Development of aptamer-based inhibitors for CRISPR/Cas system

Abstract

The occurrence of accidental mutations or deletions caused by genome editing with CRISPR/Cas9 system remains a critical unsolved problem of the technology. Blocking excess or prolonged Cas9 activity in cells is considered as one means of solving this problem. Here, we report the development of an inhibitory DNA aptamer against Cas9 by means of in vitro selection (systematic evolution of ligands by exponential enrichment) and subsequent screening with an in vitro cleavage assay. The inhibitory aptamer could bind to Cas9 at low nanomolar affinity and partially form a duplex with CRISPR RNA, contributing to its inhibitory activity. We also demonstrated that improving the inhibitory aptamer with locked nucleic acids efficiently suppressed Cas9-directed genome editing in cells and reduced off-target genome editing. The findings presented here might enable the development of safer and controllable genome editing for biomedical research and gene therapy.

An Update of Nucleic Acids Aptamers Theranostic Integration with CRISPR/Cas Technology

“…Zhao et al utilized in vitro selection to generate an inhibitory DNA aptamer against Cas9. This inhibitory aptamer could bind to Cas9 in low nanomolar affinity [ 55 ]. This high-affinity binding interaction lowered the Cas-9 directed genome editing within cells and reduced off-target effects.…”

Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas

“…As an alternative, several regulation systems have been developed to control the expression and activity of the CRISPR-Cas module. Examples include the use of inducible promoters ( 6–8 ), inducible intein splicing ( 9 , 10 ), split Cas proteins ( 11 , 12 ), inducible conformation change ( 13 ), inducible inhibition through aptamers ( 14 ) and inducible translation through riboswitches ( 15 ). Other approaches focused on the inducible guide RNA functionality using ribozymes ( 16 ), riboswitches ( 17 , 18 ) and photocaging ( 19–22 ).…”

“…The catalytic and/or regulatory functionality of these RNA molecules relies on their primary, secondary and tertiary structures, making them great candidates for developing universal tools for regulating gene expression, without the use of proteins ( 23–27 ). To this end, several studies used ribozymes and riboswitches to control the expression of a gene of interest (GOI), but also for regulating the activity and function of CRISPR-Cas ( 14–18 , 24 ). Nevertheless, these approaches leave room for improvement.…”

“…(2019) ( 17 ) and Zhao et al. (2020) ( 14 ) rely on the secondary structure of the Cas9 single guide RNA (sgRNA), which rules out the use of other CRISPR-Cas systems. Lastly, the RiboCas technology developed by Cañadas et al.…”

SIBR-Cas enables host-independent and universal CRISPR genome engineering in bacteria

“…As an alternative, several regulation systems have been developed to control the expression and activity of the CRISPR-Cas module. Examples include the use of inducible promoters, inducible intein splicing 3 , split Cas proteins 4,5 , inducible conformation change 6 , inducible inhibition through aptamers 7 and inducible translation through riboswitches 8 . Other approaches focused on the inducible guide RNA functionality using ribozymes 9 , riboswitches 10,11 and photocaging 12 .…”