Proposed field: inherited retinal dystrophies and gene therapy Title of project: Developing CRISPR gRNA delivery strategies for the treatment of retin

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The discovery of CRISPR systems has revolutionised our ability to manipulate the genome. CRISPR-Cas constructs require a guide RNA (gRNA) to direct editing to a target sequence, which can be delivered via an AAV. However, the use of AAVs come with constraints, including packaging limitations, long-term Cas over-expression, and cost-effectiveness due to the need for a unique AAV for each target. Intravitreally delivered chemically modified RNA antisense oligonucleotides have been shown to redirect splicing in photoreceptors. If a Cas-gRNA can be similarly modified and delivered, this would allow us to deliver multiple gRNAs independently, allowing simultaneous or staggered silencing of multiple targets, with applications in research and therapy. The aim of this DPhil is to investigate the separate delivery of chemically modified gRNAs to the retina by an intravitreal route and to examine the impact of the dosage, timing, and modification of these gRNAs on their efficacy. To this end, the key goals of the project are: 1) In vitro knockdown of EGFP: a. Screen gRNAs targeting EGFP in HEK293TdEGFP cells b. Test free modified (with 2'O-methyl and phosphorothioated bonds at the 3 nucleotides at the 3' and 5' ends), unmodified EGFP-targeting and scrambled gRNA in HEK293TdEGFP cells. 3' and 5' modifications were chosen given their high editing efficacy, potential toxicity of excessive modification, and wide commercial availability. c. Investigate efficacy of knockdown with TIDE, fluorescence, western blot, and qPCR 2) In vivo investigation of gRNA behaviour, delivery, and efficacy a. Characterise the SpCas9EGFP and dSpCas9KRAB/Nrl.EGFP mouse b. Deliver free modified and unmodified EGFP-targeting and scrambled gRNA intravitreally to the spCas9 mouse. c. Investigate toxicity using OCT imaging for retinal inflammation and thickness d. Examine penetration of the gRNA in the retinal layers by qPCR e. Examine effects of EGFP knockdown using TIDE, in vivo fluorescence, IHC, Western blot and qPCR 3) Development of self-targeting SaCas9 and spCas9 gRNAs a. Screening of gRNAs targeting SaCas9 and spCas9 in a HEK293T cell line b. As per step 1 above, testing chemically modified and unmodified gRNAs in HEK293TdEGFP cells. c. Investigate efficacy of knockdown with TIDE and western blot. 4) Development of a gRNA targeting EGFP to be used with SaCas9. a. Steps as per step 1 above. The aim of this is to use this guide with an SaCas9 mouse expressing SaCas9 under a GRK1 promoter, crossed with an Nrl.EGFP mouse line to create a mouse with robust SaCas9 and EGFP expression in photoreceptors, to ensure that chemically modified gRNAs are equally active with SaCas9. This step is also proposed because characterisation of the SpCas9 mouse lines showed unexpectedly poor SpCas9 expression in the photoreceptor layer, thus a mouse with more robust photoreceptor Cas9 expression was required. 5) In vivo investigation of Sa gRNA targeting EGFP a. As per step 2 above but using the SaCas9 mouse line 6) In vivo investigation of self-targeting gRNA behaviour a. In both Cas9 mouse lines, investigating the timing and dosage of delivery of a self-targeting gRNA to silence Cas9 expression while maintaining initial good EGFP knockdown.