are less favorable regulators than small molecules [189]. A particularly interesting case was recently reported by Renzl et al, who incorporated aptamers to the photoreceptor PAL into gRNAs and demonstrated 546-fold regulation of mRNA levels in response to light in HeLa cells.…”

Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR–Cas12a gRNA switch

“…This simplified the sensing of natural RNAs and, through the variation of sub-domain length, demonstrated the first example of modulating a CRISPR gRNA switch response to a given RNA trigger. Recent work has also highlighted ways to separate the guide and trigger RNA sequences for Cas9 sgRNA switches, including a design framework paralleling the one employed here ( 47 ) or involving three separate RNAs ( 48 ).…”

Controllable DNA strand displacement by independent metal–ligand complexation

“…In the past two decades, with the employment of toehold-mediated strand-displacement reactions, dynamic nucleic acid technology has been widely applied in the information technology (DNA computation), 26,27 biomedical analysis, [34][35][36] drug delivery and release [37][38][39][40] and synthetic biology. [41][42][43][44][45][46][47][48] Unlike the direct strand displacement, the toehold-mediated reaction is both thermodynamically and kinetically favored. In order to achieve a full potential for the displacement kinetics, the minimal binding ability between the invader strand and the toehold is needed, with a typical length of single-stranded DNA over 6-nt for the conventional toehold (Figure 1a).…”