CRISPR-Switch: Precisely regulated sgRNA for tight, safe and efficient genome editing

Challenge

CRISPR-Cas9 is a widely used, efficient and versatile genome editing tool. Some CRISPR-Cas9 applications, e.g. conditional gene knock-out, essential gene phenotyping or sgRNA library screening require strict temporal and/or spatial control. Multiple inducible systems regulating CRISPR-Cas9 activity exist, but largely suffer from leakiness, reduced activity and lack of specificity.

Technology

CRISPR-Switch (sgRNA with induction/termination by Cre homologous recombination) refers to novel, precisely-controlled sgRNA expression cassettes based on well-established recombinase (Cre-Lox, Flp-FRT) activity. Switch-ON setup facilitates controlled, rapid induction of sgRNA, maintaining its full activity. Alternatively, Switch-OFF setup allows for termination of editing, limiting off-target effects without deletion of the guiding sequence for easy sgRNA target identification in CRISPR screens. Sequential CRISPR-Switch (termed Switch-OVER) edits two loci in a strictly programmable manner, and allows to investigate effects of mutagenic events in a defined order to study e.g. synthetic lethality, temporal order of lesions in tumor progression etc. Importantly, CRISPR switch is compatible with variations of CRISPR such as CRISPR-a, CRISPR-i and others.

Commercial Opportunity

The proprietary technology is available for in-licensing and co-development. CRISPR-Switch can be utilized in many systems and products involving e.g.:

- Screening: Tight Switch-OFF allows for the timing control of CRISPR-Cas9 activity limiting the off-targeting risk while keeping the sgRNA’s guiding sequence, and thus facilitating subsequent target identification in screens. Tight ON switch provides an ideal sample control, especially in randomized or pooled library screens.

- In vivo genome editing: CRISPR ON switch allows for an inducible and/or tissue-specific expression of sgRNA, facilitating a specific spatial and temporal regulation of e.g. gene knock-outs and knock-ins.

- Synthetic lethality and tumor addiction studies: Consecutive genome editing via Switch-OVER allows the system to switch from one sgRNA to another.

- Therapeutic uses: CRISPR-Switch provides high activity and specificity, which represent prerequisites for therapeutic uses.

Development Status

CRISPR-Switch was successfully tested in numerous applications in vitro and in vivo. In all studies conducted, the system showed tight control, minimal leakiness, rapid induction and high editing activity. The technology is ready to be used in basic and applied research.

Patent Situation

IMBA and VBCF have filed a priority-establishing EP application in 2016, and a PCT application in 2017. The IPRs, covering CRISPR-Switch expression cassettes for conditional expression of sgRNA and uses thereof, are currently pending in EP and the US.

Further Reading

Chylinski et al. (2019) CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci. Nat. Comm. 10, Article No.: 5454 (2019)