SELF-LIMITING Cas9 CIRCUITRY FOR ENHANCED SAFETY (SLiCES) PLASMID AND LENTIVIRAL SYSTEM THEREOF

Abstract

The present invention describes a Self-Limiting Cas9 circuitry for Enhanced Safety (SLiCES) which consists of an expression unit for the Streptococcus pyogenes Cas9 (SpCas9), a first Cas9 self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self limiting circuit, by controlling Cas9 levels, results in increased genome editing specificity. For its in vivo utilization, SLiCES was integrated into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Following its delivery into target cells, the lentiSLiCES circuit is switched on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, the present hit and go system increases safety margins for genome editing.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an expression unit for CRISP / Cas9 technology and related lentiviral particles. Background technique [0002] The in vivo application of CRISPR / Cas9 technology is still limited by unwanted genome cleavage by Cas9. Long-term expression of Cas9 increases the number of genomic loci that are nonspecifically cleaved by nucleases. [0003] Genome editing by CRISPR / Cas9 technology has great potential for both basic and clinical applications because of its simplicity, target design plasticity, and multiple targeting capabilities. A major limitation of the use of CRISPR / Cas9 is the mutation induced at a site different from the intended target. This is critical for in vivo applications, as unwanted changes can lead to unfavorable clinical outcomes. [0004] An important factor affecting the amount of off-target modifications is the amount and persistence of SpCas9 expression in target cells:...

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Problems solved by technology

Nonetheless, methods reported to date suffer from nuclease cleavage (Wright, A.V. et al., Proc. Natl. Acad. 2015, 291 Sci. U.S.A. 112, 2984–2989) or chemical modification (Davis, K.M. et al., Nat. Chem. Biol. 2015, 11, 316–318) leading to a reduction of editing activity to background activity (Nihongaki, Y. et al., Nat. Biotechnol. 2015, 33, 755–760) or a prolonged time to induction (Zetsche, B. et al., Many limitations of Nat. Biotechnol. 2015, 33, 139–142)

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