23 April 2020

April 2020

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

CRISPR/Cas9 is a novel genome editing tool, that consists of two parts: The single-guide RNA (sgRNA) is a 20-nucleotide string, that can be designed to target any region within the genome. It guides the Cas9 enzyme to the desired gene locus, where Cas9 introduces a cut. This cut is then repaired by cell-internal repair mechanisms and can result in random changes or carefully designed alterations of the gene. With the possibility to alter any desired gene, CRISPR/Cas9 is not only important for basic research but might also pave the way for new therapeutic applications. However, the efficiency with which the genome editing machinery is delivered to cells is often limited. In addition, potential off-target effects are a major safety concern. The term describes the risk of introducing unintended cuts in genes that show high similarity with the target region.


Here, Peng and colleagues introduce a new near-infrared (NIR) inducible CRISPR/Cas9 nanomachine, called LACM. It combines efficient delivery with spatio-temporal controllable CRISPR/Cas9 activity. The LACM consists of a gold nanoparticle as carrier. This carrier can enter the cell without the need for special reagents and be combined with many DNA particles. Those DNA particles consist of the sgRNA, carefully designed to target the desired gene locus, and a protector DNA. The protector DNA binds to the sgRNA and the gold particle and is thus essential to form the LACM. But it is also important to control the gene editing activity: The protector DNA binds to the sgRNA and thus keeps it inactive. When the cells are irradiated with a NIR laser, the gold particle generates heat, leading to the release (and thus activation) of the sgRNA. The protector DNA is designed to form a hairpin structure upon sgRNA release. This prevents the sgRNA from re-binding to the nanomachine. Thus, the LACM combines easy and efficient uptake into the cells, with controllability of the CRISPR/Cas9 editing by activation with a NIR laser.


The LACM was tested in A549 cells with stable expression of Cas9 and green fluorescent protein (GFP). The LACM was designed to target the GFP gene, so that successful gene editing results in decreased fluorescence. Treating cells with the LACM in combination with NIR irradiation resulted in a significant decrease of fluorescent signal. Analysis of the mutational frequency demonstrated that the LACM was comparable to already established methods. An additional LACM was designed to evaluate effects for an endogenous target as well as 4 potential off-targets. Successful gene editing was only detected for the target gene. The LACM was also successfully used for gene-editing in HEK293T cells.


The authors have thus introduced a new approach to deliver CRISPR/Cas9 efficiently to cells and control its activity by irradiation with a NIR laser. Notably, the irradiation time did not have effects on cell viability and does not require special reagents to allow the CRISPR machinery to enter the cell. It has further been shown that the usage is not restricted to one particular cell line.