CRISPR has ushered in an era of precision editing of the genome, enabling researchers to develop new therapies and biomedicines. While CRISPR applications are abundant, it bodes well to remember that this technique is less than a decade old. Therefore, researchers are still optimizing and fine-tuning the method to ensure maximum safety and efficacy before it can be applied in therapy. Synthego has always been at the forefront of improving CRISPR technology and spearheading innovation in the field.

One of the primary concerns with unleashing molecular scissors inside cells is the possibility of cutting at unintended sites i.e., off-targets. Wouldn’t it be great to have a switch to control CRISPR editing in cells at will?

We are excited to report that Synthego has developed a new technique, CRISPRoffTM, that enables light-based inactivation of CRISPR editing in different cell lines across multiple gene targets. Synthego also successfully tuned on-target to off-target ratios using temporal optimization of the switch-off mechanism.

This work, published in Nature Communications, lays the foundation for more precise gene editing with controlled CRISPR editing for future therapeutics.

CRISPRoff Mechanism: Light at the End of an Edit

Methods for controlling CRISPR editing in cells, such as anti-CRISPR proteins or modified nucleases, do exist. However, they require additional components and laborious engineering steps. An ideal switch should be simple, easy to control, and universally applicable.

Figure 1. Schematic of CRISPRoff mechanism. Transfecting cells with ribonucleoprotein formed using DBsgRNA (sgRNA modified with light-responsive nucleotides) will result in editing at the target locus, similar to standard CRISPR reaction. Exposure to light breaks the DBsgRNA at specific sites, rendering it non-functional, thus “switching off” the editing.

As single guide RNA (sgRNA) is the most easily programmable component of a CRISPR system, o-nitrobenzyl groups, which are sensitive to cleavage when exposed to ultraviolet light, are added at two optimized locations on sgRNA. This dual-breakage sgRNA (DBsgRNA) is prone to fragmentation on illumination with light of a specific wavelength. This means that once the DBsgRNA is transfected in cells with Cas9 in a ribonucleoprotein (RNP) format, it can edit normally, but exposure to light results in fragmentation of the guides and halts further CRISPR editing.

CRISPRoff Controls CRISPR Editing in Cells

We first verified that DBsgRNA breaks on exposure to light of a specific wavelength, as expected, using a fragment analyzer. Next, we compared editing at a target locus with DBsgRNAs and standard sgRNAs in cells. On analyzing the genomic sequence using ICE, we noted comparable indels in the absence of light stimulation. In the samples exposed to light of specific wavelength post transfection, we found that the DBsgRNAs were no longer able to induce indels, whereas the standard sgRNA remained functional and unaffected.

Figure 2: CRISPRoff halts editing following exposure to light. Data shows the editing efficiency at target locus in HEK293 cells under different conditions. DBsgRNA and sgRNa show comparable editing efficiencies in the absence of light. After exposure to light, editing efficiency drops drastically in DBsgRNA samples, whereas the editing prowess of standard sgRNA remains unaffected. (n=2 paired experimental replicates, data is presented as mean).

Synthego successfully demonstrated that light-controlled CRISPR editing in cells using CRISPRoff is applicable across different gene targets and cell lines.

Minimizing off-targets is one of the primary goals of any CRISPR control system, due to the potential of unintended, harmful secondary effects that may occur. On-target editing has been shown to occur faster than off-target edits in other studies. Synthego, therefore, exposed samples to light at different time points and noted the on target to off-target ratio in each sample. Data showed that illuminating cells at early time points resulted in higher on target to off-target ratio in DBsgRNAs, offering a convenient way to minimize off-targets.

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CRISPRoff Highlights: Why This Light-Control Technology is Not to Be Taken Lightly

CRISPR genome editing soared in popularity, primarily because of the convenience and precision it offered to researchers. However, concerns about promiscuous editing and guaranteed safety have begun to be discussed.

CRISPRoff is a programmable DBsgRNA that can be integrated into existing CRISPR workflows without additional need for optimization or special experiments. While Synthego demonstrated the proof of concept using Cas9 in this publication, the basic idea can be expanded to other nucleases, and the photosensitive groups are also amenable to variations.

Importantly, CRISPR can be a useful tool in next-generation cell and gene therapy trials, if the off-target concerns are addressed. A future where clinical-grade sgRNA amenable to easily being switched off is available off-the-shelf is not far away.

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