CRISPR Switch: Inducible sgRNA expression for tightly regulated, efficient and safer CRISPR/Cas9 genome editing
Keywords
CRISPR/CAS9, genome editing, sgRNA expression cassettes, CRE/lox, recombination, ON/OFF switch, regulation, off-target, safety
Invention Novelty
CRISPR/Cas9 provides an easy, efficient, and affordable tool for genome editing, which has, however, certain limitations when it comes to efficiency, regulation, and off-target effects. Proven tools for achieving tightly regulated gene expression are recombinase-based systems, in which inducible recombinases (e.g., CRE) mediate either activation (ON switch) or inactivation (OFF switch) of a gene of interest. Critical, in both cases, is the presence of recombinase recognition sites (e.g., loxP), which must not interfere with the gene of interest's function and/or expression. For sgRNAs, such placement of the recombinase recognition sites is far from trivial, because sgRNAs lack introns, and the required RNA polymerase III is extremely sensitive to disruptions of the native organization. CRISPR Switch features optimized sgRNA expression cassettes that tolerate the presence of recombinase recognition sites, this way facilitating tightly regulated ON/OFF switches of sgRNA expression.
Value Proposition
Few technologies have advanced the fields of genetics and medicine as much as CRISPR/Cas did and still does. However, the outcome of corresponding experiments doese not onl dependent on the efficiency of Cas-mediated gene editing, but is likewise influenced by e.g., the efficiency of transduction, as well as the timing and location of the editing. These latter aspects are of particular importance when it comes to heterogenous system. To obtain meaningful results in organoids and in vivo, it is essential to decouple transduction and editing, and to take control of where, when, and how long the gene editing is taking place. CRISPR Switch has been designed and developed to provide this control.
Technology Description
CRISPR Switch refers to optimized sgRNA expression cassettes that tolerate the presence of recombinase recognition sites, allowing tightly regulated ON/OFF switches of sgRNA expression. In this regard, the corresponding sgRNA expression cassettes possess optimized sgRNA scaffolds for efficient genome editing. Furthermore, the sgRNAs` guiding sequences are preserved in case of OFF switches, this way facilitating subsequent sgRNA target identification. The technology can be broadly used for all kinds of genome editing applications, including functional genomic in vivo screening (see also TO 44-00013), as well as biomedical and therapeutic uses. One impressive example is CRISPR Switch Over, which allows for strictly sequential gene editing. Here, the sgRNA is not only interrupted by a stop cassette, but also the 5’-region (including promoter) of another guide RNA. In the non-induced state, this assembly results in an active sgRNA 1, while recombination removes the stop cassette and the 5’ region of guide 1, allowing for the expression of guide RNA 2. As shown by Chylinski et al. (2019), this set-up can be exploited, among others, to study tumorigenesis in mice (see Figure).
Commercial Opportunity
CRISPR/Switch is availablefor in-licensing.
Development Status
Practicability of all aspects of CRISPR Switch have been broadly validated in vitro. In all studies, the system showed tight control, minimal leakiness, rapid induction, and high editing activity. The system has been further developed into dependent CRISPR StAR technology, which facilitates reliable and reproducible in vivo screens (see TO 44-00013).
Patent Situation
CRISPR switch is protected by national/regional patent applications in US and EP (based on WO 2017/158154).
Further Reading
Chylinski et al. (2019) Nature Commun. 10: 5454.
Licensing Contact
Technology Manager
T: +49 40 1888 43 48
stachelhaus@ascenion.de
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