Screening DNA aptamers that control the DNA cleavage, homology-directed repair, and transcriptional regulation of the CRISPR-(d)Cas9 system

Abstract

Accurate genome editing based on various molecular tools has always been the focus of gene-editing research and the primary goal for therapeutic application. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a well-established gene-editing method that is preferred due to its simplicity and high efficiency. In this study, a group of single-stranded DNA aptamers with high affinity and high specificity for the Cas9 protein were obtained by the systematic evolution of ligands through the exponential enrichment method. Their binding affinity and possible binding domains to theCas9 protein were analyzed. In addition, we demonstrated the effectiveness of aptamers in regulating dCas9-modulated gene transcription, in terms of both transcriptional activationand repression. Additionally, the aptamers successfully reduced the off-target effect and improved the efficiency of gene homologous recombination repair mediated by CRISPR-Cas9. The findings suggest a potential method to better control precise gene editing and enrich the diversity of modulating tools for the CRISPR-Cas9 system.