A Genome-Editing Nanomachine Constructed with a Clustered Regularly Interspaced Short Palindromic Repeats System and Activated by Near-Infrared Illumination

Abstract

The RNA-guided CRISPR/Cas9 system is a powerful genome-editing technology with broad applications. Improving delivery efficiency and controllable activity of the CRISPR/Cas9 system is an area of intense research. We report the design, construction, and application of a CRISPR/Cas9 nanomachine (LACM), activated by a near-infrared (NIR) laser, which enables efficient delivery of single-guide RNA (sgRNA) into living cells and achieves controlled release of the sgRNA for the CRISPR/Cas9 activity. The LACM was constructed using a gold nanorod (AuNR) as a carrier that was decorated with dozens of protector DNAs stably hybridizing with the target binding domain of sgRNA. The DNA assembly on the AuNR protected the sgRNA. Irradiation with a NIR laser generated heat on the AuNR, resulting in controlled release of sgRNA, which guided the CRISPR/Cas9 genome editing. Successful editing of the <i>EGFP</i> and <i>EMX1</i> genes in A549 and HEK293T cells, as well as knocking down of the <i>PLK1</i> gene to induce apoptosis of the target cells, highlights the promising potential of the LACM for diverse applications.