Spatio-Temporal Control of CRISPR Editing With CRISPRoff

A team of scientists from Silicon Valley-based genome engineering company Synthego recently developed CRISPRoff, a revolutionary light-controllable CRISPR tool that makes it possible and easy to control temporal and spatial editing, while also increasing the ratio of on: off target editing events.

Robert Deans, PhD and Chief Scientific Officer at Synthego, highlights the significance of CRISPRoff: »We wanted to invent a technology that could control temporal and spatial CRISPR editing in cells, that was simple, easy to control, and universally applicable, and this is exactly what we’ve gotten with CRISPRoff«.

One of the obvious advantages of using an optically-controlled system such as CRISPRoff is the potential to study precise spatial control of CRISPR editing. The possibility to study the effects of gene editing in specific tissue regions is a game-changer in many areas of research and medicine.

CRISPRoff Guide RNAs

Despite advances in precision, targeting range and applicability of CRISPR to therapeutics in recent years, tools to temporally and spatially control CRISPR editing are lagging behind. Without this control, editing can continue freely in cells until the machinery i.e. Cas endonuclease and single guide RNAs (sgRNA) degrades through cellular processes. Uncontrolled CRISPR editing carries the risk of off-target edits that may result from prolonged or uncontrolled Cas activity.

Recognising that the sgRNA is the most easily programmable component of the CRISPR system, Robert Deans and his team set out to synthesise sgRNAs that could be deactivated in the presence of light.

Jared Carlson-Stevermer, lead author on the study that was recently published in Nature Communications explains the principle behind CRISPRoff:

»We added o-nitrobenzyl groups, which are sensitive to cleavage when exposed to ultraviolet light, at two optimised locations on the sgRNA. This dual breakage sgRNA (DBsgRNA) is prone to fragmentation upon illumination with light of a specific wavelength. We rationalised that once the DBsgRNA is transfected in cells with Cas9 in RNP format, it would edit normally, but exposure to light would result in fragmentation of the sgRNAs and halt further CRISPR editing«.

The team synthesised the modified DBsgRNAs, known as CRISPRoffs using solid phase synthesis, which allowed them to develop a universal sgRNA template that is photocleavable and still retains gene-editing capacity comparable to their standard sgRNA counterparts.

They first generated a variety of CRISPRoff molecules in which fixed positions along the backbone were replaced by photocleavable residues, and evaluated the fragmentation patterns of the modified guides upon exposure to broad-spectrum light. The team then selected promising CRISPRoffs to form RNP complexes that were assessed for editing activity in vitro in human cell lines.

These experiments revealed that it was possible to replace two sites on the sgRNA backbone with photocleavable residues and still retain good editing efficiency.

Universal Control Across Human Cells Lines and Genomic Targets

With the groundwork in place, the team then found the system to be efficient in several human cell lines and most of the 26 genomic targets tested. Indels (off-target insertions and deletions) were observed at low and comparable rates across all sgRNAs and CRISPRoffs tested.

In addition, they observed that photocleavage at either residue deactivates the CRISPRoff, increasing the likelihood that at least one site is cleaved upon illumination and thus strengthening the safety of CRISPRoff.

Improved On: Off Target Event Ratios

While testing their panel of CRISPRoffs against genomic targets, the team included a known cytotoxic sgRNA and corresponding CRISPRoff, and remarkably they observed greater cell survival rates when this CRISPRoff was exposed to UV light. This hinted at an increase in the ratio of on: off target events, which the team was able to verify in subsequent experiments, revealing that controlling DSB formation with the CRISPRoff system reduces off-target CRISPR editing.

On the topic of safety, Jared Carlson-Stevermer also told us: »Our research was not focused on explicitly testing toxicity in vivo, but we can confirm that we did not observe cellular toxicity in our experiments.«

While most of the CRISPRoffs responded to light, the effects of editing efficiency was variable. The team found some evidence that this could be related to individual editing kinetics at each target site.

Jared Carlson-Stevermer shared some additional thoughts on this observation: »Editing efficiency can be different across cell lines and target sites for a variety of reasons, such as local chromatin context, cell cycle state, and the related, predominant repair pathway. This is an interesting area for further study, as we continue to invent fully-controlled editing mechanisms.«

Spatial Patterning With CRISPRoff

The team explored the potential for spatial control with CRISPRoff using a GFP-expressing cell line as a proof of concept and found that it was possible to selectively disrupt GFP expression and thus create a visual patterning effect in distinct regions of a thin layer of cells.

»Our data demonstrates that CRISPRoff is effective and can be immediately useful across multiple genomic targets in multiple cell lines with the potential to optimise on-target editing events and the ability to spatially pattern cells in vitro«, said Robert Deans.